Stimulation by Erwinia carotovora of the synthesis of ethylene in cauliflower tissue

PubMed Central

Lund, Barbara M.; Mapson, L. W.

1970-01-01

The synthesis of ethylene by cauliflower floret tissue was increased when the tissue was inoculated with the soft-rot bacterium Erwinia carotovora. This effect was clearly associated with the production of pectic enzymes by the micro-organism. These enzymes, acting together with the plant enzymes, stimulated the production of ethylene from methionine. The increased synthetic activity was due to the release and increased activity of a glucose oxidase enzyme apparently attached to plant cell-wall material and liberated by the action of pectic enzymes of the bacterium. ImagesPLATE 1 PMID:5488914

Changes in digestive enzyme activities during larval development of Chinese loach Paramisgurnus dabryanus (Dabry de Thiersant, 1872).

PubMed

Zhang, Yun-Long; Wu, Qiao-Wan; Hu, Wei-Hua; Wang, Fan; Zhao, Zhong-Bo; He, Hui; Shao, Wei-Han; Fan, Qi-Xue

2015-12-01

The digestive physiology of Chinese loach (Paramisgurnus dabryanus) was studied by assessing the specific and total activities of different pancreatic (trypsin, chymotrypsin, amylase and lipase), gastric (pepsin) and intestinal (alkaline phosphatase and leucine-aminopeptidase) enzymes from hatching to 40 days after hatching (DAH). Larvae were reared at 24.4 ± 0.4 °C and fed with rotifers from mouth opening (4 DAH) to 15 DAH, from 10 to 35 DAH with Cladocera and from 30 to 40 DAH with compound diet. Enzyme activities for trypsin, chymotrypsin, amylase and lipase were detected before the onset of exogenous feeding, indicating that these enzymes were genetically pre-programmed. Most of the pancreatic enzyme specific activities increased until 20 DAH and decreased thereafter. The pepsin activity of Chinese loach was firstly detected at 30 DAH, indicating the appearance of functional gastric gland. Alkaline phosphatase specific activity was detected from hatching onward, showed marked increase and reached the second peak at 20 DAH, while a gradual increase in specific leucine-aminopeptidase activity was observed until the end of the experiment. Accordingly, the larvae of Chinese loach possess a functional digestive system before the onset of exogenous feeding and the digestive capacity gradually increases as development progresses. The abrupt increase in intestinal enzyme activities between 10 and 20 DAH demonstrates onset of juvenile-like digestive mode in Chinese loach larvae. The increase in pepsin activity after 30 DAH indicates the shift from alkaline to acidic digestion in Chinese loach larvae, which may be considered as the onset of weaning.

Effect of benzo[a]pyrene on detoxification and the activity of antioxidant enzymes of marine microalgae

NASA Astrophysics Data System (ADS)

Shen, Chen; Miao, Jingjing; Li, Yun; Pan, Luqing

2016-04-01

The objective of this study was to examine the effect of benzo[a]pyrene (BaP) on the detoxification and antioxidant systems of two microalgae, Isochrysis zhanjiangensis and Platymonas subcordiformis. In our study, these two algae were exposed to BaP for 4 days at three different concentrations including 0.5 μg L-1 (low), 3 μg L-1 (mid) and 18 μg L-1 (high). The activity of detoxification enzymes, ethoxyresorufin O-deethylase (EROD) and glutathione S-transferase (GST) increased in P. subcordiformis in all BaP-treated groups. In I. zhanjiangensis, the activity of these two enzymes increased at the beginning of exposure, and then decreased in the groups treated with mid- and high BaP. The activity of antioxidant enzyme superoxide dismutase (SOD) increased in I. zhanjiangensis in all BaP-treated groups, and then decreased in high BaP-treated group, while no significant change was observed in P. subcordiformis. The activity of antioxidant enzyme catalase (CAT) increased in I. zhanjiangensis and P. subcordiformis in all BaPtreated groups. The content of malondialdehyde (MDA) in Isochrysis zhanjiangensis increased first, and then decreased in high BaP-treated group, while no change occurred in P. subcordiformis. These results demonstrated that BaP significantly influenced the activity of detoxifying and antioxidant enzymes in microalgae. The metabolic related enzymes (EROD, GST and CAT) may serve as sensitive biomarkers of measuring the contamination level of BaP in marine water.

Activation of immobilized enzymes by acoustic wave resonance oscillation.

PubMed

Nishiyama, Hiroshi; Watanabe, Tomoya; Inoue, Yasunobu

2014-12-01

Acoustic wave resonance oscillation has been used successfully in the development of methods to activate immobilized enzyme catalysts. In this study, resonance oscillation effects were demonstrated for enzyme reactions on galactose oxidase (GAD), D-amino acid oxidase (DAAO), and L-amino acid oxidase (LAAO), all of which were immobilized covalently on a ferroelectric lead zirconate titanate (PZT) device that could generate thickness-extensional resonance oscillations (TERO) of acoustic waves. For galactose oxidation on immobilized GAD in a microreactor, TERO generation immediately increased enzyme activity 2- to 3-fold. Eliminating TERO caused a slight decrease in the activity, with ∼90% of the enhanced activity retained while the reaction proceeded. Contact of the enhanced enzyme with a galactose-free solution caused almost complete reversion of the activity to the original low level before TERO generation, indicating that, not only TERO-induced GAD activation, but also preservation of the increased activity, required a galactose substrate. Similar activity changes with TERO were observed for enzyme reactions on DAAO and LAAO. Kinetic analysis demonstrated that TERO helped strengthen the interactions of the immobilized enzyme with the reactant substrate and promoted formation of an activation complex. Copyright © 2014 Elsevier Inc. All rights reserved.

Is Liver Enzyme Release Really Associated with Cell Necrosis Induced by Oxidant Stress?

PubMed

Contreras-Zentella, Martha Lucinda; Hernández-Muñoz, Rolando

2016-01-01

Hepatic diseases are a major concern worldwide. Increased specific plasma enzyme activities are considered diagnostic features for liver diseases, since enzymes are released into the blood compartment following the deterioration of the organ. Release of liver mitochondrial enzymes is considered strong evidence for hepatic necrosis, which is associated with an increased production of ROS, often leading to greater hepatic lipid peroxidation. Lipotoxic mediators and intracellular signals activated Kupffer cells, which provides evidence strongly suggesting the participation of oxidant stress in acute liver damage, inducing the progression of liver injury to chronic liver damage. Elevated transaminase activities are considered as an index marker of hepatotoxicity, linked to oxidant stress. However, a drastic increase of serum activities of liver enzyme markers ought not necessarily to reflect liver cell death. In fact, increased serum levels of cytoplasmic enzymes have readily been observed after partial hepatectomy (PH) in the regenerating liver of rats. In this regard, we are now showing that in vitro modifications of the oxidant status affect differentially the release of liver enzymes, indicating that this release is a strictly controlled event and not directly related to the onset of oxidant stress of the liver.

[Hepatic allopurinol oxidizing enzyme in mice].

PubMed

Huh, K; Iwata, H; Yamamoto, I

1975-03-01

The relationship between allopurinol oxidizing enzyme and aldehyde oxidase was investaged in mice. The oxidation of both N-methylnicotinamide and allopurinol appears to be catalized by a single enzyme, aldehyde oxidase (aldehyde-oxygen oxidoreductase EC, 1.2.3.1.). This conclusion is based on the following evidence; The postnatal changes of allopurinol and N-methylnicotinamide oxidizing activities were similar during growth and the levels of both activities increased in a parallel fashion upon the attainment of sexual maturity. The rates of loss of the activities of both enzymes by heat denaturation as well as dexamethasone administration were similar. The inhibitors of allopurinol oxidizing enzyme also suppressed N-methylnicotinamide oxidation. Competition of N-methylnicotineamide and allopurinol for oxidation was demonstrated. The rate of increase of the activities in both enzymes was almost parallel during each step of the purification from mouse liver supernatant. It was ascertained that xanthine oxidase in the enzyme preparation does not influence allopurinol oxidation.

Effects of non-starch polysaccharides enzymes on pancreatic and small intestinal digestive enzyme activities in piglet fed diets containing high amounts of barley.

PubMed

Li, Wei-Fen; Feng, Jie; Xu, Zi-Rong; Yang, Cai-Mei

2004-03-15

To investigate effects of non-starch polysaccharides(NSP) enzymes on pancreatic and small intestinal digestive enzyme activities in piglet fed diets containing high amounts of barley. Sixty crossbred piglets averaging 13.5 kg were randomly assigned to two treatment groups with three replications (pens) based on sex and mass. Each group was fed on the diet based on barley with or without added NSP enzymes (0.15%) for a 40-d period. At the end of the experiment the pigs were weighed. Three piglets of each group were chosen and slaughtered. Pancreas, digesta from the distal end of the duodenum and jejunal mucosa were collected for determination. Activities of the digestive enzymes trypsin, chymotrypsin, amylase and lipase were determined in the small intestinal sections as well as in homogenates of pancreatic tissue. Maltase, sucrase, lactase and gamma-glutamyl transpeptidase (gamma-GT) activities were analyzed in jejunal mucosa. Supplementation with NSP enzymes improved growth performance of piglets. It showed that NSP enzymes had no effect on digestive enzyme activities in pancreas, but decreased the activities of proteolytic enzyme, trypsin, amylase and lipase in duodenal contents by 57.56%, 76.08%, 69.03% and 40.22%(P<0.05) compared with control, and increased gamma-GT activities in jejunal mucosa by 118.75%(P<0.05). Supplementation with NSP enzymes in barley based diets could improve piglets' growth performance, decrease activities of proteolytic enzyme, trypsin, amylase and lipase in duodenal contents and increase gamma-GT activities in jejunal mucosa.

Process for preparing multilayer enzyme coating on a fiber

DOEpatents

Kim, Jungbae [Richland, WA; Kwak, Ja Hun [Richland, WA; Grate, Jay W [West Richland, WA

2009-11-03

A process for preparing high stability, high activity biocatalytic materials is disclosed and processes for using the same. The process involves coating of a material or fiber with enzymes and enzyme aggregate providing a material or fiber with high biocatalytic activity and stability useful in heterogeneous environments. In one illustrative approach, enzyme "seeds" are covalently attached to polymer nanofibers followed by treatment with a reagent that crosslinks additional enzyme molecules to the seed enzymes forming enzyme aggregates thereby improving biocatalytic activity due to increased enzyme loading and enzyme stability. This approach creates a useful new biocatalytic immobilized enzyme system with potential applications in bioconversion, bioremediation, biosensors, and biofuel cells.

Alteration of extracellular enzyme activity and microbial abundance by biochar addition: Implication for carbon sequestration in subtropical mangrove sediment.

PubMed

Luo, Ling; Gu, Ji-Dong

2016-11-01

Biochar has attracted more and more attention due to its essential role in adsorbing pollutants, improving soil fertility, and modifying greenhouse gas emission. However, the influences of biochar on extracellular enzyme activity and microbial abundance are still lack and debatable. Currently, there is no information about the impact of biochar on the function of mangrove ecosystems. Therefore, we explored the effects of biochar on extracellular enzyme activity and microbial abundance in subtropical mangrove sediment, and further estimated the contribution of biochar to C sequestration. In this study, sediments were amended with 0 (control), 0.5, 1.0 and 2.0% of biochar and incubated at 25 °C for 90 days. After incubation, enzyme activities, microbial abundance and the increased percentage of sediment organic C content were determined. Both increase (phenol oxidase and β-glucosidase) and decrease (peroxidase, N-acetyl-glucosaminidase and acid phosphatase) of enzyme activities were observed in biochar treatments, but only peroxidase activity showed statistical significance (at least p < 0.01) compared to the control. Moreover, the activities of all enzymes tested were significantly related to the content of biochar addition (at least p < 0.05). On the other hand, bacterial and fungal abundance in biochar treatments were remarkably lower than control (p < 0.001), and the significantly negative relationship (p < 0.05) between bacterial abundance and the content of biochar was found. Additionally, the increased percentage of organic C gradually increased with biochar addition rate, which provided evidence for applying biochar to mitigate climate change. Given the importance of microorganisms and enzyme activities in sediment organic matter decomposition, the increased C sequestration might be explained by the large decrease of microbial abundance and enzyme activity after biochar intervention. Copyright © 2016 Elsevier Ltd. All rights reserved.

Effects of protease and non-starch polysaccharide enzyme on performance, digestive function, activity and gene expression of endogenous enzyme of broilers

PubMed Central

Wang, Mingfa; Zhang, Xiaotu; Wang, Zhixiang

2017-01-01

Three hundred one-day-old male broiler chickens (Ross-308) were fed corn-soybean basal diets containing non-starch polysaccharide (NSP) enzyme and different levels of acid protease from 1 to 42 days of age to investigate the effects of exogenous enzymes on growth performance, digestive function, activity of endogenous digestive enzymes in the pancreas and mRNA expression of pancreatic digestive enzymes. For days 1-42, compared to the control chickens, average daily feed intake (ADFI) and average daily gain (ADG) were significantly enhanced by the addition of NSP enzyme in combination with protease supplementation at 40 or 80 mg/kg (p<0.05). Feed-to-gain ratio (FGR) was significantly improved by supplementation with NSP enzymes or NSP enzyme combined with 40 or 80 mg/kg protease compared to the control diet (p<0.05). Apparent digestibility of crude protein (ADCP) was significantly enhanced by the addition of NSP enzyme or NSP enzyme combined with 40 or 80 mg/kg protease (p<0.05). Cholecystokinin (CCK) level in serum was reduced by 31.39% with NSP enzyme combined with protease supplementation at 160 mg/kg (p<0.05), but the CCK level in serum was increased by 26.51% with NSP enzyme supplementation alone. After 21 days, supplementation with NSP enzyme and NSP enzyme combined with 40 or 80 mg/kg protease increased the activity of pancreatic trypsin by 74.13%, 70.66% and 42.59% (p<0.05), respectively. After 42 days, supplementation with NSP enzyme and NSP enzyme combined with 40 mg/kg protease increased the activity of pancreatic trypsin by 32.45% and 27.41%, respectively (p<0.05). However, supplementation with NSP enzyme and 80 or 160 mg/kg protease decreased the activity of pancreatic trypsin by 10.75% and 25.88%, respectively (p<0.05). The activities of pancreatic lipase and amylase were significantly higher in treated animals than they were in the control group (p<0.05). Supplementation with NSP enzyme, NSP enzyme combined with 40 or 80 mg/kg protease increased pancreatic trypsin mRNA levels by 40%, 44% and 28%, respectively. Supplementation with NSP enzyme and 160 mg/kg protease decreased pancreatic trypsin mRNA levels by 13%. Pancreatic lipase and amylase mRNA expression were significantly elevated in treated animals compared to the control group (p<0.05). These results suggest that the amount of NSP enzyme and acid protease in the diet significantly affects digestive function, endogenous digestive-enzyme activity and mRNA expression in broilers. PMID:28323908

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jiang, Yu Ping; Cheng, Fei; Zhou, Yan Hong

Highlights: Black-Right-Pointing-Pointer Activity of certain Calvin cycle enzymes and CO{sub 2} assimilation are induced by BRs. Black-Right-Pointing-Pointer BRs upregulate the activity of the ascorbate-glutathione cycle in the chloroplasts. Black-Right-Pointing-Pointer BRs increase the chloroplast thiol reduction state. Black-Right-Pointing-Pointer A BR-induced reducing environment increases the stability of photosynthetic enzymes. -- Abstract: Brassinosteroids (BRs) play important roles in plant growth, development, photosynthesis and stress tolerance; however, the mechanism underlying BR-enhanced photosynthesis is currently unclear. Here, we provide evidence that an increase in the BR level increased the quantum yield of PSII, activities of Rubisco activase (RCA) and fructose-1,6-bisphosphatase (FBPase), and CO{sub 2} assimilation.more » BRs upregulated the transcript levels of genes and activity of enzymes involved in the ascorbate-glutathione cycle in the chloroplasts, leading to an increased ratio of reduced (GSH) to oxidized (GSSG) glutathione in the chloroplasts. An increased GSH/GSSG ratio protected RCA from proteolytic digestion and increased the stability of redox-sensitive enzymes in the chloroplasts. These results strongly suggest that BRs are capable of regulating the glutathione redox state in the chloroplasts through the activation of the ascorbate-glutathione cycle. The resulting increase in the chloroplast thiol reduction state promotes CO{sub 2} assimilation, at least in part, by enhancing the stability and activity of redox-sensitive photosynthetic enzymes through post-translational modifications.« less

Acute exercise induces biphasic increase in respiratory mRNA in skeletal muscle

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ikeda, Shin-ichi; Kizaki, Takako; Haga, Shukoh

2008-04-04

Peroxisome proliferator-activated receptor {gamma} coactivator-1{alpha} (PGC-1{alpha}) promotes the expression of oxidative enzymes in skeletal muscle. We hypothesized that activation of the p38 MAPK (mitogen-activated protein kinase) in response to exercise was associated with exercise-induced PGC-1{alpha} and respiratory enzymes expression and aimed to demonstrate this under the physiological level. We subjected mice to a single bout of treadmill running and found that the exercise induced a biphasic increase in the expression of respiratory enzymes mRNA. The second phase of the increase was accompanied by an increase in PGC-1{alpha} protein, but the other was not. Administration of SB203580 (SB), an inhibitor ofmore » p38 MAPK, suppressed the increase in PGC-1{alpha} expression and respiratory enzymes mRNA in both phases. These data suggest that p38 MAPK is associated with the exercise-induced expression of PGC-1{alpha} and biphasic increase in respiratory enzyme mRNAs in mouse skeletal muscle under physiological conditions.« less

ENZYME ACTIVITIES DURING THE ASEXUAL CYCLE OF NEUROSPORA CRASSA

PubMed Central

Stine, G. J.

1968-01-01

Three enzymes, (a) nicotinamide adenine diphosphate-dependent glutamic dehydrogenase (NAD enzyme), (b) nictoinamide adenine triphosphate-dependent glutamic dehydrogenase (NADP enzyme), and (c) nicotinamide-adenine dinucleotidase (NADase), were measured in separate extracts of Neurospora crassa grown in Vogel's medium N and medium N + glutamate. Specific activities and total units per culture of each enzyme were determined at nine separate intervals phased throughout the asexual cycle. The separate dehydrogenases were lowest in the conidia, increased slowly during germination, and increased rapidly during logarithmic mycelial growth. The amounts of these enzymes present during germination were small when compared with those found later during the production of the conidiophores. The NAD enzyme may be necessary for pregermination synthesis. The NADP-enzyme synthesis was associated with the appearance of the germ tube. Although higher levels of the dehydrogenases in the conidiophores resulted in more enzyme being found in the differentiated conidia, the rate of germination was uneffected. The greatest activity for the NADase enzyme was associated with the conidia, early phases of germination, and later production of new conidia. NADase decreased significantly with the onset of logarithmic growth, remained low during the differentiation of conidiophores, and increased considerably as the conidiophores aged. PMID:4384627

Thermophysical properties of enzyme clarified Lime (Citrus aurantifolia L) juice at different moisture contents.

PubMed

Manjunatha, S S; Raju, P S; Bawa, A S

2014-11-01

Thermophysical properties of enzyme clarified lime (Citrus aurantifolia L.) juice were evaluated at different moisture contents ranging from 30.37 % to 89.30 % (wet basis) corresponding to a water activity range of 0.835 to 0.979. The thermophysical properties evaluated were density, Newtonian viscosity, thermal conductivity, specific heat and thermal diffusivity. The investigation showed that density and Newtonian viscosity of enzyme clarified lime juice decreased significantly (p < 0.05) with increase in moisture content and water activity, whereas thermal conductivity and specific heat increased significantly (p < 0.05) with increase in moisture content and water activity and the thermal diffusivity increased marginally. Empirical mathematical models were established relating to thermophysical properties of enzyme clarified lime juice with moisture content/water activity employing regression analysis by the method of least square approximation. Results indicated the existence of strong correlation between thermophysical properties and moisture content/water activity of enzyme clarified lime juice, a significant (p < 0.0001) negative correlation between physical and thermal properties was observed.

Increased collagenase and dipeptidyl peptidase I activity in leucocytes from healthy elderly people

PubMed Central

Llorente, L; Richaud-Patin, Y; Díaz-Borjón, A; Jakez-Ocampo, J; Alvarado-De La Barrera, C

1999-01-01

The incidence of infectious diseases increases with ageing. The enzymatic activity of leucocytes may have a relevant role in the morbidity and mortality due to infections in the elderly. In this study we have compared the activity of enzymes involved in the inflammatory response in leucocytes from young and elderly women. A total of 35 healthy females was studied, 20 volunteers aged 78–98 years (mean 89.1 years) and 15 young controls aged 19–34 years (mean 26 years). All of them were in good clinical condition, without any acute or chronic disease. Intracellular enzyme activity was analysed by flow cytometry in leucocytes from young and elderly women. The enzyme substrates employed were for oxidative burst, l-aminopeptidase, collagenase, cathepsin B, C, D and, G and dipeptidyl peptidase I. The intracellular enzyme activity assessed by flow cytometry in leucocytes from young and elderly women was similar, as far as oxidative burst, l-aminopeptidase, cathepsin B, C, D and G are concerned. An increased collagenase activity was detected in granulocytes from elders. The mean fluorescence channels for this enzyme corresponded to 86 ± 23 and 60 ± 15 in cells from elders and controls, respectively (P = 0.01224). An increased dipeptidyl peptidase I activity was detected in lymphocytes from elderly women. The corresponding values for this enzyme in elders and the young were 65.9 ± 43.3 and 17.3 ± 5, respectively (P = 0.0036). The proper functional activity of intracellular enzymes involved in inflammatory responses is likely to be determinant for successful ageing. PMID:10361229

The role of certain oxidative enzymes, catalase, and beta-glucosidase on virulence of Cephalosporium maydis.

PubMed

Abd-Elrazik, A; Darweish, F A; Rushdi, M H

1978-01-01

Isolates of Cephalosporium maydis varied in their pathogenicity to D.C. 67 maize cultivar from highly to weakly pathogenic. Highly pathogenic isolates showed lower activity of polyphenol oxidase, peroxidase, cytochrome oxidase, and beta-glucosidase enzymes and higher activity of catalase and dehydrogenase than weakly pathogenic isolates. Enzymes production by the tested isolates increased as the culture age increased; except in case of catalase enzyme, the reverse action was detected. The role of these enzymes in the virulence of C. maydis is suggested and discussed.

Oxidative stress and anti-oxidant enzyme activities in the trophocytes and fat cells of queen honeybees (Apis mellifera).

PubMed

Hsieh, Yu-Shan; Hsu, Chin-Yuan

2013-08-01

Trophocytes and fat cells of queen honeybees have been used for delayed cellular senescence studies, but their oxidative stress and anti-oxidant enzyme activities with advancing age are unknown. In this study, we assayed reactive oxygen species (ROS) and anti-oxidant enzymes in the trophocytes and fat cells of young and old queens. Young queens had lower ROS levels, lower superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) activities, and higher thioredoxin reductase (TR) activity compared to old queens. These results show that oxidative stress and anti-oxidant enzyme activities in trophocytes and fat cells increase with advancing age in queens and suggest that an increase in oxidative stress and a consequent increase in stress defense mechanisms are associated with the longevity of queen honeybees.

[Change in soil enzymes activities after adding biochar or straw by fluorescent microplate method].

PubMed

Zhang, Yu-Lan; Chen, Li-Jun; Duan, Zheng-Hu; Wu, Zhi-Jie; Sun, Cai-Xia; Wang, Jun-Yu

2014-02-01

The present work was aimed to study soil a-glucosidase and beta-glucosidase activities of and red soils based on fluorescence detection method combined with 96 microplates with TECAN Infinite 200 Multi-Mode Microplate Reader. We added biochar or straw (2.5 g air dry sample/50g air dry soil sample) into and red soils and the test was carried under fixed temperature and humidity condition (25 degrees C, 20% soil moisture content). The results showed that straw addition enhances soil alpha-glucosidase and beta-glucosidase activities, beta-glucosidase activity stimulated by rice straw treatment was higher than that of corn straw treatment, and activity still maintains strong after 40 days, accounting for increasing soil carbon transformation with straw inputting. Straw inputting increased soil nutrients contents and may promote microbial activity, which also lead to the increase oin enzyme Straw inputting increased soil nutrients contents and may promote microbial activity, which also lead to the increase oin enzyme activities. Different effects of straw kinds may be related to material source that needs further research. However, biochar inputting has little effect on soil alpha-glucosidase and beta-glucosidase activity. Biochar contains less available nutrients than straw and have degradation-resistant characteristics. Compared with the conventional spectrophotometric method, fluorescence microplate method is more sensitive to soil enzyme activities in suspension liquid, which can be used in a large number of samples. In brief, fluorescence microplate method is fast, accurate, and simple to determine soil enzymes activities.

Deletion of creB in Aspergillus oryzae increases secreted hydrolytic enzyme activity.

PubMed

Hunter, A J; Morris, T A; Jin, B; Saint, C P; Kelly, J M

2013-09-01

Aspergillus oryzae has been used in the food and beverage industry for centuries, and industrial strains have been produced by multiple rounds of selection. Targeted gene deletion technology is particularly useful for strain improvement in such strains, particularly when they do not have a well-characterized meiotic cycle. Phenotypes of an Aspergillus nidulans strain null for the CreB deubiquitinating enzyme include effects on growth and repression, including increased activity levels of various enzymes. We show that Aspergillus oryzae contains a functional homologue of the CreB deubiquitinating enzyme and that a null strain shows increased activity levels of industrially important secreted enzymes, including cellulases, xylanases, amylases, and proteases, as well as alleviated inhibition of spore germination on glucose medium. Reverse transcription-quantitative PCR (RT-qPCR) analysis showed that the increased levels of enzyme activity in both Aspergillus nidulans and Aspergillus oryzae are mirrored at the transcript level, indicating transcriptional regulation. We report that Aspergillus oryzae DAR3699, originally isolated from soy fermentation, has a similar phenotype to that of a creB deletion mutant of the RIB40 strain, and it contains a mutation in the creB gene. Collectively, the results for Aspergillus oryzae, Aspergillus nidulans, Trichoderma reesei, and Penicillium decumbens show that deletion of creB may be broadly useful in diverse fungi for increasing production of a variety of enzymes.

Deletion of creB in Aspergillus oryzae Increases Secreted Hydrolytic Enzyme Activity

PubMed Central

Hunter, A. J.; Morris, T. A.; Jin, B.; Saint, C. P.

2013-01-01

Aspergillus oryzae has been used in the food and beverage industry for centuries, and industrial strains have been produced by multiple rounds of selection. Targeted gene deletion technology is particularly useful for strain improvement in such strains, particularly when they do not have a well-characterized meiotic cycle. Phenotypes of an Aspergillus nidulans strain null for the CreB deubiquitinating enzyme include effects on growth and repression, including increased activity levels of various enzymes. We show that Aspergillus oryzae contains a functional homologue of the CreB deubiquitinating enzyme and that a null strain shows increased activity levels of industrially important secreted enzymes, including cellulases, xylanases, amylases, and proteases, as well as alleviated inhibition of spore germination on glucose medium. Reverse transcription-quantitative PCR (RT-qPCR) analysis showed that the increased levels of enzyme activity in both Aspergillus nidulans and Aspergillus oryzae are mirrored at the transcript level, indicating transcriptional regulation. We report that Aspergillus oryzae DAR3699, originally isolated from soy fermentation, has a similar phenotype to that of a creB deletion mutant of the RIB40 strain, and it contains a mutation in the creB gene. Collectively, the results for Aspergillus oryzae, Aspergillus nidulans, Trichoderma reesei, and Penicillium decumbens show that deletion of creB may be broadly useful in diverse fungi for increasing production of a variety of enzymes. PMID:23835170

Carbon-Degrading Enzyme Activities Stimulated by Increased Nutrient Availability in Arctic Tundra Soils

PubMed Central

Koyama, Akihiro; Wallenstein, Matthew D.; Simpson, Rodney T.; Moore, John C.

2013-01-01

Climate-induced warming of the Arctic tundra is expected to increase nutrient availability to soil microbes, which in turn may accelerate soil organic matter (SOM) decomposition. We increased nutrient availability via fertilization to investigate the microbial response via soil enzyme activities. Specifically, we measured potential activities of seven enzymes at four temperatures in three soil profiles (organic, organic/mineral interface, and mineral) from untreated native soils and from soils which had been fertilized with nitrogen (N) and phosphorus (P) since 1989 (23 years) and 2006 (six years). Fertilized plots within the 1989 site received annual additions of 10 g N⋅m-2⋅year-1 and 5 g P⋅m-2⋅year-1. Within the 2006 site, two fertilizer regimes were established – one in which plots received 5 g N⋅m-2⋅year-1 and 2.5 g P⋅m-2⋅year-1 and one in which plots received 10 g N⋅m-2⋅year-1 and 5 g P⋅m-2⋅year-1. The fertilization treatments increased activities of enzymes hydrolyzing carbon (C)-rich compounds but decreased phosphatase activities, especially in the organic soils. Activities of two enzymes that degrade N-rich compounds were not affected by the fertilization treatments. The fertilization treatments increased ratios of enzyme activities degrading C-rich compounds to those for N-rich compounds or phosphate, which could lead to changes in SOM chemistry over the long term and to losses of soil C. Accelerated SOM decomposition caused by increased nutrient availability could significantly offset predicted increased C fixation via stimulated net primary productivity in Arctic tundra ecosystems. PMID:24204773

Antioxidative capacity and enzyme activity in Haematococcus pluvialis cells exposed to superoxide free radicals

NASA Astrophysics Data System (ADS)

Liu, Jianguo; Zhang, Xiaoli; Sun, Yanhong; Lin, Wei

2010-01-01

The antioxidative capacity of astaxanthin and enzyme activity of reactive oxygen eliminating enzymes such as superoxide dismutase (SOD), peroxidase (POD), catalase (CAT) and ascorbate peroxidase (APX) were studied in three cell types of Haematococcus pluvialis exposed to high concentrations of a superoxide anion radical (O{2/-}). The results show that defensive enzymes and astaxanthin-related mechanisms were both active in H. pluvialis during exposure to reactive oxygen species (ROS) such as O{2/-}. Astaxanthin reacted with ROS much faster than did the protective enzymes, and had the strongest antioxidative capacity to protect against lipid peroxidation. The defensive mechanisms varied significantly between the three cell types and were related to the level of astaxanthin that had accumulated in those cells. Astaxanthin-enriched red cells had the strongest antioxidative capacity, followed by brown cells, and astaxanthin-deficient green cells. Although there was no significant increase in expression of protective enzymes, the malondialdehyde (MDA) content in red cells was sustained at a low level because of the antioxidative effect of astaxanthin, which quenched O{2/-} before the protective enzymes could act. In green cells, astaxanthin is very low or absent; therefore, scavenging of ROS is inevitably reliant on antioxidative enzymes. Accordingly, in green cells, these enzymes play the leading role in scavenging ROS, and the expression of these enzymes is rapidly increased to reduce excessive ROS. However, because ROS were constantly increased in this study, the enhance enzyme activity in the green cells was not able to repair the ROS damage, leading to elevated MDA content. Of the four defensive enzymes measured in astaxanthin-deficient green cells, SOD eliminates O{2/-}, POD eliminates H2O2, which is a by-product of SOD activity, and APX and CAT are then initiated to scavenge excessive ROS.

Activation of PAF-synthesizing enzymes in rat brain stem slices after LTP induction in the medial vestibular nuclei.

PubMed

Francescangeli, Ermelinda; Grassi, Silvarosa; Pettorossi, Vito E; Goracci, Gianfrancesco

2002-11-01

LysoPAF acetyltransferase (lysoPAF-AT) and PAF-synthesizing phosphocholinetransferase (PAF-PCT) are the two enzymes which catalyze the final reactions for the synthesis of PAF. Their activities, assayed in the homogenate of rat brain stem slices and under their optimal conditions, increased 5 min after high frequency stimulation of vestibular afferents, inducing LTP in the medial vestibular nuclei. The activity of phosphatidylcholine-synthesizing phosphocholinetransferase, was not affected. Sixty minutes from the induction of LTP, PAF-PCT activity, but not that of lysoPAF-AT, was still significantly higher with respect to 5 min test stimulated control. We used AP-5 to verify whether this increase was strictly dependent upon LTP induction, which requires NMDA receptor activation. In AP-5 treated slices, lysoPAF-acetyltransferase and PAF-synthesizing phosphocholinetransferase activities increased, but they were reduced after high frequency stimulation under AP-5. In conclusion, we have demonstrated that the activities of PAF-synthesizing enzymes are activated soon after the induction of LTP and that this effect is linked to the activation of NMDA-receptors. We suggest that the enzyme activation by AP-5, preventing LTP, might be due to glutamate enhancement but, in neurons showing LTP and under normal conditions, the activation of potentiation mechanisms is critical for the enhancement of enzyme activities.

[Effects of snow pack on soil nitrogen transformation enzyme activities in a subalpine Abies faxioniana forest of western Sichuan, China].

PubMed

Xiong, Li; Xu, Zhen-Feng; Wu, Fu-Zhong; Yang, Wan-Qin; Yin, Rui; Li, Zhi-Ping; Gou, Xiao-Lin; Tang, Shi-Shan

2014-05-01

This study characterized the dynamics of the activities of urease, nitrate reductase and nitrite reductase in both soil organic layer and mineral soil layer under three depths of snow pack (deep snowpack, moderate snowpack and shallow snowpack) over the three critical periods (snow formed period, snow stable period, and snow melt period) in the subalpine Abies faxoniana forest of western Sichuan in the winter of 2012 and 2013. Throughout the winter, soil temperature under deep snowpack increased by 46.2% and 26.2%, respectively in comparison with moderate snowpack and shallow snowpack. In general, the three nitrogen-related soil enzyme activities under shallow snowpack were 0.8 to 3.9 times of those under deep snowpack during the winter. In the beginning and thawing periods of seasonal snow pack, shallow snowpack significantly increased the activities of urease, nitrate and nitrite reductase enzyme in both soil organic layer and mineral soil layer. Although the activities of the studied enzymes in soil organic layer and mineral soil layer were observed to be higher than those under deep- and moderate snowpacks in deep winter, no significant difference was found under the three snow packs. Meanwhile, the effects of snowpack on the activities of the measured enzymes were related with season, soil layer and enzyme type. Significant variations of the activities of nitrogen-related enzymes were found in three critical periods over the winter, and the three measured soil enzymes were significantly higher in organic layer than in mineral layer. In addition, the activities of the three measured soil enzymes were closely related with temperature and moisture in soils. In conclusion, the decrease of snow pack induced by winter warming might increase the activities of soil enzymes related with nitrogen transformation and further stimulate the process of wintertime nitrogen transformation in soils of the subalpine forest.

Digestive enzyme activities of turbot (Scophthalmus maximus L.) during early developmental stages under culture condition.

PubMed

Tong, X H; Xu, S H; Liu, Q H; Li, J; Xiao, Z Z; Ma, D Y

2012-06-01

Digestive enzyme activities were analysed in turbot (Scophthalmus maximus) from hatching until 60 days after hatching (DAH). Trypsin sharply increased to the climax at 17 DAH and decreased until 31 DAH followed by a stable level thereafter. Amylase was determined at 4 DAH, reached the maximum value at 19 DAH and declined sharply to 39 DAH and remained at a low level thereafter, suggesting the carbohydrate component should remain at a low level in formulated diets. Pepsin was detected at 9 DAH and increased to 34 DAH and then remained at a stable level. The above results revealed pancreatic enzymes are no longer main enzymes for food digestion after the formation of functional stomach. Leucine-alanine peptidase (Leu-ala) and alkaline phosphatase (AP) and leucine aminopeptidase N (LAP) were found in newly hatched larvae. Both AP and LAP activities markedly increased to 23 DAH, decreased abruptly to 50 DAH and increased gradually to 60 DAH. Leu-ala reached the plateau from 23 to 39 DAH, followed by a decline to 46 DAH and an increase until 60 DAH. The brush border membrane (BBM)-bound enzyme activities increased from 30% at 31 DAH to 81% at 38 DAH of the total activities, indicating the maturation of intestinal tract.

Effects of low light on photosynthetic properties, antioxidant enzyme activity, and anthocyanin accumulation in purple pak-choi (Brassica campestris ssp. Chinensis Makino).

PubMed

Zhu, Hongfang; Li, Xiaofeng; Zhai, Wen; Liu, Yang; Gao, Qianqian; Liu, Jinping; Ren, Li; Chen, Huoying; Zhu, Yuying

2017-01-01

Anthocyanins are secondary metabolites that contribute to red, blue, and purple colors in plants and are affected by light, but the effects of low light on the physiological responses of purple pak-choi plant leaves are still unclear. In this study, purple pak-choi seedlings were exposed to low light by shading with white gauze and black shading in a phytotron. The responses in terms of photosynthetic properties, carbohydrate metabolism, antioxidant enzyme activity, anthocyanin biosynthetic enzyme activity, and the relative chlorophyll and anthocyanin content of leaves were measured. The results showed that chlorophyll b, intracellular CO2 content, stomatal conductance and antioxidant activities of guaiacol peroxidase, catalase and superoxide dismutase transiently increased in the shade treatments at 5 d. The malondialdehyde content also increased under low light stress, which damages plant cells. With the extension of shading time (at 15 d), the relative chlorophyll a, anthocyanin and soluble protein contents, net photosynthetic rate, transpiration rate, stomata conductance, antioxidant enzyme activities, and activities of four anthocyanin biosynthetic enzymes decreased significantly. Thus, at the early stage of low light treatment, the chlorophyll b content increased to improve photosynthesis. When the low light treatment was extended, antioxidant enzyme activity and the activity of anthocyanin biosynthesis enzymes were inhibited, causing the purple pak-choi seedlings to fade from purple to green. This study provides valuable information for further deciphering genetic mechanisms and improving agronomic traits in purple pak-choi under optimal light requirements.

Microbial dynamics and enzyme activities in tropical Andosols depending on land use and nutrient inputs

NASA Astrophysics Data System (ADS)

Mganga, Kevin; Razavi, Bahar; Kuzyakov, Yakov

2015-04-01

Microbial decomposition of soil organic matter is mediated by enzymes and is a key source of terrestrial CO2 emissions. Microbial and enzyme activities are necessary to understand soil biochemical functioning and identify changes in soil quality. However, little is known about land use and nutrients availability effects on enzyme activities and microbial processes, especially in tropical soils of Africa. This study was conducted to examine how microbial and enzyme activities differ between different land uses and nutrient availability. As Andosols of Mt. Kilimanjaro are limited by nutrient concentrations, we hypothesize that N and P additions will stimulate enzyme activity. N and P were added to soil samples (0-20 cm) representing common land use types in East Africa: (1) savannah, (2) maize fields, (3) lower montane forest, (4) coffee plantation, (5) grasslands and (6) traditional Chagga homegardens. Total CO2 efflux from soil, microbial biomass and activities of β-glucosidase, cellobiohydrolase, chitinase and phosphatase involved in C, N and P cycling, respectively was monitored for 60 days. Total CO2 production, microbial biomass and enzyme activities varied in the order forest soils > grassland soils > arable soils. Increased β-glucosidase and cellobiohydrolase activities after N addition of grassland soils suggest that microorganisms increased N uptake and utilization to produce C-acquiring enzymes. Low N concentration in all soils inhibited chitinase activity. Depending on land use, N and P addition had an inhibitory or neutral effect on phosphatase activity. We attribute this to the high P retention of Andosols and low impact of N and P on the labile P fractions. Enhanced CO2 production after P addition suggests that increased P availability could stimulate soil organic matter biodegradation in Andosols. In conclusion, land use and nutrients influenced soil enzyme activities and microbial dynamics and demonstrated the decline in soil quality after landuse change. Key words: Andosols, β-glucosidase, Cellobiohydrolase, Chitinase, Phosphatase, Mt. Kilimanjaro

Enzymes of the Phenylpropanoid Pathway in Soybean Infected with Meloidogyne incognita or Heterodera glycines.

PubMed

Edens, R M; Anand, S C; Bolla, R I

1995-09-01

Transcription of genes encoding several enzymes and the activity of some of these enzymes of the phenylpropanoid pathway leading to synthesis of chemical and physical barriers for defense of plants against root pathogens was estimated in susceptible and resistant soybean infected with Heterodera glycines race 3 or with Meloidogyne incognita race 3. Transcription of genes encoding phenylalanine ammonia lyase (PAL) and the activity of this enzyme increased in resistant, but not susceptible, soybean cultivars after nematode infection. Likewise, transcription of the gene encoding 4-coumaryl CoA ligase and activity of this enzyme were enhanced in resistant, but not susceptible, soybean cultivars after nematode infection. Activity of PAL decreased in susceptible soybean after H. glycines or M. incognita infection. Transcription of enzymes later in the phenylpropanoid pathway leading to glyceollin synthesis increased in both resistant and susceptible soybean in response to nematode infection; the increase was greater in resistant cultivars. These results suggest possible reasons for the rapid induction of glyceollin synthesis immediately after infection of resistant soybean cultivars with H. glycines or M. incognita and the failure of this response in infected, susceptible soybean cultivars. Nematode infection had no effect on the activity of enzymes in the branch of the pathway leading to lignin synthesis.

A reusable multipurpose magnetic nanobiocatalyst for industrial applications.

PubMed

Perwez, Mohammad; Ahmad, Razi; Sardar, Meryam

2017-10-01

A multipurpose magnetic nanobiocatalyst is developed by conjugating Pectinex 3XL (a commercial enzyme containing pectinase, xylanase and cellulase activities) on 3-aminopropyl triethoxysilane activated magnetic nanoparticles. The nanobiocatalyst retained 87% of pectinase, 69% of xylanase and 58% of cellulase activity after conjugation on modified nanoparticles as compared to their soluble counterparts. Thermal stability data at 70°C showed increase in enzyme stability after conjugation to nanoparticles and the kinetic parameters (K m and V max ) remain unaltered after immobilization. The immobilized enzyme system can be successfully used upto 5th cycle after that slight decrease in enzyme activities was observed. The nanobiocatalyst retained high pectinase activities in organic solvents and chemical reagents as compared to free enzymes. DLS data shows that the nanoparticles size increases from 63nm to 86nm after immobilization. Atomic Force Microscopy data confirms the deposition of enzymes on the nanoparticles. The nanobiocatalyst was used for the clarification of pine apple and orange juice and was also used for the production of bioethanol. Hydrolysis of pretreated wheat straw produced 1.39g/l and 1.59g/l after treatment with free Pectinex 3xL and nanobiocatalyst respectively. The concentration of bioethanol also increases by 1.4 fold as compared to the free enzyme. Copyright © 2017 Elsevier B.V. All rights reserved.

Construction of Mutant Glucose Oxidases with Increased Dye-Mediated Dehydrogenase Activity

PubMed Central

Horaguchi, Yohei; Saito, Shoko; Kojima, Katsuhiro; Tsugawa, Wakako; Ferri, Stefano; Sode, Koji

2012-01-01

Mutagenesis studies on glucose oxidases (GOxs) were conducted to construct GOxs with reduced oxidase activity and increased dehydrogenase activity. We focused on two representative GOxs, of which crystal structures have already been reported—Penicillium amagasakiense GOx (PDB ID; 1gpe) and Aspergillus niger GOx (PDB ID; 1cf3). We constructed oxygen-interacting structural models for GOxs, and predicted the residues responsible for oxidative half reaction with oxygen on the basis of the crystal structure of cholesterol oxidase as well as on the fact that both enzymes are members of the glucose/methanol/choline (GMC) oxidoreductase family. Rational amino acid substitution resulted in the construction of an engineered GOx with drastically decreased oxidase activity and increased dehydrogenase activity, which was higher than that of the wild-type enzyme. As a result, the dehydrogenase/oxidase ratio of the engineered enzyme was more than 11-fold greater than that of the wild-type enzyme. These results indicate that alteration of the dehydrogenase/oxidase activity ratio of GOxs is possible by introducing a mutation into the putative functional residues responsible for oxidative half reaction with oxygen of these enzymes, resulting in a further increased dehydrogenase activity. This is the first study reporting the alteration of GOx electron acceptor preference from oxygen to an artificial electron acceptor. PMID:23203056

Immobilization Increases the Stability and Reusability of Pigeon Pea NADP+ Linked Glucose-6-Phosphate Dehydrogenase.

PubMed

Singh, Siddhartha; Singh, Amit Kumar; Singh, M Chandrakumar; Pandey, Pramod Kumar

2017-02-01

Immobilization of enzymes is valuably important as it improves the stability and hence increases the reusability of enzymes. The present investigation is an attempt for immobilization of purified glucose-6-phosphate dehydrogenase from pigeon pea on different matrix. Maximum immobilization was achieved when alginate was used as immobilization matrix. As compared to soluble enzyme the alginate immobilized enzyme exhibited enhanced optimum pH and temperature. The alginate immobilized enzyme displayed more than 80% activity up to 7 continuous reactions and more than 50% activity up to 11 continuous reactions.

LECITHINASE AND LYSOLECITHINASE ACTIVITY OF RAT INTESTINAL MUCOSA AFTER WHOLE-BODY X-IRRADIATION

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ottolenghi, A.; Bernheim, F.

1961-11-01

Twenty-four hours after whole-body x irradiation the lecithinase activity of rat intestinal mucosa has markedly decreased and the lysolecithinase activity has decreasecp to a lesser extent. Addition of normal mucosa or chyi'otrypsin to the irradiated mucosa restores the activity of both enzymes. This indicates that irradiation eithei produces an inhibitor or inactivates a mechanism necessarly to convert pro-enzymes into active enzymes. Since chymo trypsin can increase to some extent the activity of the enzymes in normal mucosa, the second possibility seems more probable. (auth)

Processing of poultry feathers by alkaline keratin hydrolyzing enzyme from Serratia sp. HPC 1383.

PubMed

Khardenavis, Anshuman A; Kapley, Atya; Purohit, Hemant J

2009-04-01

The present study describes the production and characterization of a feather hydrolyzing enzyme by Serratia sp. HPC 1383 isolated from tannery sludge, which was identified by the ability to form clear zones around colonies on milk agar plates. The proteolytic activity was expressed in terms of the micromoles of tyrosine released from substrate casein per ml per min (U/mL min). Induction of the inoculum with protein was essential to stimulate higher activity of the enzyme, with 0.03% feathermeal in the inoculum resulting in increased enzyme activity (45U/mL) that further increased to 90U/mL when 3d old inoculum was used. The highest enzyme activity, 130U/mL, was observed in the presence of 0.2% yeast extract. The optimum assay temperature and pH for the enzyme were found to be 60 degrees C and 10.0, respectively. The enzyme had a half-life of 10min at 60 degrees C, which improved slightly to 18min in presence of 1mM Ca(2+). Inhibition of the enzyme by phenylmethyl sulfonyl fluoride (PMSF) indicated that the enzyme was a serine protease. The enzyme was also partially inhibited (39%) by the reducing agent beta-mercaptoethanol and by divalent metal ions such as Zn(2+) (41% inhibition). However, Ca(2+) and Fe(2+) resulted in increases in enzyme activity of 15% and 26%, respectively. The kinetic constants of the keratinase were found to be 3.84 microM (K(m)) and 108.7 microM/mLmin (V(max)). These results suggest that this extracellular keratinase may be a useful alternative and eco-friendly route for handling the abundant amount of waste feathers or for applications in other industrial processes.

E2 potentializes benzo(a)pyrene-induced hepatic cytochrome P450 enzyme activities in Nile tilapia at high concentrations.

PubMed

Rodrigues, Aline Cristina Ferreira; Moneró, Tatiana de Oliveira; Frighetto, Rosa Toyoko Shiraishi; de Almeida, Eduardo Alves

2015-11-01

In the aquatic environment, biotransformation enzymes are established biomarkers for assessing PAH exposure in fish, but little is known about the effect of 17β-estradiol (E2) on these enzymes during exposure to benzo(a)pyrene (BaP). In this study, Nile tilapia (Oreochromis niloticus) were exposed for 3, 5, and 10 days to BaP (300 μg L(-1)) and E2 (5 μg L(-1)). These substances were applied isolated or mixed. In the mixture experiment, fish were analyzed pre- and postexposure in order to better understand whether preexposure to the hormone masks the responses activated by PAH or vice versa. Phase I enzymes ethoxyresorufin-O-deethylase (EROD), pentoxyresorufin-O-depenthylase (PROD), and benzyloxyresorufin-O-debenzylase (BROD) activities as well as the phase II enzyme glutathione S-transferase (GST) were analyzed. Isolated E2 treatment decreased EROD activity after 3 days, but this enzyme activity returned to control values after 5 and 10 days of exposure. Isolated BaP treatment significantly induced EROD activity after 3 and 5 days, and the activity returned to control levels after ten exposure days. Combined treatment (E2 + Bap) significantly increased EROD activity, both in the pre- and postexposure. This increase was even higher than in the isolated BaP treatment, suggesting a synergism between these two compounds. When E2 and BaP were used singly, they did not change BROD and PROD activities. However, combined treatment (E2 + Bap) significantly increased PROD activity. Isolated BaP treatment increased GST activity after 10 days. However, this response was not observed in the mixture treatment, suggesting that E2 suppressed the GST induction modulated by BaP. The results put together indicated that E2 altered the biotransformation pathway regarding enzymes activated by BaP in Nile tilapia.

Towards Understanding the Catalytic Mechanism of Human Paraoxonase 1: Experimental and In Silico Mutagenesis Studies.

PubMed

Tripathy, Rajan K; Aggarwal, Geetika; Bajaj, Priyanka; Kathuria, Deepika; Bharatam, Prasad V; Pande, Abhay H

2017-08-01

Human paraoxonase 1 (h-PON1) is a ~45-kDa serum enzyme that can hydrolyze a variety of substrates, including organophosphate (OP) compounds. It is a potential candidate for the development of antidote against OP poisoning in humans. However, insufficient OP-hydrolyzing activity of native enzyme affirms the urgent need to develop improved variant(s) having enhanced OP-hydrolyzing activity. The crystal structure of h-PON1 remains unsolved, and the molecular details of how the enzyme catalyses hydrolysis of different types of substrates are also not clear. Understanding the molecular details of the catalytic mechanism of h-PON1 is essential to engineer better variant(s) of enzyme. In this study, we have used a random mutagenesis approach to increase the OP-hydrolyzing activity of recombinant h-PON1. The mutants not only showed a 10-340-fold increased OP-hydrolyzing activity against different OP substrates but also exhibited differential lactonase and arylesterase activities. In order to investigate the mechanistic details of the effect of observed mutations on the hydrolytic activities of enzyme, molecular docking studies were performed with selected mutants. The results suggested that the observed mutations permit differential binding of substrate/inhibitor into the enzyme's active site. This may explain differential hydrolytic activities of the enzyme towards different substrates.

Expression, purification and immobilization of tannase from Staphylococcus lugdunensis MTCC 3614.

PubMed

Chaitanyakumar, Amballa; Anbalagan, M

2016-12-01

Enzymes find their applications in various industries, due to their error free conversion of substrate into product. Tannase is an enzyme used by various industries for degradation of tannin. Biochemical characterization of a specific enzyme from one organism to other is one of the ways to search for enzymes with better traits for industrial applications. Here, tannase encoding gene from Staphylococcus lugdunensis was cloned and suitability of the enzyme in various conditions was analysed to find its application in various industry. The recombinant protein was expressed with 6× His tag and purified using nickel affinity beads. The enzyme was purified up to homogeneity, with approximate molecular weight of 66 kDa. Purified tannase exhibited specific activity of about 716 U/mg. Optimum enzyme activity was found to be 40 °C at pH 7.0. Biochemical characterization revealed; metal ions such as Zn 2+ , Fe 2+ , Fe 3+ and Mn 2+ inhibited tannase activity, and SDS at lower concentration, increased tannase activity. Non polar organic solvents increased the tannase activity and polar solvents inhibited the tannase activity. Tannase immobilization studies show protection of the enzyme under wide range of pH and temperature. Also in this study we report a method for recovery and repeated use of the tannase.

A Chaperone Enhances Blood α-Glucosidase Activity in Pompe Disease Patients Treated With Enzyme Replacement Therapy

PubMed Central

Parenti, Giancarlo; Fecarotta, Simona; la Marca, Giancarlo; Rossi, Barbara; Ascione, Serena; Donati, Maria Alice; Morandi, Lucia Ovidia; Ravaglia, Sabrina; Pichiecchio, Anna; Ombrone, Daniela; Sacchini, Michele; Pasanisi, Maria Barbara; De Filippi, Paola; Danesino, Cesare; Della Casa, Roberto; Romano, Alfonso; Mollica, Carmine; Rosa, Margherita; Agovino, Teresa; Nusco, Edoardo; Porto, Caterina; Andria, Generoso

2014-01-01

Enzyme replacement therapy is currently the only approved treatment for Pompe disease, due to acid α-glucosidase deficiency. Clinical efficacy of this approach is variable, and more effective therapies are needed. We showed in preclinical studies that chaperones stabilize the recombinant enzyme used for enzyme replacement therapy. Here, we evaluated the effects of a combination of enzyme therapy and a chaperone on α-glucosidase activity in Pompe disease patients. α-Glucosidase activity was analyzed by tandem-mass spectrometry in dried blood spots from patients treated with enzyme replacement therapy, either alone or in combination with the chaperone N-butyldeoxynojirimycin given at the time of the enzyme infusion. Thirteen patients with different presentations (3 infantile-onset, 10 late-onset) were enrolled. In 11 patients, the combination treatment resulted in α-glucosidase activities greater than 1.85-fold the activities with enzyme replacement therapy alone. In the whole patient population, α-glucosidase activity was significantly increased at 12 hours (2.19-fold, P = 0.002), 24 hours (6.07-fold, P = 0.001), and 36 hours (3.95-fold, P = 0.003). The areas under the curve were also significantly increased (6.78-fold, P = 0.002). These results suggest improved stability of recombinant α-glucosidase in blood in the presence of the chaperone. PMID:25052852

Threonine deaminase from extremely halophilic bacteria - Cooperative substrate kinetics and salt dependence.

NASA Technical Reports Server (NTRS)

Lieberman, M. M.; Lanyi, J. K.

1972-01-01

The effect of salt on the activity, stability, and allosteric properties of catabolic threonine deaminase from Halobacterium cutirubrum was studied. The enzyme exhibits sigmoidal kinetics with the substrate, threonine. The Hill slope is 1.55 at pH 10. The enzyme is activated by ADP at low substrate concentrations. In the presence of this effector, sigmoidal kinetics are no longer observed. At pH 10, in the absence of ADP, enzyme activity increases with increasing NaCl concentration from 0 to 4 M.

Optimization of process variables by central composite design for the immobilization of urease enzyme on functionalized gold nanoparticles for various applications.

PubMed

Talat, Mahe; Singh, Ashwani Kumar; Srivastava, O N

2011-08-01

In the present study, enzyme urease has been immobilized on amine-functionalized gold nanoparticles (AuNPs). AuNPs were synthesized using natural precursor, i.e., clove extract and amine functionalized through 0.004 M L: -cysteine. Enzyme (urease) was extracted and purified from the vegetable waste, i.e., seeds of pumpkin to apparent homogeneity (sp. activity 353 U/mg protein). FTIR spectroscopy and transmission electron microscopy was used to characterize the immobilized enzyme. The immobilized enzyme exhibited enhanced activity as compared with the enzyme in the solution, especially, at lower enzyme concentration. Based on the evaluation of activity assay of the immobilized enzyme, it was found that the immobilized enzyme was quite stable for about a month and could successfully be used even after eight cycles having enzyme activity of about 47%. In addition to this central composite design (CCD) with the help of MINITAB version 15 Software was utilized to optimize the process variables viz., pH and temperature affecting the enzyme activity upon immobilization on AuNPs. The results predicted by the design were found in good agreement (R2 = 96.38%) with the experimental results indicating the applicability of proposed model. The multiple regression analysis and ANOVA showed the individual and cumulative effect of pH and temperature on enzyme activity indicating that the activity increased with the increase of pH up to 7.5 and temperature 75 °C. The effects of each variables represented by main effect plot, 3D surface plot, isoresponse contour plot and optimized plot were helpful in predicting results by performing a limited set of experiments.

Aβ degradation or cerebral perfusion? Divergent effects of multifunctional enzymes.

PubMed

Miners, J Scott; Palmer, Jennifer C; Tayler, Hannah; Palmer, Laura E; Ashby, Emma; Kehoe, Patrick G; Love, Seth

2014-01-01

There is increasing evidence that deficient clearance of β-amyloid (Aβ) contributes to its accumulation in late-onset Alzheimer disease (AD). Several Aβ-degrading enzymes, including neprilysin (NEP), endothelin-converting enzyme (ECE), and angiotensin-converting enzyme (ACE) reduce Aβ levels and protect against cognitive impairment in mouse models of AD. In post-mortem human brain tissue we have found that the activity of these Aβ-degrading enzymes rise with age and increases still further in AD, perhaps as a physiological response that helps to minimize the build-up of Aβ. ECE-1/-2 and ACE are also rate-limiting enzymes in the production of endothelin-1 (ET-1) and angiotensin II (Ang II), two potent vasoconstrictors, increases in the levels of which are likely to contribute to reduced blood flow in AD. This review considers the possible interdependence between Aβ-degrading enzymes, ischemia and Aβ in AD: ischemia has been shown to increase Aβ production both in vitro and in vivo, whereas increased Aβ probably enhances ischemia by vasoconstriction, mediated at least in part by increased ECE and ACE activity. In contrast, NEP activity may help to maintain cerebral perfusion, by reducing the accumulation of Aβ in cerebral blood vessels and lessening its toxicity to vascular smooth muscle cells. In assessing the role of Aβ-degrading proteases in the pathogenesis of AD and, particularly, their potential as therapeutic agents, it is important to bear in mind the multifunctional nature of these enzymes and to consider their effects on other substrates and pathways.

Increasing the Thermostable Sugar-1-Phosphate Nucleotidylyltransferase Activities of the Archaeal ST0452 Protein through Site Saturation Mutagenesis of the 97th Amino Acid Position.

PubMed

Honda, Yuki; Zang, Qian; Shimizu, Yasuhiro; Dadashipour, Mohammad; Zhang, Zilian; Kawarabayasi, Yutaka

2017-02-01

The ST0452 protein is a bifunctional protein exhibiting sugar-1-phosphate nucleotidylyltransferase (sugar-1-P NTase) and amino-sugar-1-phosphate acetyltransferase activities and was isolated from the thermophilic archaeon Sulfolobus tokodaii Based on the previous observation that five single mutations increased ST0452 sugar-1-P NTase activity, nine double-mutant ST0452 proteins were generated with the intent of obtaining enzymes exhibiting a further increase in catalysis, but all showed less than 15% of the wild-type N-acetyl-d-glucosamine-1-phosphate uridyltransferase (GlcNAc-1-P UTase) activity. The Y97A mutant exhibited the highest activity of the single-mutant proteins, and thus site saturation mutagenesis of the 97th position (Tyr) was conducted. Six mutants showed both increased GlcNAc-1-P UTase and glucose-1-phosphate uridyltransferase activities, eight mutants showed only enhanced GlcNAc-1-P UTase activity, and six exhibited higher GlcNAc-1-P UTase activity than that of the Y97A mutant. Kinetic analyses of three typical mutants indicated that the increase in sugar-1-P NTase activity was mainly due to an increase in the apparent k cat value. We hypothesized that changing the 97th position (Tyr) to a smaller amino acid with similar electronic properties would increase activity, and thus the Tyr at the corresponding 103rd position of the Escherichia coli GlmU (EcGlmU) enzyme was replaced with the same residues. The Y103N mutant EcGlmU showed increased GlcNAc-1-P UTase activity, revealing that the Tyr at the 97th position of the ST0452 protein (103rd position in EcGlmU) plays an important role in catalysis. The present results provide useful information regarding how to improve the activity of natural enzymes and how to generate powerful enzymes for the industrial production of sugar nucleotides. It is typically difficult to increase enzymatic activity by introducing substitutions into a natural enzyme. However, it was previously found that the ST0452 protein, a thermostable enzyme from the thermophilic archaeon Sulfolobus tokodaii, exhibited increased activity following single amino acid substitutions of Ala. In this study, ST0452 proteins exhibiting a further increase in activity were created using a site saturation mutagenesis strategy at the 97th position. Kinetic analyses showed that the increased activities of the mutant proteins were principally due to increased apparent k cat values. These mutant proteins might suggest clues regarding the mechanism underlying the reaction process and provide very important information for the design of synthetic improved enzymes, and they can be used as powerful biocatalysts for the production of sugar nucleotide molecules. Moreover, this work generated useful proteins for three-dimensional structural analysis clarifying the processes underlying the regulation and mechanism of enzymatic activity. Copyright © 2017 American Society for Microbiology.

Changes in Activities of Respiratory Enzymes in Lungs of Guinea-pigs Exposed to Silica Dust: II. Comparison of the Effects of Quartz Dust and Lampblack on the Succinate Oxidase System

PubMed Central

Breyer, Maria G.; Kilroe-Smith, T. A.; Prinsloo, H.

1964-01-01

Kilroe-Smith and Breyer (1963) reported that in the early stages of silicosis in guinea-pigs exposed to the inhalation of quartz dust, before the formation of collagen, there were increases in the specific activities of the complete succinate oxidase system and succinate dehydrogenase. The effects on these enzymes of quartz dust have now been compared with the effects of the fibrogenically `inert' lampblack. Lampblack causes a slight increase in the specific activities of these enzymes but the effects are small compared to those caused by quartz. Lampblack also causes a much smaller increase in lung weight than quartz, thus the enzyme increases are roughly parallel to the rise in lung weight. It appears that the effects observed on the enzymes are part of the general pattern associated with the early stages of the development of silicosis. PMID:14106132

Purification and characterization of the enzyme cholesterol oxidase from a new isolate of Streptomyces sp.

PubMed

Praveen, Vandana; Srivastava, Akanksha; Tripathi, C K M

2011-11-01

An extracellular cholesterol oxidase (cho) enzyme was isolated from the Streptomyces parvus, a new source and purified 18-fold by ion exchange and gel filtration chromatography. Specific activity of the purified enzyme was found to be 20 U/mg with a 55 kDa molecular mass. The enzyme was stable at pH 7.2 and 50 °C. The enzyme activity was inhibited in the presence of Pb(2+), Ag(2+), Hg(2+), and Zn(2+) and enhanced in the presence of Mn(2+). The enzyme activity was inhibited by the thiol-reducing reagents (DTT, β-mercaptoethanol), suggesting that disulfide linkage is essential for the enzyme activity. The enzyme activity was found to be maximum in the presence of Triton X-100 and X-114 detergents whereas sodium dodecyl sulfate fully inactivated the enzyme. The enzyme showed moderate stability towards all organic solvents except acetone, benzene, chloroform and the activity increased in the presence of isopropanol and ethanol. The K(m) value for the oxidation of cholesterol by this enzyme was 0.02 mM.

Changes in the biological activity of chestnut soils upon the long-term application of fertilizers in a rotation with oil-bearing crops

NASA Astrophysics Data System (ADS)

Eleshev, R. E.; Bakenova, Z. B.

2012-11-01

Experimental studies showed that irrigated chestnut soils on the piedmont of the Zailiiskiy Alatau Range are characterized by the moderate activity of the hydrolytic and redox enzymes. The use of these soils in the crop rotation system increases the hydrolytic activity of the enzymes (invertase, urease, and ATP synthase) by 30% in comparison with the monoculture; at the same time, it does not have a significant impact on the changes in the biological activity of the redox enzymes (catalase and dehydrogenase). The hydrolytic activity of the soils is activated to a greater extent in the crop rotation and in the monoculture against the background application of organic fertilizers. In this case, the recommended rates of mineral fertilizers do not inhibit the activity of the hydrolytic and redox enzymes. An increase in the hydrolytic activity of the enzymes directly affects the yield of oilseed flax. Therefore, indices of the hydrolytic activity of soils can be used as a test for the diagnostics of the efficiency of fertilizers both in crop rotation and monoculture systems.

Evaluation of 2-Thioxo-2,3,5,6,7,8-hexahydropyrimido[4,5-d]pyrimidin-4(1H)-one analogues as GAA Activators

PubMed Central

Marugan, Juan J.; Zheng, Wei; Motabar, Omid; Southall, Noel; Goldin, Ehud; Sidransky, Ellen; Aungst, Ronald A.; Liu, Ke; Sadhukhan, Subir Kumar; Austin, Christopher P.

2010-01-01

Pompe disease is a lysosomal storage disease (LSD) caused by a deficiency in the lysosomal enzyme acid α-glucosidase. In several LSDs, enzyme inhibitors have been used as small molecule chaperones to facilitate and increase the translocation of mutant protein from the endoplasmic reticulum to the lysosome. Enzyme activators with chaperone activity would be even more desirable as they would not inhibit the enzyme after translocation and might potentiate the activity of the enzyme that is successfully translocated. Herein we report our initial findings of a new series of acid α-glucosidase activators. PMID:20206419

Evaluation of 2-thioxo-2,3,5,6,7,8-hexahydropyrimido[4,5-d]pyrimidin-4(1H)-one analogues as GAA activators.

PubMed

Marugan, Juan J; Zheng, Wei; Motabar, Omid; Southall, Noel; Goldin, Ehud; Sidransky, Ellen; Aungst, Ronald A; Liu, Ke; Sadhukhan, Subir Kumar; Austin, Christopher P

2010-05-01

Pompe disease is a lysosomal storage disease (LSD) caused by a deficiency in the lysosomal enzyme acid alpha-glucosidase. In several LSDs, enzyme inhibitors have been used as small molecule chaperones to facilitate and increase the translocation of mutant protein from the endoplasmic reticulum to the lysosome. Enzyme activators with chaperone activity would be even more desirable as they would not inhibit the enzyme after translocation and might potentiate the activity of the enzyme that is successfully translocated. Herein we report our initial findings of a new series of acid alpha-glucosidase activators.

Response of soil physicochemical properties and enzyme activities to long-term reclamation of coastal saline soil, Eastern China.

PubMed

Xie, Xuefeng; Pu, Lijie; Wang, Qiqi; Zhu, Ming; Xu, Yan; Zhang, Meng

2017-12-31

Soil enzyme activity during different years of reclamation and land use patterns could indicate changes in soil quality. The objective of this research is to explore the dynamics of 5 soil enzyme activities (dehydrogenase, amylase, urease, acid phosphatase and alkaline phosphatase) involved in C, N, and P cycling and their responses to changes in soil physicochemical properties resulting from long-term reclamation of coastal saline soil. Soil samples from a total of 55 sites were collected from a coastal reclamation area with different years of reclamation (0, 7, 32, 40, 63a) in this study. The results showed that both long-term reclamation and land use patterns have significant effects on soil physicochemical properties and enzyme activities. Compared with the bare flat, soil water content, soil bulk density, pH and electrical conductivity showed a decreasing trend after reclamation, whereas soil organic carbon, total nitrogen and total phosphorus tended to increase. Dehydrogenase, amylase and acid phosphatase activities initially increased and then decreased with increasing years of reclamation, whereas urease and alkaline phosphatase activities were characterized by an increase-decrease-increase trend. Moreover, urease, acid phosphatase and alkaline phosphatase activities exhibited significant differences between coastal saline soil with 63years of reclamation and bare flat, whereas dehydrogenase and amylase activities remained unchanged. Aquaculture ponds showed higher soil water content, pH and EC but lower soil organic carbon, total nitrogen and total phosphorus than rapeseed, broad bean and wheat fields. Rapeseed, broad bean and wheat fields displayed higher urease and alkaline phosphatase activities and lower dehydrogenase, amylase and acid phosphatase activities compared with aquaculture ponds. Redundancy analysis revealed that the soil physicochemical properties explained 74.5% of the variation in soil enzyme activities and that an obvious relationship existed between soil nutrients and soil enzyme activities. These results will assist governmental evaluation of the quality of reclaimed coastal soil. Copyright © 2017 Elsevier B.V. All rights reserved.

Effect of elevated CO2 on the interaction between invasive thrips, Frankliniella occidentalis, and its host kidney bean, Phaseolus vulgaris.

PubMed

Qian, Lei; He, Shuqi; Liu, Xiaowei; Huang, Zujin; Chen, Fajun; Gui, Furong

2018-05-08

Elevated CO 2 can alter the leaf damage caused by insect herbivores. Frankliniella occidentalis (Pergande) is highly destructive invasive pest in crop production worldwide. To investigate how elevated CO 2 affects F. occidentalis fed with Phaseolus vulgaris and in particularly, the interaction between plant defense and thrips anti-defense, nutrients content and antioxidant enzymes activity of P. vulgaris have been measured, as well as the detoxifying enzymes activity of adult thrips. Elevated CO 2 increased soluble sugar, soluble protein, and free amino acids content in non-thrips plants, and decreased SOD and POD activity in these plants. Thrips feeding reduced the nutrients content in plants, and increased their SOD, CAT and POD activity. Variation of nutrients content and antioxidant enzymes activity in plants showed an opposite tendency over thrips feeding time. After feeding, AchE, CarE, and MFO activity in thrips increased to against plant defense. More thrips densities induced stronger plant defense, in return, detoxifying enzymes in thrips increased over thrips number. Our study revealed that F. occidentalis can induce not only antioxidant-associated plant defense, but also the thrips detoxifying enzymes. Elevated CO 2 might not only enhance plant defense to thrips attack, but also increase thrips anti-defense against plant defense. This article is protected by copyright. All rights reserved.

Defense reactions of bean genotypes to bacterial pathogens in controlled conditions

NASA Astrophysics Data System (ADS)

Uysal, B.; Bastas, K. K.

2018-03-01

This study was focused on the role of antioxidant enzymes and total protein in imparting resistance against common bacterial blight caused by Xanthomonas axonopodis pv. phaseoli (Xap) and halo blight caused by Pseudomonas syringae pv. phaseolicola (Psp) in bean. Activities of Ascorbate peroxidase (APX), Catalase (CAT) and total protein were studied in resistant and susceptible bean genotypes. Five-day-old seedlings were inoculated with a bacterial suspension (108 CFU ml-1) and harvested at different time intervals (0, 12, 24 and 36 up to 72 h) under controlled growing conditions and assayed for antioxidant enzymes and total protein. Temporal increase of CAT, APX enzymes activities showed maximum activity at 12 h after both pathogens inoculation (hpi) in resistant cultivar, whereas in susceptible it increased at 72 h after both pathogens inoculation for CAT and 12, 24 h for APX enzymes. Maximum total protein activities were observed at 12 h and 24 h respectively after Xap, Psp inoculation (hpi) in resistant and maximum activities were observed at 24 h and 72 h respectively after Xap, Psp inoculation (hpi) in susceptible. Increase of antioxidant enzyme and total protein activities might be an important component in the defense strategy of resistance and susceptible bean genotypes against the bacterial infection. These findings suggest that disease protection is proportional to the amount of enhanced CAT, APX enzyme and total protein activity.

Six different plasma enzymes in bald eagles (Haliaeetus leucocephalus) and their usefulness in pathological diagnosis

USGS Publications Warehouse

Dieter, M.P.; Wiemeyer, Stanley N.

1978-01-01

1. Activities of creatine phosphokinase, glutamic oxalacetic transaminase, glutamic pyruvic transaminase, lactate dehydrogenase, fructose diphosphate aldolase and cholinesterase were measured in plasma of bald eagles.2. There were no sex differences in the plasma enzyme activities.3. An acute dieldrin dosage (10 mg/kg) of a female bald eagle resulted in 400% increases in activities of plasma creatine phosphokinase and glutamic oxalacetic transaminase and 250% increases in activities of lactate dehydrogenase and glutamic pyruvic transaminase.4. At 11 days post-dosage all but one of the plasma enzyme activities had returned to normal; glutamic oxalacetic transaminase activity remained 100% above pre-dosage values.5. Plasma enzyme assays constitute a non-destrcutive procedure that can be used in valuable wildlife species to screen for the presence and prevalence of environmental contaminants.

Effect of seasonality and Cr(VI) on starch-sucrose partitioning and related enzymes in floating leaves of Salvinia minima.

PubMed

Rosa, Mariana; Prado, Carolina; Chocobar-Ponce, Silvana; Pagano, Eduardo; Prado, Fernando

2017-09-01

Effects of seasonality and increasing Cr(VI) concentrations on leaf starch-sucrose partitioning, sucrose- and starch-related enzyme activities, and carbon allocation toward leaf development were analyzed in fronds (floating leaves) of the floating fern Salvinia minima. Carbohydrates and enzyme activities of Cr-exposed fronds showed different patterns in winter and summer. Total soluble sugars, starch, glucose and fructose increased in winter fronds, while sucrose was higher in summer ones. Starch and soluble carbohydrates, except glucose, increased under increasing Cr(VI) concentrations in winter fronds, while in summer ones only sucrose increased under Cr(VI) treatment. In summer fronds starch, total soluble sugars, fructose and glucose practically stayed without changes in all assayed Cr(VI) concentrations. Enzyme activities related to starch and sucrose metabolisms (e.g. ADPGase, SPS, SS and AI) were higher in winter fronds than in summer ones. Total amylase and cFBPase activities were higher in summer fronds. Cr(VI) treatment increased enzyme activities, except ADPGase, in both winter and summer fronds but no clear pattern changes were observed. Data of this study show clearly that carbohydrate metabolism is differently perturbed by both seasonality and Cr(VI) treatment in summer and winter fronds, which affects leaf starch-sucrose partitioning and specific leaf area (SLA) in terms of carbon investment. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Dipeptidyl peptidase IV in angiotensin-converting enzyme inhibitor associated angioedema.

PubMed

Byrd, James Brian; Touzin, Karine; Sile, Saba; Gainer, James V; Yu, Chang; Nadeau, John; Adam, Albert; Brown, Nancy J

2008-01-01

Angioedema is a potentially life-threatening adverse effect of angiotensin-converting enzyme inhibitors. Bradykinin and substance P, substrates of angiotensin-converting enzyme, increase vascular permeability and cause tissue edema in animals. Studies indicate that amino-terminal degradation of these peptides, by aminopeptidase P and dipeptidyl peptidase IV, may be impaired in individuals with angiotensin-converting enzyme inhibitor-associated angioedema. This case-control study tested the hypothesis that dipeptidyl peptidase IV activity and antigen are decreased in sera of patients with a history of angiotensin-converting enzyme inhibitor-associated angioedema. Fifty subjects with a history of angiotensin-converting enzyme inhibitor-associated angioedema and 176 angiotensin-converting enzyme inhibitor-exposed control subjects were ascertained. Sera were assayed for angiotensin-converting enzyme activity, aminopeptidase P activity, aminopeptidase N activity, dipeptidyl peptidase IV activity, and antigen and the ex vivo degradation half-lives of bradykinin, des-Arg(9)-bradykinin, and substance P in a subset. The prevalence of smoking was increased and of diabetes decreased in case versus control subjects. Overall, dipeptidyl peptidase IV activity (26.6+/-7.8 versus 29.6+/-7.3 nmol/mL per minute; P=0.026) and antigen (465.8+/-260.8 versus 563.1+/-208.6 ng/mL; P=0.017) were decreased in sera from individuals with angiotensin-converting enzyme inhibitor-associated angioedema compared with angiotensin-converting enzyme inhibitor-exposed control subjects without angioedema. Dipeptidyl peptidase IV activity (21.5+/-4.9 versus 29.8+/-6.7 nmol/mL per minute; P=0.001) and antigen (354.4+/-124.7 versus 559.8+/-163.2 ng/mL; P=0.003) were decreased in sera from cases collected during angiotensin-converting enzyme inhibition but not in the absence of angiotensin-converting enzyme inhibition. The degradation half-life of substance P correlated inversely with dipeptidyl peptidase IV antigen during angiotensin-converting enzyme inhibition. Environmental or genetic factors that reduce dipeptidyl peptidase IV activity may predispose individuals to angioedema.

Dipeptidyl Peptidase IV in Angiotensin-Converting Enzyme Inhibitor–Associated Angioedema

PubMed Central

Byrd, James Brian; Touzin, Karine; Sile, Saba; Gainer, James V.; Yu, Chang; Nadeau, John; Adam, Albert; Brown, Nancy J.

2009-01-01

Angioedema is a potentially life-threatening adverse effect of angiotensin-converting enzyme inhibitors. Bradykinin and substance P, substrates of angiotensin-converting enzyme, increase vascular permeability and cause tissue edema in animals. Studies indicate that amino-terminal degradation of these peptides, by aminopeptidase P and dipeptidyl peptidase IV, may be impaired in individuals with angiotensin-converting enzyme inhibitor–associated angioedema. This case-control study tested the hypothesis that dipeptidyl peptidase IV activity and antigen are decreased in sera of patients with a history of angiotensin-converting enzyme inhibitor–associated angioedema. Fifty subjects with a history of angiotensin-converting enzyme inhibitor–associated angioedema and 176 angiotensin-converting enzyme inhibitor–exposed control subjects were ascertained. Sera were assayed for angiotensin-converting enzyme activity, aminopeptidase P activity, aminopeptidase N activity, dipeptidyl peptidase IV activity, and antigen and the ex vivo degradation half-lives of bradykinin, des-Arg9-bradykinin, and substance P in a subset. The prevalence of smoking was increased and of diabetes decreased in case versus control subjects. Overall, dipeptidyl peptidase IV activity (26.6±7.8 versus 29.6±7.3 nmol/mL per minute; P=0.026) and antigen (465.8±260.8 versus 563.1±208.6 ng/mL; P=0.017) were decreased in sera from individuals with angiotensin-converting enzyme inhibitor–associated angioedema compared with angiotensin-converting enzyme inhibitor–exposed control subjects without angioedema. Dipeptidyl peptidase IV activity (21.5±4.9 versus 29.8±6.7 nmol/mL per minute; P=0.001) and antigen (354.4±124.7 versus 559.8±163.2 ng/mL; P=0.003) were decreased in sera from cases collected during angiotensin-converting enzyme inhibition but not in the absence of angiotensin-converting enzyme inhibition. The degradation half-life of substance P correlated inversely with dipeptidyl peptidase IV antigen during angiotensin-converting enzyme inhibition. Environmental or genetic factors that reduce dipeptidyl peptidase IV activity may predispose individuals to angioedema. PMID:18025295

Remarkable Reproducibility of Enzyme Activity Profiles in Tomato Fruits Grown under Contrasting Environments Provides a Roadmap for Studies of Fruit Metabolism1[W][OPEN

PubMed Central

Biais, Benoît; Bénard, Camille; Beauvoit, Bertrand; Colombié, Sophie; Prodhomme, Duyên; Ménard, Guillaume; Bernillon, Stéphane; Gehl, Bernadette; Gautier, Hélène; Ballias, Patricia; Mazat, Jean-Pierre; Sweetlove, Lee; Génard, Michel; Gibon, Yves

2014-01-01

To assess the influence of the environment on fruit metabolism, tomato (Solanum lycopersicum ‘Moneymaker’) plants were grown under contrasting conditions (optimal for commercial, water limited, or shaded production) and locations. Samples were harvested at nine stages of development, and 36 enzyme activities of central metabolism were measured as well as protein, starch, and major metabolites, such as hexoses, sucrose, organic acids, and amino acids. The most remarkable result was the high reproducibility of enzyme activities throughout development, irrespective of conditions or location. Hierarchical clustering of enzyme activities also revealed tight relationships between metabolic pathways and phases of development. Thus, cell division was characterized by high activities of fructokinase, glucokinase, pyruvate kinase, and tricarboxylic acid cycle enzymes, indicating ATP production as a priority, whereas cell expansion was characterized by enzymes involved in the lower part of glycolysis, suggesting a metabolic reprogramming to anaplerosis. As expected, enzymes involved in the accumulation of sugars, citrate, and glutamate were strongly increased during ripening. However, a group of enzymes involved in ATP production, which is probably fueled by starch degradation, was also increased. Metabolites levels seemed more sensitive than enzymes to the environment, although such differences tended to decrease at ripening. The integration of enzyme and metabolite data obtained under contrasting growth conditions using principal component analysis suggests that, with the exceptions of alanine amino transferase and glutamate and malate dehydrogenase and malate, there are no links between single enzyme activities and metabolite time courses or levels. PMID:24474652

Microbial Enzyme Activity and Carbon Cycling in Grassland Soil Fractions

NASA Astrophysics Data System (ADS)

Allison, S. D.; Jastrow, J. D.

2004-12-01

Extracellular enzymes are necessary to degrade complex organic compounds present in soils. Using physical fractionation procedures, we tested whether old soil carbon is spatially isolated from degradative enzymes across a prairie restoration chronosequence in Illinois, USA. We found that carbon-degrading enzymes were abundant in all soil fractions, including macroaggregates, microaggregates, and the clay fraction, which contains carbon with a mean residence time of ~200 years. The activities of two cellulose-degrading enzymes and a chitin-degrading enzyme were 2-10 times greater in organic matter fractions than in bulk soil, consistent with the rapid turnover of these fractions. Polyphenol oxidase activity was 3 times greater in the clay fraction than in the bulk soil, despite very slow carbon turnover in this fraction. Changes in enzyme activity across the restoration chronosequence were small once adjusted for increases in soil carbon concentration, although polyphenol oxidase activity per unit carbon declined by 50% in native prairie versus cultivated soil. These results are consistent with a `two-pool' model of enzyme and carbon turnover in grassland soils. In light organic matter fractions, enzyme production and carbon turnover both occur rapidly. However, in mineral-dominated fractions, both enzymes and their carbon substrates are immobilized on mineral surfaces, leading to slow turnover. Soil carbon accumulation in the clay fraction and across the prairie restoration chronosequence probably reflects increasing physical isolation of enzymes and substrates on the molecular scale, rather than the micron to millimeter scale.

Malabsorption syndrome observed in the common octopus Octopus vulgaris infected with Aggregata octopiana (Protista: Apicomplexa).

PubMed

Gestal, C; Páez de la Cadena, M; Pascual, S

2002-08-15

Octopus vulgaris infected with Aggregata octopiana were collected from an open-water culture system in the Ría of Aldán (NW Spain). Digestive tract infection values were determined with the use of a Neubauer chamber by counting the number of A. octopiana sporocysts. After determining enzyme activity values by the colorimetric Api-Zym system Biomerieux, one representative enzyme of glycosidases, peptid hydrolases and phosphoric hydrolases showing high activity was spectrophotometrically analysed. The enzymes were maltase and leucine-aminopeptidase (LAP) involved in the absorption process, and acid phosphatase, a lysosomic enzyme, respectively. Enzymatic activity of maltase and LAP decreased significantly, with increased sporocyst counts. However, acid phosphatase activity increased with severity of infection, indicating the presence of degradative enzymes from phagocytic cells in the infected area. A detrimental effect on gastrointestinal function may result from a decrease or malfunction of absorption enzymes. The results suggest a malabsorption syndrome resulting from parasitic infection.

Immobilization of pectin degrading enzyme from Bacillus licheniformis KIBGE IB-21 using agar-agar as a support.

PubMed

Rehman, Haneef Ur; Aman, Afsheen; Zohra, Raheela Rahmat; Qader, Shah Ali Ul

2014-02-15

Pectinase from Bacillus licheniformis KIBGE IB-21 was immobilized in agar-agar matrix using entrapment technique. Effect of different concentrations of agar-agar on pectinase immobilization was investigated and it was found that maximum immobilization was achieved at 3.0% agar-agar with 80% enzyme activity. After immobilization, the optimum temperature of enzyme increased from 45 to 50 °C and reaction time from 5 to 10 minutes as compared to free enzyme. Due to the limited diffusion of high molecular weight substrate, K(m) of immobilized enzyme slightly increased from 1.017 to 1.055 mg ml(-1), while Vmax decreased from 23,800 to 19,392 μM min(-1) as compared to free enzyme. After 120 h entrapped pectinase retained their activity up to 82% and 71% at 30 °C and 40 °C, respectively. The entrapped pectinase showed activity until 10th cycle and maintain 69.21% activity even after third cycle. Copyright © 2013 Elsevier Ltd. All rights reserved.

Activation of phenylalanine hydroxylase by phenylalanine does not require binding in the active site.

PubMed

Roberts, Kenneth M; Khan, Crystal A; Hinck, Cynthia S; Fitzpatrick, Paul F

2014-12-16

Phenylalanine hydroxylase (PheH), a liver enzyme that catalyzes the hydroxylation of excess phenylalanine in the diet to tyrosine, is activated by phenylalanine. The lack of activity at low levels of phenylalanine has been attributed to the N-terminus of the protein's regulatory domain acting as an inhibitory peptide by blocking substrate access to the active site. The location of the site at which phenylalanine binds to activate the enzyme is unknown, and both the active site in the catalytic domain and a separate site in the N-terminal regulatory domain have been proposed. Binding of catecholamines to the active-site iron was used to probe the accessibility of the active site. Removal of the regulatory domain increases the rate constants for association of several catecholamines with the wild-type enzyme by ∼2-fold. Binding of phenylalanine in the active site is effectively abolished by mutating the active-site residue Arg270 to lysine. The k(cat)/K(phe) value is down 10⁴ for the mutant enzyme, and the K(m) value for phenylalanine for the mutant enzyme is >0.5 M. Incubation of the R270K enzyme with phenylalanine also results in a 2-fold increase in the rate constants for catecholamine binding. The change in the tryptophan fluorescence emission spectrum seen in the wild-type enzyme upon activation by phenylalanine is also seen with the R270K mutant enzyme in the presence of phenylalanine. Both results establish that activation of PheH by phenylalanine does not require binding of the amino acid in the active site. This is consistent with a separate allosteric site, likely in the regulatory domain.

Activation of Phenylalanine Hydroxylase by Phenylalanine Does Not Require Binding in the Active Site

PubMed Central

2015-01-01

Phenylalanine hydroxylase (PheH), a liver enzyme that catalyzes the hydroxylation of excess phenylalanine in the diet to tyrosine, is activated by phenylalanine. The lack of activity at low levels of phenylalanine has been attributed to the N-terminus of the protein’s regulatory domain acting as an inhibitory peptide by blocking substrate access to the active site. The location of the site at which phenylalanine binds to activate the enzyme is unknown, and both the active site in the catalytic domain and a separate site in the N-terminal regulatory domain have been proposed. Binding of catecholamines to the active-site iron was used to probe the accessibility of the active site. Removal of the regulatory domain increases the rate constants for association of several catecholamines with the wild-type enzyme by ∼2-fold. Binding of phenylalanine in the active site is effectively abolished by mutating the active-site residue Arg270 to lysine. The kcat/Kphe value is down 104 for the mutant enzyme, and the Km value for phenylalanine for the mutant enzyme is >0.5 M. Incubation of the R270K enzyme with phenylalanine also results in a 2-fold increase in the rate constants for catecholamine binding. The change in the tryptophan fluorescence emission spectrum seen in the wild-type enzyme upon activation by phenylalanine is also seen with the R270K mutant enzyme in the presence of phenylalanine. Both results establish that activation of PheH by phenylalanine does not require binding of the amino acid in the active site. This is consistent with a separate allosteric site, likely in the regulatory domain. PMID:25453233

Role of active site rigidity in activity: MD simulation and fluorescence study on a lipase mutant.

PubMed

Kamal, Md Zahid; Mohammad, Tabrez Anwar Shamim; Krishnamoorthy, G; Rao, Nalam Madhusudhana

2012-01-01

Relationship between stability and activity of enzymes is maintained by underlying conformational flexibility. In thermophilic enzymes, a decrease in flexibility causes low enzyme activity while in less stable proteins such as mesophiles and psychrophiles, an increase in flexibility is associated with enhanced enzyme activity. Recently, we identified a mutant of a lipase whose stability and activity were enhanced simultaneously. In this work, we probed the conformational dynamics of the mutant and the wild type lipase, particularly flexibility of their active site using molecular dynamic simulations and time-resolved fluorescence techniques. In contrast to the earlier observations, our data show that active site of the mutant is more rigid than wild type enzyme. Further investigation suggests that this lipase needs minimal reorganization/flexibility of active site residues during its catalytic cycle. Molecular dynamic simulations suggest that catalytically competent active site geometry of the mutant is relatively more preserved than wild type lipase, which might have led to its higher enzyme activity. Our study implies that widely accepted positive correlation between conformation flexibility and enzyme activity need not be stringent and draws attention to the possibility that high enzyme activity can still be accomplished in a rigid active site and stable protein structures. This finding has a significant implication towards better understanding of involvement of dynamic motions in enzyme catalysis and enzyme engineering through mutations in active site.

Effects of 2,4,6-trinitrotoluene (TNT) on phase I and phase II biotransformation enzymes in European eel Anguilla anguilla (Linnaeus, 1758).

PubMed

Della Torre, Camilla; Corsi, Ilaria; Arukwe, Augustine; Alcaro, Luigi; Amato, Ezio; Focardi, Silvano

2008-07-01

The aim of this study was to investigate effects of the explosive 2,4,6-trinitrotoluene (TNT) on liver drug metabolizing genes and enzymes in the European eel Anguilla anguilla as a model fish species. Eels were exposed in vivo for 6h and 24h to 0.5, 1 and 2.5mg/L nominal concentrations of TNT. Expression of CYP1A, glutathione-S-transferase (pi-class; GST) and uridine-diphosphate glucuronosyltransferase (1-family) (UDPGT) genes was investigated by RT-PCR, and 7-ethoxy- and 7-methoxyresorufin-O-dealkylases (EROD, MROD), NADPH cyt c reductase (NADPH red), UDPGT and GST enzyme activities were measured by biochemical assays. An in vitro study was also performed, measuring only EROD activity. TNT exposure produced no modulation of CYP1A transcript expression while a significant inhibition of EROD enzyme activity was observed and confirmed in vitro. UDPGT transcript increased dose-dependently only at 6h while the UDPGT activity tended to increase dose-dependently at 24h. GST gene expression increased after 24h and significant increases of GST activity were observed both at 6 and 24h only at the highest TNT concentration. An increase of NADPH red activity was observed at 24h. Our results seem to indicate an inhibitory effect of TNT on CYP1A-dependent catalytic activities and a possible involvement of phase II enzymes as well as NADPH red in TNT metabolism in eels.

Nanoarmoring of Enzymes by Interlocking in Cellulose Fibers With Poly(Acrylic Acid).

PubMed

Riccardi, Caterina M; Kasi, Rajeswari M; Kumar, Challa V

2017-01-01

A simple method for interlocking glucose oxidase (GOx) and horseradish peroxidase (HRP) in cellulose fibers using poly(acrylic acid) (PAA) as an armor around the enzyme, without any need for activation of the cellulose support, is reported here. The resulting enzyme paper is an inexpensive, stable, simple, wearable, and washable biosensor. PAA functions as a multifunctional tether to interlock the enzyme molecules around the paper fibers so that the enzymes are protected against thermal/chemical denaturation and not released from the paper when washed with a detergent. The decreased conformational entropy of the interlocked enzyme protected by the nanoarmor is likely responsible for increased enzyme stability to heat and chemical denaturants (retained ≥70 percent enzyme activity after washing with urea or SDS for 30min), and the polymer protects the enzyme against inactivation by proteases, bacteria, inhibitors, etc. The kinetics of the interlocked enzyme were similar to that of the enzyme in solution. The V max was 6(±0.5)mM per minute before washing, then increased slightly to 9(±1.4)mM per minute after washing with water. The K m was 22(±6.4mM), which was slightly higher compared to GOx in solution (25-27mM). Because the surface area of the paper does not limit the enzyme loading, about 20% of enzyme was successfully loaded onto the paper (0.2g enzyme per gram of paper), and ≥95% of the enzyme was retained after washing. Interlocking works with other enzymes such as laccase, where ≥60% of the enzyme activity is retained. This novel methodology provides a low cost, simple, modular approach of achieving high enzyme loadings in ordinary filter paper, not limited by cellulose surface area, and there has been no need for complex methods of enzyme engineering or toxic methods of activation of the solid support to prepare highly active biocatalysts. © 2017 Elsevier Inc. All rights reserved.

Differential expression of glucose-metabolizing enzymes in multiple sclerosis lesions.

PubMed

Nijland, Philip G; Molenaar, Remco J; van der Pol, Susanne M A; van der Valk, Paul; van Noorden, Cornelis J F; de Vries, Helga E; van Horssen, Jack

2015-12-04

Demyelinated axons in multiple sclerosis (MS) lesions have an increased energy demand in order to maintain conduction. However, oxidative stress-induced mitochondrial dysfunction likely alters glucose metabolism and consequently impairs neuronal function in MS. Imaging and pathological studies indicate that glucose metabolism is altered in MS, although the underlying mechanisms and its role in neurodegeneration remain elusive. We investigated expression patterns of key enzymes involved in glycolysis, tricarboxylic acid (TCA) cycle and lactate metabolism in well-characterized MS tissue to establish which regulators of glucose metabolism are involved in MS and to identify underlying mechanisms. Expression levels of glycolytic enzymes were increased in active and inactive MS lesions, whereas expression levels of enzymes involved in the TCA cycle were upregulated in active MS lesions, but not in inactive MS lesions. We observed reduced expression and production capacity of mitochondrial α-ketoglutarate dehydrogenase (αKGDH) in demyelinated axons, which correlated with signs of axonal dysfunction. In inactive lesions, increased expression of lactate-producing enzymes was observed in astrocytes, whereas lactate-catabolising enzymes were mainly detected in axons. Our results demonstrate that the expression of various enzymes involved in glucose metabolism is increased in both astrocytes and axons in active MS lesions. In inactive MS lesions, we provide evidence that astrocytes undergo a glycolytic shift resulting in enhanced astrocyte-axon lactate shuttling, which may be pivotal for the survival of demyelinated axons. In conclusion, we show that key enzymes involved in energy metabolism are differentially expressed in active and inactive MS lesions. Our findings imply that, in addition to reduced oxidative phosphorylation activity, other bioenergetic pathways are affected as well, which may contribute to ongoing axonal degeneration in MS.

Impaired small-bowel barrier integrity in the presence of lumenal pancreatic digestive enzymes leads to circulatory shock.

PubMed

Kistler, Erik B; Alsaigh, Tom; Chang, Marisol; Schmid-Schönbein, Geert W

2012-08-01

In bowel ischemia, impaired mucosal integrity may allow intestinal pancreatic enzyme products to become systemic and precipitate irreversible shock and death. This can be attenuated by pancreatic enzyme inhibition in the small-bowel lumen. It is unresolved, however, whether ischemically mediated mucosal disruption is the key event allowing pancreatic enzyme products systemic access and whether intestinal digestive enzyme activity in concert with increased mucosal permeability leads to shock in the absence of ischemia. To test this possibility, the small intestinal lumen of nonischemic rats was perfused for 2 h with either digestive enzymes, a mucin disruption strategy (i.e., mucolytics) designed to increase mucosal permeability, or both, and animals were observed for shock. Digestive enzymes perfused included trypsin, chymotrypsin, elastase, amylase, and lipase. Control (n = 6) and experimental animals perfused with pancreatic enzymes only (n = 6) or single enzymes (n = 3 for each of the five enzyme groups) maintained stable hemodynamics. After mucin disruption using a combination of enteral N-acetylcysteine, atropine, and increased flow rates, rats (n = 6) developed mild hypotension (P < 0.001 compared with groups perfused with pancreatic enzymes only after 90 min) and increased intestinal permeability to intralumenally perfused fluorescein isothiocyanate-dextran 20 kd (P < 0.05) compared with control and enzyme-only groups, but there were no deaths. All animals perfused with both digestive enzymes and subjected to mucin disruption (n = 6) developed hypotension and increased intestinal permeability (P < 0.001 after 90 min). Pancreatic enzymes were measured in the intestinal wall of both groups subjected to mucin disruption, but not in the enzyme-only or control groups. Depletion of plasma protease inhibitors was found only in animals perfused with pancreatic enzymes plus mucin disruption, implicating increased permeability and intralumenal pancreatic enzyme egress in this group. These experiments demonstrate that increased bowel permeability via mucin disruption in the presence of pancreatic enzymes can induce shock and increase systemic protease activation in the absence of ischemia, implicating bowel mucin disruption as a key event in early ischemia. Digestive enzymes and their products, if allowed to penetrate the gut wall, may trigger multiorgan failure and death.

IMPAIRED SMALL BOWEL BARRIER INTEGRITY IN THE PRESENCE OF LUMENAL PANCREATIC DIGESTIVE ENZYMES LEADS TO CIRCULATORY SHOCK

PubMed Central

Kistler, Erik B.; Alsaigh, Tom; Chang, Marisol; Schmid-Schönbein, Geert W.

2012-01-01

In bowel ischemia, impaired mucosal integrity may allow intestinal pancreatic enzyme products to become systemic and precipitate irreversible shock and death. This can be attenuated by pancreatic enzyme inhibition in the small bowel lumen. It is unresolved, however, whether ischemically-mediated mucosal disruption is the key event allowing pancreatic enzyme products systemic access, and whether intestinal digestive enzyme activity in concert with increased mucosal permeability leads to shock in the absence of ischemia. To test this possibility, the small intestinal lumen of non-ischemic rats was perfused for two hours with either digestive enzymes, a mucin disruption strategy (i.e., mucolytics) designed to increase mucosal permeability, or both, and animals were observed for shock. Digestive enzymes perfused included trypsin, chymotrypsin, elastase, amylase and lipase. Control (n=6) and experimental animals perfused with pancreatic enzymes only (n=6) or single enzymes (n=3 for each of the five enzyme groups) maintained stable hemodynamics. After mucin disruption using a combination of enteral N-acetylcysteine, atropine, and increased flow rates, rats (n=6) developed mild hypotension (p<0.001 compared to groups perfused with pancreatic enzymes only after 90 minutes) and increased intestinal permeability to intralumenally perfused FITC-dextrans-20kD (p<0.05) compared to control and enzyme-only groups, but there were no deaths. All animals perfused with both digestive enzymes and subjected to mucin disruption (n=6) developed hypotension and increased intestinal permeability (p<0.001 after 90 minutes). Pancreatic enzymes were measured in the intestinal wall of both groups subjected to mucin disruption, but not in the enzyme-only or control groups. Depletion of plasma protease inhibitors was found only in animals perfused with pancreatic enzymes plus mucin disruption, implicating increased permeability and intralumenal pancreatic enzyme egress in this group. These experiments demonstrate that increased bowel permeability via mucin disruption in the presence of pancreatic enzymes can induce shock and increase systemic protease activation in the absence of ischemia, implicating bowel mucin disruption as a key event in early ischemia. Digestive enzymes and their products, if allowed to penetrate the gut wall may trigger multiorgan failure and death. PMID:22576000

Activity changes of antioxidant and detoxifying enzymes in Tenebrio molitor (Coleoptera: Tenebrionidae) larvae infected by the entomopathogenic nematode Heterorhabditis beicherriana (Rhabditida: Heterorhabditidae).

PubMed

Li, Xingyue; Liu, Qizhi; Lewis, Edwin E; Tarasco, Eustachio

2016-12-01

Entomopathogenic nematodes (EPNs) of the genera Steinernema and Heterorhabditis are lethal parasites of many insect species. To investigate defensive mechanisms towards EPNs in relation to antioxidative and detoxifying enzymes, we chose Tenebrio molitor (Coleoptera: Tenebrionidae) as experimental insect. We studied the activity changes of superoxide dismutases (SODs), peroxidases (PODs), and catalases (CATs), as well as tyrosinase (TYR), acetylcholinesterase (AChE), carboxylesterase (CarE), and glutathione S-transferase (GSTs) for 40 h in T. molitor larvae infected with Heterorhabditis beicherriana infective juveniles (IJs) at 5 rates (0, 20, 40, 80, and 160 IJs/larva). We found that when T. molitor larvae infected with H. beicherriana at higher rates (80 and 160 IJs/larva), SOD activity quickly increased to more than 70 % higher than that control levels. The activities of POD and CAT increased after 24 h. TYR activity increased slowly at lower rates of infection for 16 h, followed by a slight decrease, and then increasing from 32 to 40 h. The other detoxifying enzymes (GST, CarE, and AChE) were enhanced at lower infection rates, but were inhibited at higher rates. Our results suggested that host antioxidative response and detoxification reactions played a central role in the defensive reaction to EPNs, and that this stress which was reflected by the higher level enzymes activity contributed to the death of hosts. Further study should explore the exact function of these enzymes using different species of EPNs and investigate the links between enzyme activity and host susceptibility to EPNs.

[CSF enzyme activities in patients with head injury--especially on GOT, GPT, LDH, and CPK (AUTHOR'S TRANSL)].

PubMed

Nakamura, H; Mizuno, T; Kawamura, K; Kamino, T

1976-08-01

In our studies on patients with head injury, it was noted that there are some correlations between their clinical courses and the urinary excretion of creatine (cr), creatinine (Crn), 17-ketosteroid and 17-hydroxycorticosteroid. We observed the high urinary excretion of Cr in patients with severe head injury while almost negative in a mild case. We reported those facts in 1974. Also noted in patients with head injury is the relationship between the enzyme-activities (GOT, GPT, LDH and CPK) in the cerebrospinal fluid and their clinical courses. In this paper, we reported 34 cases of head injured patients (simple type: 2, concussion: 9, contusion: 8, acute intracranial hematoma: 7 and chronic intra-cranial hematoma: 8). The control values of CSF enzyme-activities were determined in these 14 cases (simple head injury, whip-lash injury and osteoma of the skull) as GOT less that 15, GPT less than 7, LDH less than 12 and CPK less than 8 units. In the moderate cases, a slight increase in activities of 4 enzymes in CSF were observed, while in severe or comatose cases, the enzyme-activities (especially LDH and CPK) were greater than in the controls. In the dead cases these values were five times as high as the normal case. In the patients recovering from a serious stage, these activities decreased to normal. High CSF enzyme-levels tend to indicate a poor prognosis and low levels a favorable progrosis. In the patients with a significant elevation of CSF enzymes, a high urinary excretion of Cr [normal range: 0-150 (ca. 50)mg/day] was often observed. There was no apparent correlation between the enzyme level in CSF and that in serum and the increase or decrease of these 4 enzymes are not always proprotionate with each other. As reported by Green (1958) and Lending (1961), cerebral cell necrosis and increased permeability of BLB, BBB or cerebral cell membrane can be related to the increase of enzymeactivities. With these observations, it can be considered that severe head injury gives influence on metabolic function in the hypothalamus and may cause in the levels of CSF enzymes and/or the urinary excretions of Cr, Crn and corticosteroids. And the examinations of enzyme activities in the patients with head injury may become a useful aid to make an outlook of their clinical coure and prognosis.

Effects of dietary lead acetate on hepatic detoxication enzyme activity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wagstaff, D.J.

1979-12-01

Lead-containing compounds usually inhibit enzymic and metabolic processes. This inhibition is presumed to be the mechanism of intoxication by these compounds. Inhibition of detoxication activities of liver microsomal enzymes could be particularly detrimental because the toxicity of many different substances would be increased. Exposure of experimental animals to lead compounds in several studies has been associated with depressed activity of hepatic microsomal enzymes, reduced levels of hepatic cytochrome P-450, reduced levels of hepatic microsomal protein, and prolonged hexobarbital sleep times. The present report contains observations that under certain experimental conditions there is stimulated hepatic meicrosomal enzyme activity in rats fedmore » lead acetate.« less

Defying the activity-stability trade-off in enzymes: taking advantage of entropy to enhance activity and thermostability.

PubMed

Siddiqui, Khawar Sohail

2017-05-01

The biotechnological applications of enzymes are limited due to the activity-stability trade-off, which implies that an increase in activity is accompanied by a concomitant decrease in protein stability. This premise is based on thermally adapted homologous enzymes where cold-adapted enzymes show high intrinsic activity linked to enhanced thermolability. In contrast, thermophilic enzymes show low activity around ambient temperatures. Nevertheless, genetically and chemically modified enzymes are beginning to show that the activity-stability trade-off can be overcome. In this review, the origin of the activity-stability trade-off, the thermodynamic basis for enhanced activity and stability, and various approaches for escaping the activity-stability trade-off are discussed. The role of entropy in enhancing both the activity and the stability of enzymes is highlighted with a special emphasis placed on the involvement of solvent water molecules. This review is concluded with suggestions for further research, which underscores the implications of these findings in the context of productivity curves, the Daniel-Danson equilibrium model, catalytic antibodies, and life on cold planets.

Stoichiometry constrains microbial response to root exudation - insights from a model and a field experiment in a temperate forest

NASA Astrophysics Data System (ADS)

Drake, J. E.; Darby, B. A.; Giasson, M.-A.; Kramer, M. A.; Phillips, R. P.; Finzi, A. C.

2012-06-01

Healthy plant roots release a wide range of chemicals into soils. This process, termed root exudation, is thought to increase the activity of microbes and the exo-enzymes they synthesize, leading to accelerated rates of carbon (C) mineralization and nutrient cycling in rhizosphere soils relative to bulk soils. The causal role of exudation, however, is difficult to isolate with in-situ observations, given the complex nature of the rhizosphere environment. We investigated the potential effects of root exudation on microbial and exo-enzyme activity using a theoretical model of decomposition and a field experiment, with a specific focus on the stoichiometric constraint of nitrogen (N) availability. The field experiment isolated the effect of exudation by pumping solutions of exudate mimics through microlysimeter "root simulators" into intact forest soils over two 50-day periods. Using a combined model-experiment approach, we tested two hypotheses: (1) exudation alone is sufficient to stimulate microbial and exo-enzyme activity in rhizosphere soils, and (2) microbial response to C-exudates (carbohydrates and organic acids) is constrained by N-limitation. Experimental delivery of exudate mimics containing C and N significantly increased microbial respiration, microbial biomass, and the activity of exo-enzymes that decompose labile components of soil organic matter (SOM, e.g., cellulose, amino sugars), while decreasing the activity of exo-enzymes that degrade recalcitrant SOM (e.g., polyphenols, lignin). However, delivery of C-only exudates had no effect on microbial biomass or overall exo-enzyme activity, and only increased microbial respiration. The theoretical decomposition model produced complementary results; the modeled microbial response to C-only exudates was constrained by limited N supply to support the synthesis of N-rich microbial biomass and exo-enzymes, while exuding C and N together elicited an increase in modeled microbial biomass, exo-enzyme activity, and decomposition. Thus, hypothesis (2) was supported, while hypothesis (1) was only supported when C and N compounds were exuded together. This study supports a cause-and-effect relationship between root exudation and enhanced microbial activity, and suggests that exudate stoichiometry is an important and underappreciated driver of microbial activity in rhizosphere soils.

Cellulase stability, adsorption/desorption profiles and recycling during successive cycles of hydrolysis and fermentation of wheat straw.

PubMed

Rodrigues, Ana Cristina; Felby, Claus; Gama, Miguel

2014-03-01

The potential of enzymes recycling after hydrolysis and fermentation of wheat straw under a variety of conditions was investigated, monitoring the activity of the enzymes in the solid and liquid fractions, using low molecular weight substrates. A significant amount of active enzymes could be recovered by recycling the liquid phase. In the early stage of the process, enzyme adsorb to the substrate, then gradually returning to the solution as the saccharification proceeds. At 50°C, normally regarded as an acceptable operational temperature for saccharification, the enzymes (Celluclast) significantly undergo thermal deactivation. The hydrolysis yield and enzyme recycling efficiency in consecutive recycling rounds can be increased by using high enzyme loadings and moderate temperatures. Indeed, the amount of enzymes in the liquid phase increased with its thermostability and hydrolytic efficiency. This study contributes towards developing effective enzymes recycling strategies and helping to reduce the enzyme costs on bioethanol production. Copyright © 2014 Elsevier Ltd. All rights reserved.

Serum prolidase enzyme activity in obese subjects and its relationship with oxidative stress markers.

PubMed

Aslan, Mehmet; Duzenli, Ufuk; Esen, Ramazan; Soyoral, Yasemin Usul

2017-10-01

The relationship between increased serum enzyme activity of prolidase and increased rate of collagen turnover in the arterial wall has been asserted in previous studies. Collagen reflects much of the strength to the connective tissue involved in the arterial wall. Atherosclerosis is very common vessel disease and oxidative stress plays a pivotal role in the etiopathogenesis. Our objective was to examine the serum enzyme activity of prolidase and its possible relationships with oxidative stress parameters in obese subjects. Our present study was conducted 27 obese subjects and 26 age-matched healthy control subjects. The serum enzyme activity of prolidase in all study population was evaluated spectrophotometrically. Oxidative stress levels in obese subjects were analyzed with total antioxidant capacity (TAC) and total oxidant status (TOS) as well as oxidative stress index (OSI). Obese subjects have higher serum TOS and OSI indicators as well as prolidase activity than those in control subjects (for all; p<0.001). Moreover, obese subjects have lower levels of TAC than in those in healthy subjects (p<0.001). In the Pearson's correlation analysis, enzyme activity of prolidase was positively related with TOS (p<0.001, r=0.529) and OSI (p<0.001, r=0.519) as well as BMI (p<0.001, r=0.692) and inversely related with TAC (p<0.05, r=-0.405) in obese subjects. Increased serum prolidase activity and decreased antioxidant levels are likely to be a results of increased of oxidative stress levels in obese subjects. The significantly correlation between increased oxidative stress and increased prolidase activity may play a pivotal role in etiopathogenesis of atherosclerotic cardiovascular diseases in obese subjects. Copyright © 2017 Elsevier B.V. All rights reserved.

Characterization and ontogenetic development of digestive enzymes in Pacific bluefin tuna Thunnus orientalis larvae.

PubMed

Murashita, Koji; Matsunari, Hiroyuki; Kumon, Kazunori; Tanaka, Yosuke; Shiozawa, Satoshi; Furuita, Hirofumi; Oku, Hiromi; Yamamoto, Takeshi

2014-12-01

The major digestive enzymes in Pacific bluefin tuna Thunnus orientalis larvae were characterized, and the physiological characteristics of the enzymes during early ontogeny were clarified using biochemical and molecular approaches. The maximum activity of trypsin (Try), chymotrypsin (Ct) and amylase (Amy) was observed at pH 6-11, 8-11 and 6-9, respectively. Maximum activity of Try, Ct and Amy occurred at 50 °C, that of lipase (Lip) was at 60 °C and that of pepsin (Pep) was at 40-50 °C. These pH and thermal profiles were similar to those for other fish species but differed from those previously reported for adult bluefin tuna. Enzyme activity for all enzymes assayed was found to decrease at high temperatures (Try, Ct, Amy and Pep: 50 °C; Lip: 40 °C), which is similar to findings for other fish species with one marked exception-increased Try activity was observed at 40 °C. Lip activity appeared to be dependent on bile salts under our assay conditions, resulting in a significant increase in activity in the presence of bile salts. Ontogenetic changes in pancreatic digestive enzymes showed similar gene expression patterns to those of other fish species, whereas marked temporal increases in enzyme activities were observed at 10-12 days post hatching (dph), coinciding with previously reported timing of the development of the pyloric caeca in bluefin tuna larvae. However, complete development of digestive function was indicated by the high pep gene expression from 19 dph, which contradicts the profile of Pep activity and previously reported development timing of the gastric gland. These findings contribute to the general knowledge of bluefin tuna larval digestive system development.

Enhanced enzyme stability through site-directed covalent immobilization.

PubMed

Wu, Jeffrey Chun Yu; Hutchings, Christopher Hayden; Lindsay, Mark Jeffrey; Werner, Christopher James; Bundy, Bradley Charles

2015-01-10

Breakthroughs in enzyme immobilization have enabled increased enzyme recovery and reusability, leading to significant decreases in the cost of enzyme use and fueling biocatalysis growth. However, current enzyme immobilization techniques suffer from leaching, enzyme stability, and recoverability and reusability issues. Moreover, these techniques lack the ability to control the orientation of the immobilized enzymes. To determine the impact of orientation on covalently immobilized enzyme activity and stability, we apply our PRECISE (Protein Residue-Explicit Covalent Immobilization for Stability Enhancement) system to a model enzyme, T4 lysozyme. The PRECISE system uses non-canonical amino acid incorporation and the Huisgen 1,3-dipolar cycloaddition "click" reaction to enable directed enzyme immobilization at rationally chosen residues throughout an enzyme. Unlike previous site-specific systems, the PRECISE system is a truly covalent immobilization method. Utilizing this system, enzymes immobilized at proximate and distant locations from the active site were tested for activity and stability under denaturing conditions. Our results demonstrate that orientation control of covalently immobilized enzymes can provide activity and stability benefits exceeding that of traditional random covalent immobilization techniques. PRECISE immobilized enzymes were 50 and 73% more active than randomly immobilized enzymes after harsh freeze-thaw and chemical denaturant treatments. Copyright © 2014 Elsevier B.V. All rights reserved.

The Ontogeny of Cytochrome P450 Enzyme Activity and Protein Abundance in Conventional Pigs in Support of Preclinical Pediatric Drug Research.

PubMed

Millecam, Joske; De Clerck, Laura; Govaert, Elisabeth; Devreese, Mathias; Gasthuys, Elke; Schelstraete, Wim; Deforce, Dieter; De Bock, Lies; Van Bocxlaer, Jan; Sys, Stanislas; Croubels, Siska

2018-01-01

Since the implementation of several legislations to improve pediatric drug research, more pediatric clinical trials are being performed. In order to optimize these pediatric trials, adequate preclinical data are necessary, which are usually obtained by juvenile animal models. The growing piglet has been increasingly suggested as a potential animal model due to a high degree of anatomical and physiological similarities with humans. However, physiological data in pigs on the ontogeny of major organs involved in absorption, distribution, metabolism, and excretion of drugs are largely lacking. The aim of this study was to unravel the ontogeny of porcine hepatic drug metabolizing cytochrome P450 enzyme (CYP450) activities as well as protein abundances. Liver microsomes from 16 conventional pigs (8 males and 8 females) per age group: 2 days, 4 weeks, 8 weeks, and 6-7 months were prepared. Activity measurements were performed with substrates of major human CYP450 enzymes: midazolam (CYP3A), tolbutamide (CYP2C), and chlorzoxazone (CYP2E). Next, the hepatic scaling factor, microsomal protein per gram liver (MPPGL), was determined to correct for enzyme losses during the fractionation process. Finally, protein abundance was determined using proteomics and correlated with enzyme activity. No significant sex differences within each age category were observed in enzyme activity or MPPGL. The biotransformation rate of all three substrates increased with age, comparable with human maturation of CYP450 enzymes. The MPPGL decreased from birth till 8 weeks of age followed by an increase till 6-7 months of age. Significant sex differences in protein abundance were observed for CYP1A2, CYP2A19, CYP3A22, CYP4V2, CYP2C36, CYP2E_1, and CYP2E_2. Midazolam and tolbutamide are considered good substrates to evaluate porcine CYP3A/2C enzymes, respectively. However, chlorzoxazone is not advised to evaluate porcine CYP2E enzyme activity. The increase in biotransformation rate with age can be attributed to an increase in absolute amount of CYP450 proteins. Finally, developmental changes were observed regarding the involvement of specific CYP450 enzymes in the biotransformation of the different substrates.

TISSUE ENZYME RESPONSE TO COLD AND TO HYPERPHAGIA IN THE RAT,

DTIC Science & Technology

activated glutaminase were increased. In animals with a comparable hyperphagia due to bilateral ablation of the ventromedial region of the hypothalamus...concluded that changes of enzyme activities in cold-exposed rats are not simply an adaptation to the increased nutrient flow consequent upon the hyperphagia induced. (Author)

Cost-efficient entrapment of β-glucosidase in nanoscale latex and silicone polymeric thin films for use as stable biocatalysts.

PubMed

Javed, Muhammad Rizwan; Buthe, Andreas; Rashid, Muhammad Hamid; Wang, Ping

2016-01-01

β-Glucosidase is an ubiquitous enzyme which has enormous biotechnological applications. Its deficiency in natural enzyme preparations is often overcome by exogenous supplementation, which further increases the enzyme utilization cost. Enzyme immobilization offers a potential solution through enzyme recycling and easy recovery. In the present work Aspergillus niger β-glucosidase is immobilized within nanoscale polymeric materials (polyurethane, latex and silicone), through entrapment, and subsequently coated onto a fibrous support. Highest apparent activity (90 U g(-1) polymer) was observed with latex, while highest entrapment efficiency (93%) was observed for the silicone matrix. Immobilization resulted in the thermo-stabilization of the β-glucosidase with an increase in optimum temperature and activation energy for cellobiose hydrolysis. Supplementation to cellulases also resulted in an increased cellulose hydrolysis, while retaining more than 70% functional stability. Hence, the current study describes novel preparations of immobilized β-glucosidase as highly stable and active catalysts for industrial food- and bio-processing applications. Copyright © 2015 Elsevier Ltd. All rights reserved.

Eicosapentaenoic acid and docosahexaenoic acid increase the degradation of amyloid-β by affecting insulin-degrading enzyme.

PubMed

Grimm, Marcus O W; Mett, Janine; Stahlmann, Christoph P; Haupenthal, Viola J; Blümel, Tamara; Stötzel, Hannah; Grimm, Heike S; Hartmann, Tobias

2016-12-01

Omega-3 polyunsaturated fatty acids (PUFAs) have been proposed to be highly beneficial in Alzheimer's disease (AD). AD pathology is closely linked to an overproduction and accumulation of amyloid-β (Aβ) peptides as extracellular senile plaques in the brain. Total Aβ levels are not only dependent on its production by proteolytic processing of the amyloid precursor protein (APP), but also on Aβ-clearance mechanisms, including Aβ-degrading enzymes. Here we show that the omega-3 PUFAs eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) increase Aβ-degradation by affecting insulin-degrading enzyme (IDE), the major Aβ-degrading enzyme secreted into the extracellular space of neuronal and microglial cells. The identification of the molecular mechanisms revealed that EPA directly increases IDE enzyme activity and elevates gene expression of IDE. DHA also directly stimulates IDE enzyme activity and affects IDE sorting by increasing exosome release of IDE, resulting in enhanced Aβ-degradation in the extracellular milieu. Apart from the known positive effect of DHA in reducing Aβ production, EPA and DHA might ameliorate AD pathology by increasing Aβ turnover.

Status epilepticus-induced changes in the subcellular distribution and activity of calcineurin in rat forebrain.

PubMed

Kurz, Jonathan E; Rana, Annu; Parsons, J Travis; Churn, Severn B

2003-12-01

This study was performed to determine the effect of prolonged status epilepticus on the activity and subcellular location of a neuronally enriched, calcium-regulated enzyme, calcineurin. Brain fractions isolated from control animals and rats subjected to pilocarpine-induced status epilepticus were subjected to differential centrifugation. Specific subcellular fractions were tested for both calcineurin activity and enzyme content. Significant, status epilepticus-induced increases in calcineurin activity were found in homogenates, nuclear fractions, and crude synaptic membrane-enriched fractions isolated from both cortex and hippocampus. Additionally, significant increases in enzyme levels were observed in crude synaptic fractions as measured by Western analysis. Immunohistochemical studies revealed a status epilepticus-induced increase in calcineurin immunoreactivity in dendritic structures of pyramidal neurons of the hippocampus. The data demonstrate a status epilepticus-induced increase in calcineurin activity and concentration in the postsynaptic region of forebrain pyramidal neurons.

The Effects of Ibogaine on Uterine Smooth Muscle Contractions: Relation to the Activity of Antioxidant Enzymes.

PubMed

Oreščanin-Dušić, Zorana; Tatalović, Nikola; Vidonja-Uzelac, Teodora; Nestorov, Jelena; Nikolić-Kokić, Aleksandra; Mijušković, Ana; Spasić, Mihajlo; Paškulin, Roman; Bresjanac, Mara; Blagojević, Duško

2018-01-01

Ibogaine is an indole alkaloid originally extracted from the root bark of the African rainforest shrub Tabernanthe iboga . It has been explored as a treatment for substance abuse because it interrupts drug addiction and relieves withdrawal symptoms. However, it has been shown that ibogaine treatment leads to a sharp and transient fall in cellular ATP level followed by an increase of cellular respiration and ROS production. Since contractile tissues are sensitive to changes in the levels of ATP and ROS, here we investigated an ibogaine-mediated link between altered redox homeostasis and uterine contractile activity. We found that low concentrations of ibogaine stimulated contractile activity in spontaneously active uteri, but incremental increase of doses inhibited it. Inhibitory concentrations of ibogaine led to decreased SOD1 and elevated GSH-Px activity, but doses that completely inhibited contractions increased CAT activity. Western blot analyses showed that changes in enzyme activities were not due to elevated enzyme protein concentrations but posttranslational modifications. Changes in antioxidant enzyme activities point to a vast concentration-dependent increase in H 2 O 2 level. Knowing that extracellular ATP stimulates isolated uterus contractility, while H 2 O 2 has an inhibitory effect, this concentration-dependent stimulation/inhibition could be linked to ibogaine-related alterations in ATP level and redox homeostasis.

The Effects of Ibogaine on Uterine Smooth Muscle Contractions: Relation to the Activity of Antioxidant Enzymes

PubMed Central

Paškulin, Roman

2018-01-01

Ibogaine is an indole alkaloid originally extracted from the root bark of the African rainforest shrub Tabernanthe iboga. It has been explored as a treatment for substance abuse because it interrupts drug addiction and relieves withdrawal symptoms. However, it has been shown that ibogaine treatment leads to a sharp and transient fall in cellular ATP level followed by an increase of cellular respiration and ROS production. Since contractile tissues are sensitive to changes in the levels of ATP and ROS, here we investigated an ibogaine-mediated link between altered redox homeostasis and uterine contractile activity. We found that low concentrations of ibogaine stimulated contractile activity in spontaneously active uteri, but incremental increase of doses inhibited it. Inhibitory concentrations of ibogaine led to decreased SOD1 and elevated GSH-Px activity, but doses that completely inhibited contractions increased CAT activity. Western blot analyses showed that changes in enzyme activities were not due to elevated enzyme protein concentrations but posttranslational modifications. Changes in antioxidant enzyme activities point to a vast concentration-dependent increase in H2O2 level. Knowing that extracellular ATP stimulates isolated uterus contractility, while H2O2 has an inhibitory effect, this concentration-dependent stimulation/inhibition could be linked to ibogaine-related alterations in ATP level and redox homeostasis. PMID:29599898

Substance P increases Sympathetic Activity during Combined Angiotensin Converting Enzyme and Dipeptidyl Peptidase-4 Inhibition

PubMed Central

Devin, Jessica K.; Pretorius, Mias; Nian, Hui; Yu, Chang; Billings, Frederic T.; Brown, Nancy J.

2014-01-01

Dipeptidyl peptidase-4 inhibitors prevent the degradation of incretin hormones and reduce post-prandial hyperglycemia in patients with type 2 diabetes mellitus. Dipeptidyl peptidase-4 degrades other peptides with a penultimate proline or alanine, including bradykinin and substance P, which are also substrates of angiotensin-converting enzyme. During angiotensin-converting enzyme inhibition, substance P is inactivated primarily by dipeptidyl peptidase-4, while bradykinin is first inactivated by aminopeptidase P. This study tested the hypothesis that dipeptidyl peptidase-4 inhibition potentiates vasodilator and fibrinolytic responses to substance P when angiotensin-converting enzyme is inhibited. Twelve healthy subjects participated in this randomized, double-blinded, placebo-controlled crossover study. On each study day, subjects received sitagliptin 200 mg p.o. or placebo. Substance P and bradykinin were infused via brachial artery before and during intra-arterial enalaprilat. Sitagliptin and enalaprilat each reduced forearm vascular resistance and increased forearm blood flow without affecting mean arterial pressure, but there was no interactive effect of the inhibitors. Enalaprilat increased bradykinin-stimulated vasodilation and tissue plasminogen activator release; sitagliptin did not affect these responses to bradykinin. The vasodilator response to substance P was unaffected by sitagliptin and enalaprilat, however, substance P increased heart rate and vascular release of norepinephrine during combined angiotensin-converting enzyme and dipeptidyl peptidase-4 inhibition. In women, sitagliptin diminished tissue plasminogen activator release in response to substance P both alone and during enalaprilat. Substance P increases sympathetic activity during combined angiotensin-converting enzyme and dipeptidyl peptidase-4 inhibition. PMID:24516103

Impact of heavy metal on activity of some microbial enzymes in the riverbed sediments: Ecotoxicological implications in the Ganga River (India).

PubMed

Jaiswal, Deepa; Pandey, Jitendra

2018-04-15

We studied the extracellular enzyme activity (EEA) in the riverbed sediment along a 518km gradient of the Ganga River receiving carbon and nutrient load from varied human sources. Also, we tested, together with substrate-driven stimulation, if the heavy metal accumulated in the sediment inhibits enzyme activities. Because pristine values are not available, we considered Dev Prayag, a least polluted site located 624km upstream to main study stretch, as a reference site. There were distinct increases in enzyme activities in the sediment along the study gradient from Dev Prayag, however, between-site differences were in concordance with sediment carbon(C), nitrogen (N) and phosphorus (P). Fluorescein diacetate hydrolysis (FDAase), β-glucosidase (Glu) and protease activities showed positive correlation with C, N and P while alkaline phosphatase was found negatively correlated with P. Enzyme activities were found negatively correlated with heavy metal, although ecological risk index (E R i ) varied with site and metal species. Dynamic fit curves showed significant positive correlation between heavy metal and microbial metabolic quotient (qCO 2 ) indicating a decrease in microbial activity in response to increasing heavy metal concentrations. This study forms the first report linking microbial enzyme activities to regional scale sediment heavy metal accumulation in the Ganga River, suggests that the microbial enzyme activities in the riverbed sediment were well associated with the proportion of C, N and P and appeared to be a sensitive indicator of C, N and P accumulation in the river. Heavy metal accumulated in the sediment inhibits enzyme activities, although C rich sediment showed relatively low toxicity due probably to reduced bioavailability of the metal. The study has relevance from ecotoxicological as well as from biomonitoring perspectives. Copyright © 2017 Elsevier Inc. All rights reserved.

Spatially resolved metabolic analysis reveals a central role for transcriptional control in carbon allocation to wood.

PubMed

Roach, Melissa; Arrivault, Stéphanie; Mahboubi, Amir; Krohn, Nicole; Sulpice, Ronan; Stitt, Mark; Niittylä, Totte

2017-06-15

The contribution of transcriptional and post-transcriptional regulation to modifying carbon allocation to developing wood of trees is not well defined. To clarify the role of transcriptional regulation, the enzyme activity patterns of eight central primary metabolism enzymes across phloem, cambium, and developing wood of aspen (Populus tremula L.) were compared with transcript levels obtained by RNA sequencing of sequential stem sections from the same trees. Enzymes were selected on the basis of their importance in sugar metabolism and in linking primary metabolism to lignin biosynthesis. Existing enzyme assays were adapted to allow measurements from ~1 mm3 sections of dissected stem tissue. These experiments provided high spatial resolution of enzyme activity changes across different stages of wood development, and identified the gene transcripts probably responsible for these changes. In most cases, there was a clear positive relationship between transcripts and enzyme activity. During secondary cell wall formation, the increases in transcript levels and enzyme activities also matched with increased levels of glucose, fructose, hexose phosphates, and UDP-glucose, emphasizing an important role for transcriptional regulation in carbon allocation to developing aspen wood. These observations corroborate the efforts to increase carbon allocation to wood by engineering gene regulatory networks. © The Author 2017. Published by Oxford University Press on behalf of the Society for Experimental Biology.

Increased Activity of Rhizosphere and Hyphosphere Enzymes under Elevated CO2 in a Loblolly Pine Stand

NASA Astrophysics Data System (ADS)

Meier, I.; Phillips, R.

2012-12-01

The stimulatory effect of elevated atmospheric CO2 under global climate change on forest productivity has been predicted to decrease over time as pools of available N in soil become depleted, but empirical support for such progressive N limitation has been lacking. Increased N acquisition from soil depleted in inorganic nitrogen requires stimulation of the microbial processing of organic N, possibly through increasing C supply to soil by plant roots or mycorrhizal hyphae. Increases in (mycorr)rhizosphere C fluxes could stimulate microbes to produce extra-cellular enzymes that release N from SOM, feeding back from soil microsites to ecosystem-scale processes. We investigated the influence of elevated CO2 on root exudation and soil enzyme activity at the Duke Forest FACE site, USA, where loblolly pine (Pinus taeda L.) stands have been exposed to elevated CO2 for 14 years and N fertilization for five years. In each plot, root boxes containing acetate windows were installed in 2008. Two years after installation, we collected soils adjacent to root tips (the rhizosphere), hyphal tips (the hyphosphere) and bulk soil. We measured in situ root exudation rates from intact pine roots. Study objectives were to analyze (i) the influence of atmospheric CO2 on root exudation and extra-cellular enzyme activities, (ii) the influence of soil N availability in regulating these activities, and (iii) the relationship between the activities of enzymes involved in N cycling in soils and gross N transformations at soil microsites. Elevated atmospheric CO2 significantly increased the activity of β-1-4-N-acetylglucosaminidase (NAG) in the rhizosphere by almost 2.5 times (39 to 95 nmol h-1 g-1), and 1.6fold in the hyphosphere relative to ambient plots. NAG is an enzyme involved in the degradation of chitin from the cell walls of soil organisms, releasing absorbable forms of nitrogen. The activity of peroxidase, which degrades aromatic C compounds of SOM, increased significantly in the hyphosphere of stands exposed to elevated CO2. Nitrogen fertilization diminished this effect of elevated CO2 on enzyme activities at microsites. Our results show that the metabolism of microbial communities is shifted to the decomposition of organic N under elevated atmospheric CO2, presumably stimulated by N limitation and increased root C exudation.

Stability improvement of immobilized lactoperoxidase using polyaniline polymer.

PubMed

Jafary, Fariba; Kashanian, Soheila; Sharieat, Ziadin Samsam; Jafary, Farzaneh; Omidfar, Kobra; Paknejad, Maliheh

2012-12-01

Enzyme engineering via immobilization techniques is perfectly compatible against the other chemical or biological approximate to improve enzyme functions and stability. In this study lactoperoxidase was immobilized onto polyaniline polymer activated with glutaraldehyde as a bifunctional agent, to improve enzyme properties. Polyaniline polymer was used due its unique physical and chemical properties to immobilize lactoperoxidase (LPO). The optimum activity of immobilized LPO was observed at pH 6 and 55 °C, which has been increased about 10 °C for the immobilized enzyme. The immobilized enzyme maintained absolutely active for 60 days whereas the native enzyme lost 80 % of its initial activity within this period of time. Moreover, the immobilized enzyme can be reused for several times without loss of activity. The kinetic parameter studies showed slight differences between free and immobilized enzymes. The K(m) and K(m.app) were calculated to be 0.6 and 0.4; also V(max) and V(max.app) were 1.3 and 0.9 respectively.

Evaluation of Krebs cycle enzymes in the brain of rats after chronic administration of antidepressants.

PubMed

Scaini, Giselli; Santos, Patricia M; Benedet, Joana; Rochi, Natália; Gomes, Lara M; Borges, Lislaine S; Rezin, Gislaine T; Pezente, Daiana P; Quevedo, João; Streck, Emilio L

2010-05-31

Several works report brain impairment of metabolism as a mechanism underlying depression. Citrate synthase and succinate dehydrogenase are enzymes localized within cells in the mitochondrial matrix and are important steps of Krebs cycle. In addition, citrate synthase has been used as a quantitative enzyme marker for the presence of intact mitochondria. Thus, we investigated citrate synthase and succinate dehydrogenase activities from rat brain after chronic administration of paroxetine, nortriptiline and venlafaxine. Adult male Wistar rats received daily injections of paroxetine (10mg/kg), nortriptiline (15mg/kg), venlafaxine (10mg/kg) or saline in 1.0mL/kg volume for 15 days. Twelve hours after the last administration, the rats were killed by decapitation, the hippocampus, striatum and prefrontal cortex were immediately removed, and activities of citrate synthase and succinate dehydrogenase were measured. We verified that chronic administration of paroxetine increased citrate synthase activity in the prefrontal cortex, hippocampus, striatum and cerebral cortex of adult rats; cerebellum was not affected. Chronic administration of nortriptiline and venlafaxine did not affect the enzyme activity in these brain areas. Succinate dehydrogenase activity was increased by chronic administration of paroxetine and nortriptiline in the prefrontal cortex, hippocampus, striatum and cerebral cortex of adult rats; cerebellum was not affected either. Chronic administration of venlafaxine increased succinate dehydrogenase activity in prefrontal cortex, but did not affect the enzyme activity in cerebellum, hippocampus, striatum and cerebral cortex. Considering that metabolism impairment is probably involved in the pathophysiology of depressive disorders, an increase in these enzymes by antidepressants may be an important mechanism of action of these drugs. Copyright (c) 2010 Elsevier Inc. All rights reserved.

Modulation of digestive physiology and biochemistry in Mytilus californianus in response to feeding level acclimation and microhabitat

PubMed Central

Sung, Aaron; Garcia, Nathan S.; Gracey, Andrew Y.; German, Donovan P.

2016-01-01

ABSTRACT The intertidal mussel Mytilus californianus is a critical foundation species that is exposed to fluctuations in the environment along tidal- and wave-exposure gradients. We investigated feeding and digestion in mussels under laboratory conditions and across environmental gradients in the field. We assessed whether mussels adopt a rate-maximization (higher ingestion and lower assimilation) or a yield-maximization acquisition (lower ingestion and higher assimilation) strategy under laboratory conditions by measuring feeding physiology and digestive enzyme activities. We used digestive enzyme activity to define resource acquisition strategies in laboratory studies, then measured digestive enzyme activities in three microhabitats at the extreme ends of the tidal- and wave-exposure gradients within a stretch of shore (<20 m) projected sea-ward. Our laboratory results indicated that mussels benefit from a high assimilation efficiency when food concentration is low and have a low assimilation efficiency when food concentration is high. Additionally, enzyme activities of carbohydrases amylase, laminarinase and cellulase were elevated when food concentration was high. The protease trypsin, however, did not increase with increasing food concentration. In field conditions, low-shore mussels surprisingly did not have high enzyme activities. Rather, high-shore mussels exhibited higher cellulase activities than low-shore mussels. Similarly, trypsin activity in the high-shore-wave-sheltered microhabitat was higher than that in high-shore-wave-exposed. As expected, mussels experienced increasing thermal stress as a function of reduced submergence from low to high shore and shelter from wave-splash. Our findings suggest that mussels compensate for limited feeding opportunities and thermal stress by modulating digestive enzyme activities. PMID:27402963

A novel and efficient method for the immobilization of thermolysin using sodium chloride salting-in and consecutive microwave irradiation.

PubMed

Chen, Feifei; Zhang, Fangkai; Du, Fangchuan; Wang, Anming; Gao, Weifang; Wang, Qiuyan; Yin, Xiaopu; Xie, Tian

2012-07-01

Sodium chloride salting-in and microwave irradiation were combined to drive thermolysin molecules into mesoporous support to obtain efficiently immobilized enzyme. When the concentration of sodium chloride was 3 M and microwave power was 40 W, 93.2% of the enzyme was coupled to the support by 3 min, and the maximum specific activity of the immobilized enzyme was 17,925.1 U mg(-1). This was a 4.5-fold increase in activity versus enzyme immobilized using conventional techniques, and a 1.6-fold increase versus free enzyme. Additionally, the thermal stability of the immobilized thermolysin was significantly improved. When incubated at 70°C, there was no reduction in activity by 3.5h, whereas free thermolysin lost most of its activity by 3h. Immobilization also protected the thermolysin against organic solvent denaturation. The microwave-assisted immobilization technique, combined with sodium chloride salting-in, could be applied to other sparsely soluble enzymes immobilization because of its simplicity and high efficiency. Copyright © 2011 Elsevier Ltd. All rights reserved.

[Comparison of the efficiency of polygalacturonase and beta-glucosidase enzyme preparations in stabilization of cherry plum wine material].

PubMed

Stepanova, E V; Landesman, E O; Fedorova, T V; Iakovleva, K E; Koroleva, O V

2006-01-01

Pektofoetidin and Pectinex, enzyme preparations with the highest polygalacturonase and beta-glucosidase activities, were covalently immobilized on DEAE cellulose and Aminosilochromes 10 and 30. After treatment of cherry plum wine material with the soluble and immobilized enzyme preparations, the content of phenolics increased by 26 and 40%, respectively. The increase was accompanied by a decrease in the protein content (by up to 37%), carbohydrate content (by 17% on the average), and antioxidant activity (5-37%). The most efficient treatment involved Pektofoetidin immobilized on Aminosilochrome 10. It increased the clarity of the wine material and its antioxidant activity by 100 and 10%, respectively.

Extending enzyme molecular recognition with an expanded amino acid alphabet

PubMed Central

Windle, Claire L.; Simmons, Katie J.; Ault, James R.; Trinh, Chi H.; Nelson, Adam

2017-01-01

Natural enzymes are constructed from the 20 proteogenic amino acids, which may then require posttranslational modification or the recruitment of coenzymes or metal ions to achieve catalytic function. Here, we demonstrate that expansion of the alphabet of amino acids can also enable the properties of enzymes to be extended. A chemical mutagenesis strategy allowed a wide range of noncanonical amino acids to be systematically incorporated throughout an active site to alter enzymic substrate specificity. Specifically, 13 different noncanonical side chains were incorporated at 12 different positions within the active site of N-acetylneuraminic acid lyase (NAL), and the resulting chemically modified enzymes were screened for activity with a range of aldehyde substrates. A modified enzyme containing a 2,3-dihydroxypropyl cysteine at position 190 was identified that had significantly increased activity for the aldol reaction of erythrose with pyruvate compared with the wild-type enzyme. Kinetic investigation of a saturation library of the canonical amino acids at the same position showed that this increased activity was not achievable with any of the 20 proteogenic amino acids. Structural and modeling studies revealed that the unique shape and functionality of the noncanonical side chain enabled the active site to be remodeled to enable more efficient stabilization of the transition state of the reaction. The ability to exploit an expanded amino acid alphabet can thus heighten the ambitions of protein engineers wishing to develop enzymes with new catalytic properties. PMID:28196894

Characterization of a neutral protease from lysosomes of rabbit polymorphonuclear leucocytes

PubMed Central

Davies, Philip; Rita, Giuseppe A.; Krakauer, Kathrin; Weissmann, Gerald

1971-01-01

1. The subcellular distribution has been investigated of a protease from rabbit polymorphonuclear leucocytes, obtained from peritoneal exudates. The enzyme, optimally active between pH7.0 and 7.5, hydrolyses histone but not haemoglobin, sediments almost exclusively with a granule fraction rich in other lysosomal enzymes, and is latent until the granules are disrupted by various means. 2. Enzymic analysis of specific and azurophilic granules separated by zonal centrifugation showed that neutral protease activity was confined to fractions rich in enzymes characteristic of azurophile granules. 3. Recovery of neutral protease activity from subcellular fractions was several times greater than that found in whole cells. This finding was explained by the presence of a potent inhibitor of the enzyme activity in the cytoplasm. 4. The effect of the inhibitor was reversed by increasing ionic strength (up to 2.5m-potassium chloride) and by polyanions such as heparin and dextran sulphate, but not by an uncharged polymer, dextran. 5. The enzyme was also inhibited, to a lesser extent, by 1-chloro-4-phenyl-3-l-toluene-p-sulphonamidobutan-2-one, soya-bean trypsin inhibitor and ∈-aminohexanoate (∈-aminocaproate). 6. The granule fractions failed to hydrolyse artificial substrates for trypsin and chymotrypsin. 7. Partial separation of the enzyme was achieved by Sephadex gel filtration at high ionic strength and by isoelectric focusing. The partially separated, activated enzyme showed an approximately 300-fold increase in specific activity over that in whole cells. PMID:5126908

Peroxisomal enzymes in the liver of rats with experimental diabetes mellitus type 2.

PubMed

Turecký, L; Kupčová, V; Uhlíková, E; Mojto, V

2014-01-01

Diabetes mellitus is relatively frequently associated with fatty liver disease. Increased oxidative stress probably plays an important role in the development of this hepatopathy. One of possible sources of reactive oxygen species in liver is peroxisomal system. There are several reports about changes of peroxisomal enzymes in experimental diabetes, mainly enzymes of fatty acid oxidation. The aim of our study was to investigate the possible changes of activities of liver peroxisomal enzymes, other than enzymes of beta-oxidation, in experimental diabetes mellitus type 2. Biochemical changes in liver of experimental animals suggest the presence of liver steatosis. The changes of serum parameters in experimental group are similar to changes in serum of patients with non-alcoholic fatty liver disease. We have shown that diabetes mellitus influenced peroxisomal enzymes by the different way. Despite of well-known induction of peroxisomal beta-oxidation, the activities of catalase, aminoacid oxidase and NADH-cytochrome b(5) reductase were not significantly changed and the activities of glycolate oxidase and NADP-isocitrate dehydrogenase were significantly decreased. The effect of diabetes on liver peroxisomes is probably due to the increased supply of fatty acids to liver in diabetic state and also due to increased oxidative stress. The changes of metabolic activity of peroxisomal compartment may participate on the development of diabetic hepatopathy.

Altered cytochrome P450 activities and expression levels in the liver and intestines of the monosodium glutamate-induced mouse model of human obesity.

PubMed

Tomankova, Veronika; Liskova, Barbora; Skalova, Lenka; Bartikova, Hana; Bousova, Iva; Jourova, Lenka; Anzenbacher, Pavel; Ulrichova, Jitka; Anzenbacherova, Eva

2015-07-15

Cytochromes P450 (CYPs) are enzymes present from bacteria to man involved in metabolism of endogenous and exogenous compounds incl. drugs. Our objective was to assess whether obesity leads to changes in activities and expression of CYPs in the mouse liver, small intestine and colon. An obese mouse model with repeated injection of monosodium glutamate (MSG) to newborns was used. Controls were treated with saline. All mice were sacrificed at 8 months. In the liver and intestines, levels of CYP mRNA and proteins were analyzed using RT-PCR and Western blotting. Activities of CYP enzymes were measured with specific substrates of human orthologous forms. At the end of the experiment, body weight, plasma insulin and leptin levels as well as the specific content of hepatic CYP enzymes were increased in obese mice. Among CYP enzymes, hepatic CYP2A5 activity, protein and mRNA expression increased most significantly in obese animals. Higher activities and protein levels of hepatic CYP2E1 and 3A in the obese mice were also found. No or a weak effect on CYPs 2C and 2D was observed. In the small intestine and colon, no changes of CYP enzymes were detected except for increased expression of CYP2E1 and decreased expression of CYP3A mRNAs in the colon of the obese mice. Results of our study suggest that the specific content and activities of some liver CYP enzymes (especially CYP2A5) can be increased in obese mice. Higher activity of CYP2A5 (CYP2A6 human ortholog) could lead to altered metabolism of drug substrates of this enzyme (valproic acid, nicotine, methoxyflurane). Copyright © 2015 Elsevier Inc. All rights reserved.

A rise in the plasma activities of hepatic enzymes is not a common consequence of hypoglycaemia.

PubMed

Jones, R G; Grant, P J; Brown, D; Stickland, M; Wiles, P G

1988-04-01

Eight otherwise healthy insulin-dependent diabetic patients were subjected to controlled, symptomatic hypoglycaemia for 20 min (median glucose concentration 1.7 mmol/l, range 1.0-2.6 mmol/l). Concentrations of plasma adrenaline and plasma vasopressin were significantly increased, indicating normal counter-regulatory responses for these hormones. Plasma activities of the hepatic enzymes AST, ALT, LDH, GGT, and CK did not increase during or following the period of hypoglycaemia. Thus, abnormal plasma enzyme activities noted after clinical hypoglycaemia should be fully investigated, and not disregarded as due to the hypoglycaemic episode.

Mitochondrial oxidative enzyme activity in individual fibre types in hypo- and hyperthyroid rat skeletal muscles.

PubMed

Johnson, M A; Turnbull, D M

1984-04-01

Quantitative cytochemical and biochemical techniques have been used in combination to study the response of mitochondrial oxidative enzymes in individual muscle fibre types to hypo- and hyperthyroidism. Hypothyroidism resulted in decreased activity of succinate dehydrogenase (SDH), L-glycerol-3-phosphate dehydrogenase (L-GPDH), and D-3-hydroxybutyrate dehydrogenase (D-HBDH) in all fibre types of both slow-twitch soleus and fast-twitch extensor digitorum longus (e.d.l.) muscles. In hyperthyroidism, only L-GPDH activity increased in e.d.l. but more marked increases were seen in soleus muscles, which also showed increased SDH activity. In addition to these alterations in the enzyme activity in individual fibre types the metabolic profile of the muscle is further modified by the hormone-induced interconversion of slow- to fast-twitch fibres and vice versa.

Irradiation effects on hydrases for biomedical applications

NASA Astrophysics Data System (ADS)

Furuta, Masakazu; Ohashi, Isao; Oka, Masahito; Hayashi, Toshio

2000-03-01

To apply an irradiation technique to sterilize "Hybrid" biomedical materials including enzymes, we selected papain, a well-characterized plant endopeptidase as a model to examine durability of enzyme activity under the practical irradiation condition in which limited data were available for irradiation inactivation of enzymes. Dry powder and frozen aqueous solution of papain showed significant durability against 60Co-gamma irradiation suggesting that, the commercial irradiation sterilizing method is applicable without modification. Although irradiation of unfrozen aqueous papain solution showed an unusual change of the enzymatic activity with the increasing doses, and was totally inactivated at 15 kGy, we managed to keep the residual activity more than 50% of initial activity after 30-kGy irradiation, taking such optimum conditions as increasing enzyme concentration from 10 to 100 mg/ml and purging with N 2 gas to suppress the formation of free radicals.

A six-year longitudinal study of phosphorus enrichment on soil enzymes in acidic forest soils.

NASA Astrophysics Data System (ADS)

Deforest, J. L.; Freedman, Z.

2017-12-01

Acidic nitrogen (N) deposition may be shifting the nutrient economies of forest soils from one dominated by N more towards phosphorus (P) limitation. While the short-term responses of nutrient enrichment experiments are reported, there is a lack of information on the longer-term response mediating ecosystem nutrient dynamics, especially for P. We hypothesized that long-term soil P amendments should result in the persistent suppression of P-acquiring extracellular enzymes when compared with ambient soils. Alternatively, vegetation and/or the microbial community may have acclimated to require more P (i.e., communities more suitable to the altered nutrient economy) resulting in an increase in the activity of P-acquiring enzymes relative to carbon (C) and N-acquiring enzyme activity. To test the hypothesis, P availability was indirectly and/or directly increased by raising soil pH and/or the addition of phosphate fertilizer and maintained for six years. Study sites were in two North American eastern deciduous forest regions on glaciated soils with modest P availability and unglaciated with low P availability. For the glaciated sites, C:N acquiring enzyme activity remained stable and was insensitive to 6 years of elevated pH and/or P in the, but there was modest increases in the unglaciated site. Phosphorus-acquiring enzyme activity was insensitive to the treatments in the glaciated sites. For unglaciated sites, P-acquiring enzyme activity was suppressed under P addition in year one, rebounded in the second year, and was suppressed in the subsequent years. These results suggest that the basal nutrient resources of an ecosystem will have a very strong influence on its response to nutrient enrichment. Likewise, the second-year recovery of P-acquiring enzyme activity might be evidence of acclimation, but the gradual yearly suppression of these enzymes suggests the system has not reach a steady state.

Effects of frying oil and Houttuynia cordata thunb on xenobiotic-metabolizing enzyme system of rodents

PubMed Central

Chen, Ya-Yen; Chen, Chiao-Ming; Chao, Pi-Yu; Chang, Tsan-Ju; Liu, Jen-Fang

2005-01-01

AIM: To evaluate the effects of frying oil and Houttuynia cordata Thunb (H. cordata), a vegetable traditionally consumed in Taiwan, on the xenobiotic-metabolizing enzyme system of rodents. METHODS: Forty-eight Sprague-Dawley rats were fed with a diet containing 0%, 2% or 5% H. cordata powder and 15% fresh soybean oil or 24-h oxidized frying oil (OFO) for 28 d respectively. The level of microsomal protein, total cytochrome 450 content (CYP450) and enzyme activities including NADPH reductase, ethoxyresorufin O-deethylase (EROD), pentoxyresorufin O-dealkylase (PROD), aniline hydroxylase (ANH), aminopyrine demethylase (AMD), and quinone reductase (QR) were determined. QR represented phase II enzymes, the rest of the enzymes tested represented phase I enzymes. RESULTS: The oxidized frying oil feeding produced a significant increase in phase I and II enzyme systems, including the content of CYP450 and microsomal protein, and the activities of NADPH reductase, EROD, PROD, ANH, AMD and QR in rats (P<0.05). In addition, the activities of EROD, ANH and AMD decreased and QR increased after feeding with H. cordata in OFO-fed group (P<0.05). The feeding with 2% H. cordata diet showed the most significant effect. CONCLUSION: The OFO diet induces phases I and II enzyme activity, and the 2% H. cordata diet resulted in a better regulation of the xenobiotic-metabolizing enzyme system. PMID:15637750

Effects of aqueous eucalyptus extracts on seed germination, seedling growth and activities of peroxidase and polyphenoloxidase in three wheat cultivar seedlings (Triticum aestivum L.).

PubMed

Ziaebrahimi, L; Khavari-Nejad, R A; Fahimi, H; Nejadsatari, T

2007-10-01

Evaluation of allelopathic effects of this plant on other near cultivations especially wheat is the aim of this study. Effects of water extracts of eucalyptus leaves examined on germination and growth of three wheat cultivar seeds and seedlings. Results showed that: germination percentage strongly decreased, leaf and root lengths also affected and dry and wet weights of both roots and shoots showed similar change patterns. Activities of peroxidase and polyphenoloxidase as antioxidant enzymes in roots and shoots measured. Activity of peroxidases increased in stress conditions and roots showed more increased enzyme activity than leaves. Activity of polyphenoloxidases increased only in one of three cultivars and again roots showed more activity of this enzyme in response to eucalyptus extract. Suggest that detoxification process were conducted mainly in roots of seedlings.

Enhanced activity and stability of L-arabinose isomerase by immobilization on aminopropyl glass.

PubMed

Zhang, Ye-Wang; Jeya, Marimuthu; Lee, Jung-Kul

2011-03-01

Immobilization of Bacillus licheniformis L: -arabinose isomerase (BLAI) on aminopropyl glass modified with glutaraldehyde (4 mg protein g support⁻¹) was found to enhance the enzyme activity. The immobilization yield of BLAI was proportional to the quantity of amino groups on the surface of support. Reducing particle size increased the adsorption capacity (q(m)) and affinity (k(a)). The pH and temperature for immobilization were optimized to be pH 7.1 and 33 °C using response surface methodology (RSM). The immobilized enzyme was characterized and compared to the free enzyme. There is no change in optimal pH and temperature before and after immobilization. However, the immobilized BLAI enzyme achieved 145% of the activity of the free enzyme. Correspondingly, the catalytic efficiency (k(cat)/K(m)) was improved 1.47-fold after immobilization compared to the free enzyme. The thermal stability was improved 138-fold (t₁/₂) increased from 2 to 275 h) at 50 °C following immobilization.

Induction of antioxidant enzyme activities by a phenylurea derivative, EDU.

PubMed

Stevens, T M; Boswell, G A; Adler, R; Ackerman, N R; Kerr, J S

1988-10-01

Oxygen free radicals have the potential to mediate cell injury. Defenses against such radicals include the antioxidant enzymes superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-PX). The purposes of this study were (1) to develop an in vitro model using human cells in which to investigate a potential pharmacologic agent as an inducer of these antioxidant enzymes; (2) to investigate the phenylurea derivative N-[2-(2-oxo-1-imidazolindinyl)ethyl]-N-phenylurea (EDU) in this model with paraquat (PQ) serving as the positive control; and (3) to determine if induction of the antioxidant enzymes by EDU occurs in vivo. Human gingival fibroblasts (Gin-1) were used as the target cell in vitro; PQ and EDU, an inducer of SOD and CAT activities in plants, were evaluated as antioxidant enzyme inducers. Total SOD activity in Gin-1 cells increased 2-fold (p less than 0.05) in the presence of 1.0 mM PQ for 18-48 hr compared with untreated controls. Gin-1 cells incubated with 0.25-2.0 mM PQ for 24 hr had significantly increased total SOD (1.5 to 2.0-fold; p less than 0.05). CAT activity increased with 1.0 and 2.0 mM PQ (p less than 0.05). In the presence of PQ, GSH-PX activity decreased (p less than 0.05) in a concentration-dependent manner, indicating inactivation of this enzyme. No toxicity, indicated by lactate dehydrogenase released into the incubation medium, was noted at PQ concentrations below 5.0 mM. In the presence of 0.125-2.0 mM EDU, total SOD activity in Gin-1 cells significantly increased (1.5 to 2.0-fold; p less than 0.05). CAT activity significantly increased in a dose-dependent manner (p less than 0.05), while GSH-PX activity remained constant following exposure to 0.125-2.0 mM EDU. Intraperitoneal administration of EDU to rats twice a day for 2 days at 100 mg/kg induced SOD activity in heart, liver, and lung compared to controls (p less than 0.05). CAT activity increased in the liver 56% and in the lung 36% (p less than 0.05). GSH-PX activity remained constant. Our findings indicate that Gin-1 cells are a useful model in which to study inducers of antioxidant enzymes in vitro and that the phenylurea compound EDU induces SOD and CAT activities both in vitro and in vivo.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Alhenc-Gelas, F.; Weare, J.A.; Johnson, R.L. Jr.

CE (converting enzyme) was purified from human lung, and antisera were raised in rabbits. Antisera inhibited the activity of the purified enzyme from lung and kidney and the plasma CE of normal persons and sarcoid patients. With antisera at a titer of 1:100,000, a sensitive, direct RIA was developed. CE purified from lung or kidney and CE present in normal and in sarcoid plasma gave parallel logit-log displacement lines, suggesting immunological identity. The level of CE in normal human plasma was 400 +/- 131 ng/ml. In untreated sarcoid patients, the enzyme level and activity increased in parallel. There was amore » negative correlation between enzyme level and diffusing capacity of the lung for CO in sarcoid patients. Synthetic inhibitors such as captopril or MK 421 did not interfere with the RIA, permitting enzyme levels to be monitored in patients undergoing acute inhibitor therapy. During administration of MK 421, CE activity was negligible and plasma levels of CE did not change. In contrast, renin activity increased eightfold during the inhibitor therapy.« less

Investigating the effect of gallium curcumin and gallium diacetylcurcumin complexes on the structure, function and oxidative stability of the peroxidase enzyme and their anticancer and antibacterial activities.

PubMed

Jahangoshaei, Parisa; Hassani, Leila; Mohammadi, Fakhrossadat; Hamidi, Akram; Mohammadi, Khosro

2015-10-01

Curcumin has a wide spectrum of biological and pharmacological activities including anti-inflammatory, antioxidant, antiproliferative, antimicrobial and anticancer activities. Complexation of curcumin with metals has gained attention in recent years for improvement of its stability. In this study, the effect of gallium curcumin and gallium diacetylcurcumin on the structure, function and oxidative stability of horseradish peroxidase (HRP) enzyme were evaluated by spectroscopic techniques. In addition to the enzymatic investigation, the cytotoxic effect of the complexes was assessed on bladder, MCF-7 breast cancer and LNCaP prostate carcinoma cell lines by MTT assay. Furthermore, antibacterial activity of the complexes against S. aureus and E. coli was explored by dilution test method. The results showed that the complexes improve activity of HRP and also increase its tolerance against the oxidative condition. After addition of the complexes, affinity of HRP for hydrogen peroxide substrate decreases, while the affinity increases for phenol substrate. Circular dichroism, intrinsic and synchronous fluorescence spectra showed that the enzyme structure around the catalytic heme group becomes less compact and also the distance between the heme group and tryptophan residues increases due to binding of the complexes to HRP. On the whole, it can be concluded that the change in the enzyme structure upon binding to the gallium curcumin and gallium diacetylcurcumin complexes results in an increase in the antioxidant efficiency and activity of the peroxidise enzyme. The result of anticancer and antibacterial activities suggested that the complexes exhibit the potential for cancer treatment, but they have no significant antibacterial activity.

High sensibility to reactivation by acidic lipids of the recombinant human plasma membrane Ca2+-ATPase isoform 4xb purified from Saccharomyces cerevisiae.

PubMed

Cura, Carolina I; Corradi, Gerardo R; Rinaldi, Débora E; Adamo, Hugo P

2008-12-01

The human plasma membrane Ca2+ pump (isoform 4xb) was expressed in Saccharomyces cerevisiae and purified by calmodulin-affinity chromatography. Under optimal conditions the recombinant enzyme (yPMCA) hydrolyzed ATP in a Ca2+ dependent manner at a rate of 15 micromol/mg/min. The properties of yPMCA were compared to those of the PMCA purified from human red cells (ePMCA). The mobility of yPMCA in SDS-PAGE was the expected for the hPMCA4xb protein but slightly lower than that of ePMCA. Both enzymes achieved maximal activity when supplemented with acidic phospholipids. However, while ePMCA in mixed micelles of phosphatidylcholine-detergent had 30% of its maximal activity, the yPMCA enzyme was nearly inactive. Increasing the phosphatidylcholine content of the micelles did not increase the activity of yPMCA but the activity in the presence of phosphatidylcholine improved by partially removing the detergent. The reactivation of the detergent solubilized yPMCA required specifically acidic lipids and, as judged by the increase in the level of phosphoenzyme, it involved the increase in the amount of active enzyme. These results indicate that the function of yPMCA is highly sensitive to delipidation and the restitution of acidic lipids is needed for a functional enzyme.

Increased Diels-Alderase activity through backbone remodeling guided by Foldit players.

PubMed

Eiben, Christopher B; Siegel, Justin B; Bale, Jacob B; Cooper, Seth; Khatib, Firas; Shen, Betty W; Players, Foldit; Stoddard, Barry L; Popovic, Zoran; Baker, David

2012-01-22

Computational enzyme design holds promise for the production of renewable fuels, drugs and chemicals. De novo enzyme design has generated catalysts for several reactions, but with lower catalytic efficiencies than naturally occurring enzymes. Here we report the use of game-driven crowdsourcing to enhance the activity of a computationally designed enzyme through the functional remodeling of its structure. Players of the online game Foldit were challenged to remodel the backbone of a computationally designed bimolecular Diels-Alderase to enable additional interactions with substrates. Several iterations of design and characterization generated a 24-residue helix-turn-helix motif, including a 13-residue insertion, that increased enzyme activity >18-fold. X-ray crystallography showed that the large insertion adopts a helix-turn-helix structure positioned as in the Foldit model. These results demonstrate that human creativity can extend beyond the macroscopic challenges encountered in everyday life to molecular-scale design problems.

[Study on soil enzyme activities and microbial biomass carbon in greenland irrigated with reclaimed water].

PubMed

Pan, Neng; Hou, Zhen-An; Chen, Wei-Ping; Jiao, Wen-Tao; Peng, Chi; Liu, Wen

2012-12-01

The physicochemical properties of soils might be changed under the long-term reclaimed water irrigation. Its effects on soil biological activities have received great attentions. We collected surface soil samples from urban green spaces and suburban farmlands of Beijing. Soil microbial biomass carbon (SMBC), five types of soil enzyme activities (urease, alkaline phosphatase, invertase, dehydrogenase and catalase) and physicochemical indicators in soils were measured subsequently. SMBC and enzyme activities from green land soils irrigated with reclaimed water were higher than that of control treatments using drinking water, but the difference is not significant in farmland. The SMBC increased by 60.1% and 14.2% than those control treatments in 0-20 cm soil layer of green land and farmland, respectively. Compared with their respective controls, the activities of enzymes in 0-20 cm soil layer of green land and farmland were enhanced by an average of 36.7% and 7.4%, respectively. Investigation of SMBC and enzyme activities decreased with increasing of soil depth. Significantly difference was found between 0-10 cm and 10-20 cm soil layer in green land. Soil biological activities were improved with long-term reclaimed water irrigation in Beijing.

Changes of Serum Angiotensin-Converting Enzyme Activity During Treatment of Patients with Graves’ Disease*

PubMed Central

Lee, Dong Soo; Chung, June-Key; Cho, Bo Youn; Koh, Chang-Soon; Lee, Munho

1986-01-01

Serum angiotensin-converting enzyme activity was measured spectrophotometrically, and serum thyrotropin-binding-inhibitory immunoglobulin (TBII) activity was measured by radioreceptor assay in normal subjects and in patients with Graves’ disease serially before and during treatment, and these activities were compared with each other and with thyroid hormone levels in various thyroid functional status. Correlation between serum angiotensin-converting enzyme activity and serum thyroid hormone level was pursued with relation to the changes of thyroid functional status in patients with Graves’ disease during treatment. Serum angiotensin-converting enzyme activity was significantly elevated in patients with hyperthyroid Graves’ disease before the start of treatment (35 ± 13 nmol/min/ml, n=50), and not in patients with Graves’ disease, euthyroid state during treatment with antithyroid drugs or radioactive iodine (23 ± 9 nmol/min/ml, n=12), but decreased significantly in patients with Graves’ disease, hypothyroid state transiently during treatment (15 ± 4 nmol/min/ml, n=12), respectively in comparison with normal control subjects. Serum angiotensin-converting enzyme activity was positively correlated with the log value of serum T3 concentration (r=0.62, p<0.001, n=95), and with the log value of free thyroxine index (r=0.66, p<0.001, n=91) but not statistically significantly with serum TBII activity. Serum angiotensin-converting enzyme activity was followed in 11 patients with initially increased activity and the activity decreased in proportion to serum thyroid hormone level during treatment, irrespective of treatment modality. It is suggested that thyroid hormones play a role in the increase and decrease of serum angiotensin-converting enzyme activity directly or indirectly influencing the peripheral tissues (probably reticuloendothelial cells or peripheral endothelial cells) in patients with Graves’ disease. PMID:15759385

Kalpaamruthaa ameliorates mitochondrial and metabolic alterations in diabetes mellitus induced cardiovascular damage.

PubMed

Latha, Raja; Shanthi, Palanivelu; Sachdanandam, Panchanadham

2014-12-01

Efficacy of Kalpaamruthaa on the activities of lipid and carbohydrate metabolic enzymes, electron transport chain complexes and mitochondrial ATPases were studied in heart and liver of experimental rats. Cardiovascular damage (CVD) was developed in 8 weeks after type 2 diabetes mellitus induction with high fat diet (2 weeks) and low dose of streptozotocin (2 × 35 mg/kg b.w. i.p. in 24 hr interval). In CVD-induced rats, the activities of total lipase, cholesterol ester hydrolase and cholesterol ester synthetase were increased, while lipoprotein lipase and lecithin-cholesterol acyltransferase activities were decreased. The activities of lipid-metabolizing enzymes were altered by Kalpaamruthaa in CVD-induced rats towards normal. Kalpaamruthaa modulated the activities of glycolytic enzymes (hexokinase, phosphogluco-isomerase, aldolase and glucose-6-phosphate dehydrogenase), gluconeogenic enzymes (glucose-6-phosphatase and fructose-1, 6-bisphosphatase) and glycogenolytic enzyme (glycogen phosphorylase) along with increased glycogen content in the liver of CVD-induced rats. The activities of isocitrate dehydrogenase, succinate dehydrogenase, malate dehydrogenase, α-ketoglutarate dehydrogenase, Complexes and ATPases (Na(+)/K(+)-ATPase, Ca(2+)-ATPase and Mg(2+)-ATPase) were decreased in CVD-induced rats, which were ameliorated by the treatment with Kalpaamruthaa. This study ascertained the efficacy of Kalpaamruthaa for the treatment of CVD in diabetes through the modulation of metabolizing enzymes and mitochondrial dysfunction.

Changes in element accumulation, phenolic metabolism, and antioxidative enzyme activities in the red-skin roots of Panax ginseng.

PubMed

Zhou, Ying; Yang, Zhenming; Gao, Lingling; Liu, Wen; Liu, Rongkun; Zhao, Junting; You, Jiangfeng

2017-07-01

Red-skin root disease has seriously decreased the quality and production of Panax ginseng (ginseng). To explore the disease's origin, comparative analysis was performed in different parts of the plant, particularly the epidermis, cortex, and/or fibrous roots of 5-yr-old healthy and diseased red-skin ginseng. The inorganic element composition, phenolic compound concentration, reactive oxidation system, antioxidant concentrations such as ascorbate and glutathione, activities of enzymes related to phenolic metabolism and oxidation, and antioxidative system particularly the ascorbate-glutathione cycle were examined using conventional methods. Aluminum (Al), iron (Fe), magnesium, and phosphorus were increased, whereas manganese was unchanged and calcium was decreased in the epidermis and fibrous root of red-skin ginseng, which also contained higher levels of phenolic compounds, higher activities of the phenolic compound-synthesizing enzyme phenylalanine ammonia-lyase and the phenolic compound oxidation-related enzymes guaiacol peroxidase and polyphenoloxidase. As the substrate of guaiacol peroxidase, higher levels of H 2 O 2 and correspondingly higher activities of superoxide dismutase and catalase were found in red-skin ginseng. Increased levels of ascorbate and glutathione; increased activities of l-galactose 1-dehydrogenase, ascorbate peroxidase, ascorbic acid oxidase, and glutathione reductase; and lower activities of dehydroascorbate reductase, monodehydroascorbate reductase, and glutathione peroxidase were found in red-skin ginseng. Glutathione- S -transferase activity remained constant. Hence, higher element accumulation, particularly Al and Fe, activated multiple enzymes related to accumulation of phenolic compounds and their oxidation. This might contribute to red-skin symptoms in ginseng. It is proposed that antioxidant and antioxidative enzymes, especially those involved in ascorbate-glutathione cycles, are activated to protect against phenolic compound oxidation.

A Rigidifying Salt-Bridge Favors the Activity of Thermophilic Enzyme at High Temperatures at the Expense of Low-Temperature Activity

PubMed Central

Lam, Sonia Y.; Yeung, Rachel C. Y.; Yu, Tsz-Ha; Sze, Kong-Hung; Wong, Kam-Bo

2011-01-01

Background Thermophilic enzymes are often less active than their mesophilic homologues at low temperatures. One hypothesis to explain this observation is that the extra stabilizing interactions increase the rigidity of thermophilic enzymes and hence reduce their activity. Here we employed a thermophilic acylphosphatase from Pyrococcus horikoshii and its homologous mesophilic acylphosphatase from human as a model to study how local rigidity of an active-site residue affects the enzymatic activity. Methods and Findings Acylphosphatases have a unique structural feature that its conserved active-site arginine residue forms a salt-bridge with the C-terminal carboxyl group only in thermophilic acylphosphatases, but not in mesophilic acylphosphatases. We perturbed the local rigidity of this active-site residue by removing the salt-bridge in the thermophilic acylphosphatase and by introducing the salt-bridge in the mesophilic homologue. The mutagenesis design was confirmed by x-ray crystallography. Removing the salt-bridge in the thermophilic enzyme lowered the activation energy that decreased the activation enthalpy and entropy. Conversely, the introduction of the salt-bridge to the mesophilic homologue increased the activation energy and resulted in increases in both activation enthalpy and entropy. Revealed by molecular dynamics simulations, the unrestrained arginine residue can populate more rotamer conformations, and the loss of this conformational freedom upon the formation of transition state justified the observed reduction in activation entropy. Conclusions Our results support the conclusion that restricting the active-site flexibility entropically favors the enzymatic activity at high temperatures. However, the accompanying enthalpy-entropy compensation leads to a stronger temperature-dependency of the enzymatic activity, which explains the less active nature of the thermophilic enzymes at low temperatures. PMID:21423654

A rigidifying salt-bridge favors the activity of thermophilic enzyme at high temperatures at the expense of low-temperature activity.

PubMed

Lam, Sonia Y; Yeung, Rachel C Y; Yu, Tsz-Ha; Sze, Kong-Hung; Wong, Kam-Bo

2011-03-01

Thermophilic enzymes are often less active than their mesophilic homologues at low temperatures. One hypothesis to explain this observation is that the extra stabilizing interactions increase the rigidity of thermophilic enzymes and hence reduce their activity. Here we employed a thermophilic acylphosphatase from Pyrococcus horikoshii and its homologous mesophilic acylphosphatase from human as a model to study how local rigidity of an active-site residue affects the enzymatic activity. Acylphosphatases have a unique structural feature that its conserved active-site arginine residue forms a salt-bridge with the C-terminal carboxyl group only in thermophilic acylphosphatases, but not in mesophilic acylphosphatases. We perturbed the local rigidity of this active-site residue by removing the salt-bridge in the thermophilic acylphosphatase and by introducing the salt-bridge in the mesophilic homologue. The mutagenesis design was confirmed by x-ray crystallography. Removing the salt-bridge in the thermophilic enzyme lowered the activation energy that decreased the activation enthalpy and entropy. Conversely, the introduction of the salt-bridge to the mesophilic homologue increased the activation energy and resulted in increases in both activation enthalpy and entropy. Revealed by molecular dynamics simulations, the unrestrained arginine residue can populate more rotamer conformations, and the loss of this conformational freedom upon the formation of transition state justified the observed reduction in activation entropy. Our results support the conclusion that restricting the active-site flexibility entropically favors the enzymatic activity at high temperatures. However, the accompanying enthalpy-entropy compensation leads to a stronger temperature-dependency of the enzymatic activity, which explains the less active nature of the thermophilic enzymes at low temperatures.

Effects of dietary Pb on accumulation, histopathology, and digestive enzyme activities in the digestive system of tilapia (Oreochromis niloticus).

PubMed

Dai, Wei; Du, Huahua; Fu, Linglin; Jin, Chengguan; Xu, Zirong; Liu, Huitao

2009-02-01

With the increasing occurrence of dietary lead (Pb) contamination in aquatic environment, threat of the dietary Pb toxicity to aquatic organisms attracted more attention. In this study, after being exposed to dietary Pb at concentrations of 0, 100, 400, and 800-microg/g dry weight for 60 days, the groups of tilapia (Oreochromis niloticus) were sacrificed and sampled to analyze the effects of dietary Pb on accumulation, histopathology, and digestive enzyme activities in tissues of the digestive system. The results showed that the Pb accumulation in tissues increased with the dietary Pb concentrations. Moreover, Pb accumulated in sampled tissues in the following order: intestine > stomach > liver. By observation of liver histological sections in optical microscope, lesions could be detected in the Pb-contaminated groups. It was also demonstrated that the inhibitory effect of dietary Pb on digestive enzyme activities was dietary Pb concentration dependent. Different degrees of inhibition of enzyme activities were exhibited in sampled tissues. It was indicated that digestive enzyme activities in the digestive system might be considered as the potential biomarkers of dietary Pb contamination in tilapia.

Calcium protects Trifolium repens L. seedlings against cadmium stress.

PubMed

Wang, Chang Quan; Song, Heng

2009-09-01

The effect of calcium (Ca(2+)) on Trifolium repens L. seedlings subjected to cadmium (Cd(2+)) stress was studied by investigating plant growth and changes in activity of antioxidative enzymes. Physiological analysis was carried out on seedlings cultured for 2 weeks on half-strength Hoagland medium with Cd(2+) concentrations of 0, 400 and 600 microM, and on corresponding medium supplied with CaCl(2) (5 mM). Exposure to increasing Cd(2+) reduced the fresh weight of the upper part (stems + leaves) of the seedlings more strongly than that of the root system. In both parts of T. repens seedlings H(2)O(2) level and lipid peroxidation increased. In the upper part, Cd(2+) exposure led to a significant decrease in the activity of superoxide dismutase, catalase and glutathione peroxidase and an increase in ascorbate peroxidase activity. In contrast, the roots showed an increase in the activity of antioxidative enzymes under Cd(2+) stress. Ca(2+) addition to medium reduced the Cd(2+) accumulation, and considerably reversed the Cd(2+)-induced decrease in fresh mass as well as the changes in lipid peroxidation in the both parts of T. repens seedlings. Ca(2+) application diminished the Cd(2+) effect on the activity of antioxidative enzymes in the upper part, even though it did not significantly affect these enzymes in the roots. So the possible mechanisms for the action of Ca(2+) in Cd(2+) stress were considered to reduce Cd(2+) accumulation, alleviate lipid peroxidation and promote activity of antioxidative enzymes.

Relevance of substrates and products on the desorption of lipases physically adsorbed on hydrophobic supports.

PubMed

Virgen-Ortíz, Jose J; Tacias-Pascacio, Veymar G; Hirata, Daniela B; Torrestiana-Sanchez, Beatriz; Rosales-Quintero, Arnulfo; Fernandez-Lafuente, Roberto

2017-01-01

Lipase B from Candida antarctica (CALB) has been physically immobilized on octyl-agarose via interfacial activation. The incubation of the enzyme in 80% ethanol at pH 5 and 25°C has not significant effect on enzyme activity. Moreover, the hydrolysis of 100mM tributyrin catalyzed by this biocatalyst exhibited a quite linear reaction course. However, a new cycle of tributyrin hydrolysis showed a drastic drop in the activity. SDS-PAGE gels of the supernatant and the biocatalyst showed a significant enzyme desorption after the reaction. Similar results could be appreciated using triacetin or sunflower oil, while using 300mM methyl phenyl acetate, butyl butyrate or ethyl butyrate most enzyme molecules remained immobilized. The results show that the detergent properties of some reaction products increase the enzyme release from the hydrophobic support, and this problem increased if the concentration of the reactants increased. Using 500mM tributyrin, even in fully aqueous medium, some enzyme desorption from the support may be observed. Thus, the results show a limitation of this kind of biocatalysts that should be considered in the selection of an industrial lipase biocatalyst. Copyright © 2016 Elsevier Inc. All rights reserved.

Improved production of an enzyme that hydrolyses raw yam starch by Penicillium sp. S-22 using fed-batch fermentation.

PubMed

Sun, Hai-Yan; Ge, Xiang-Yang; Zhang, Wei-Guo

2006-11-01

A newly isolated strain, Penicillium sp. S-22, was used to produce an enzyme that hydrolyses raw yam starch [raw yam starch digesting enzyme (RYSDE)]. The enzyme activity and overall enzyme productivity were respectively 16 U/ml and 0.19 U/ml h in the batch culture. The enzyme activity increased to 85 U/ml by feeding of partially hydrolyzed raw yam starch. When a mixture containing partially hydrolyzed raw yam starch and peptone was fed by a pH-stat strategy, the enzyme activity reached 366 U/ml, 23-fold of that obtained in the batch culture, and the overall productivity reached 3.4 U/ml h, which was 18-fold of that in the batch culture.

Immobilization of BSA, enzymes and cells of Bacillus stearothermophilus onto cellulose polygalacturonic acid and starch based graft copolymers containing maleic arhydride

DOE Office of Scientific and Technical Information (OSTI.GOV)

Beddows, C.G.; Gil, M.H.; Guthrie, J.T.

1986-01-01

Poly(maleic anhydride styrene) graft copolymers of cellulose, pectin polygalacturonic acid salt, calcium polygalacturonate, and starch were prepared and used to immobilize proteins. The cellulose grafts coupled quite appreciable quantities of acid phosphatase, glucose oxidase, and trypsin. However, the general retention of activity was somewhat disappointing. Further investigation with acid phosphatase showed that the amount of enzyme immobilized increased as the amount of anhydride in the graft copolymer increased but no such relationship existed for the enzymic activity. The cellulose graft copolymers were hydrolyzed and it appeared that the carboxyl group aided adsorption of the enzyme. Attempts to couple acid phosphatasemore » using CMC through the free carboxyl groups, created by hydrolysis, gave only a small increase in the extent of protein coupling. However, the unhydrolyzed system gave a useful degree of immobilization of cells of Bacillus stearothermophilus, as did a poly(maleic anhydride/styrene)-cocellulose system. Attempts to improve the activity by using grafts based on other polysaccharide supports met with mixed success. Pectin products were soluble. Polygalacturonic acid products were partially soluble and extremely high levels of enzymic activity were obtained. This was probably due in part to the hydrophilic nature of the system, which also encouraged absorption of the enzyme. Attempts were made to reduce the solubility by using the calcium pectinate salt. Immobilization of acid phosphatase and trypsin resulted in increased protein coupling but relatively poor activities were attained. Calcium polygalacturonate was used to prepare an insoluble graft copolymeric system containing acrylonitrile-comaleic anhydride. The resulting gels gave excellent coupling with acid phosphatase which had a very good retention of activity.« less

Dose of Phenobarbital and Age of Treatment at Early Life are Two Key Factors for the Persistent Induction of Cytochrome P450 Enzymes in Adult Mouse Liver

PubMed Central

Tien, Yun-Chen; Liu, Ke; Pope, Chad; Wang, Pengcheng; Ma, Xiaochao

2015-01-01

Drug treatment of neonates and infants and its long-term consequences on drug responses have emerged in recent years as a major challenge for health care professionals. In the current study, we use phenobarbital as a model drug and mouse as an in vivo model to demonstrate that the dose of phenobarbital and age of treatment are two key factors for the persistent induction of gene expression and consequential increases of enzyme activities of Cyp2b, Cyp2c, and Cyp3a in adult livers. We show that phenobarbital treatment at early life of day 5 after birth with a low dose (<100 mg/kg) does not change expression and enzyme activities of Cyp2b, Cyp2c, and Cyp3a in adult mouse liver, whereas phenobarbital treatment with a high dose (>200 mg/kg) significantly increases expression and enzyme activities of these P450s in adult liver. We also demonstrate that phenobarbital treatment before day 10 after birth, but not at later ages, significantly increases mRNAs, proteins, and enzyme activities of the tested P450s. Such persistent induction of P450 gene expression and enzyme activities in adult livers by phenobarbital treatment only occurs within a sensitive age window early in life. The persistent induction in gene expression and enzyme activities is higher in female mice than in male mice for Cyp2b10 but not for Cyp2c29 and Cyp3a11. These results will stimulate studies to evaluate the long-term impacts of drug treatment with different doses at neonatal and infant ages on drug metabolism, therapeutic efficacy, and drug-induced toxicity throughout the rest of life. PMID:26400395

Foliar and Seed Application of Amino Acids Affects the Antioxidant Metabolism of the Soybean Crop.

PubMed

Teixeira, Walquíria F; Fagan, Evandro B; Soares, Luís H; Umburanas, Renan C; Reichardt, Klaus; Neto, Durval D

2017-01-01

In recent years, the application of natural substances on crops has been intensified in order to increase the resistance and yield of the soybean crop. Among these products are included plant biostimulants that may contain algae extracts, amino acids, and plant regulators in their composition. However, there is little information on the isolated effect of each of these constituents. The objective of this research was to evaluate the effect of the application of isolated amino acids on the antioxidant metabolism of the soybean crop. Experiments were carried out in a greenhouse and in the field with the application of the amino acids glutamate, phenylalanine, cysteine, glycine in seed treatment, and foliar application at V 4 growth stage. Antioxidant metabolism constituents evaluated were superoxide dismutase, catalase, peroxidase, hydrogen peroxide content, proline, and lipid peroxidation. In addition, resistance enzymes as polyphenol oxidase and phenylalanine ammonia-lyase (PAL) were evaluated. In both experiments, the use of cysteine, only in seed treatment and in both seed treatment and foliar application increased the activity of the enzyme PAL and catalase. Also in both experiments, the use of phenylalanine increased the activity of the enzyme PAL when the application was carried out as foliar application or both in seed treatment and foliar application. In the field experiment, the application of glutamate led to an increase in the activity of the catalase and PAL enzymes for seed treatment and foliar application. The use of the set of amino acids was only efficient in foliar application, which led to a greater activity of the enzymes peroxidase, PAL, and polyphenol oxidase. The other enzymes as well as lipid peroxidation and hydrogen peroxide presented different results according to the experiment. Therefore, glutamate, cysteine, phenylalanine, and glycine can act as signaling amino acids in soybean plants, since small doses are enough to increase the activity of the antioxidant enzymes.

Differential effects of non-ionic detergents on microsomal and sarcolemmal adenylate cyclase in cardiac muscle

PubMed Central

Sulakhe, Prakash V.; Narayanan, Njanoor

1978-01-01

1. About 4 and 23% of the homogenate adenylate cyclase activity was recovered in the microsomal and sarcolemmal fractions isolated from guinea-pig heart ventricles. 2. Cardiac microsomal adenylate cyclase activity [basal as well as p[NH]ppG (guanyl-5′-yl imidodiphosphate)- and NaF-stimulated] was increased over 2-fold in the presence of Lubrol-PX (0.01–0.1%). 3. The sarcolemmal enzyme, however, showed concentration-dependent inhibition caused by the detergent under all assay conditions, except when p[NH]ppG was included in the assay. In the latter case, the detergent (0.01–0.02%) caused a modest increase (30–45%) in enzyme activity. 4. Another non-ionic detergent, Triton X-100, also stimulated the microsomal cyclase and inhibited the sarcolemmal enzyme. 5. With either membrane fraction, Lubrol-PX solubilized the enzyme when the detergent/membrane protein ratio was 2.5 (μmol of detergent/mg of protein). 6. The findings with homogenate and a washed particulate fraction resembled those obtained with sarcolemma, and those with isolated sarcoplasmic reticulum resembled those with microsomal preparations. 7. p[NH]ppG, and to some extent NaF, protected the detergent-induced inactivation of the enzyme observed at higher detergent concentrations (0.5% Lubrol-PX and 0.05–0.5% Triton X-100). 8. In the absence of detergents, p[NH]ppG increased the basal enzyme activity about 2-fold in microsomal fractions, but did not appreciably stimulate the sarcolemmal enzyme. Isoproterenol, on the other hand, increased the sarcolemmal enzyme activity (>2-fold) in the presence of p[NH]ppG and caused only moderate stimulation (31%) of the microsomal enzyme under these conditions. 9. These findings support the view that, although the bulk of adenylate cyclase resides in heart sarcolemma (plasma membrane), the microsomal activity cannot be accounted for solely by contamination of the microsomal fraction with sarcolemma, as has been suggested by others [Besch, Jones & Watanabe (1976) Circ. Res. 39, 586–595; Engelhard, Plut & Storm (1976) Biochim. Biophys. Acta 451, 48–61]. Further, the results of this study show that cardiac sarcoplasmic-reticulum membranes possess this enzyme. PMID:736892

Incorporating Peatland Plant Communities into the Enzymic 'Latch' Hypothesis: Can Vegetation Influence Carbon Storage Mechanisms?

NASA Astrophysics Data System (ADS)

Romanowicz, K. J.; Daniels, A. L.; Potvin, L. R.; Kane, E. S.; Kolka, R. K.; Chimner, R. A.; Lilleskov, E. A.

2012-12-01

High water table conditions in peatland ecosystems are known to favor plant production over decomposition and carbon is stored. Dominant plant communities change in response to water table but little is know of how these changes affect belowground carbon storage. One hypothesis known as the enzymic 'latch' proposed by Freeman et al. suggests that oxygen limitations due to high water table conditions inhibit microorganisms from synthesizing specific extracellular enzymes essential for carbon and nutrient mineralization, allowing carbon to be stored as decomposition is reduced. Yet, this hypothesis excludes plant community interactions on carbon storage. We hypothesize that the dominant vascular plant communities, sedges and ericaceous shrubs, will have inherently different effects on peatland carbon storage, especially in response to declines in water table. Sedges greatly increase in abundance following water table decline and create extensive carbon oxidation and mineralization hotspots through the production of deep roots with aerenchyma (air channels in roots). Increased oxidation may enhance aerobic microbial activity including increased enzyme activity, leading to peat subsidence and carbon loss. In contrast, ericaceous shrubs utilize enzymatically active ericoid mycorrhizal fungi that suppress free-living heterotrophs, promoting decreased carbon mineralization by mediating changes in rhizosphere microbial communities and enzyme activity regardless of water table declines. Beginning May 2010, bog monoliths were harvested, housed in mesocosm chambers, and manipulated into three vegetation treatments: unmanipulated (+sedge, +Ericaceae), sedge (+sedge, -Ericaceae), and Ericaceae (-sedge, +Ericaceae). Following vegetation manipulations, two distinct water table manipulations targeting water table seasonal profiles were implemented: (low intra-seasonal variability, higher mean water table; high intra-seasonal variability, lower mean water table). In 2012, peat cores are being assayed monthly from June - October for two oxidase enzyme activities (phenol oxidase, peroxidase) and four hydrolase enzyme activities (β-glucosidase, chitinase, cellobiohydrolase, and acid-phosphatase). Early season assays (June and July) where water table treatments did not significantly vary showed trends of decreasing oxidase activities while hydrolase activities increased. These preliminary results show no significant differences between vegetation treatments but as the season progresses (August - October), water table levels between high and low treatments will continue to experience greater dissimilarities. These water table declines within sedge and ericaceous shrub communities may have opposing effects on rhizosphere extracellular enzyme activities indicating plant communities may significantly influence belowground carbon storage mechanisms in ways not previously considered in peatland ecosystems.

Ribulose-1,5-bisphosphate Carboxylase/Oxygenase and Polyphenol Oxidase in the Tobacco Mutant Su/su and Three Green Revertant Plants 1

PubMed Central

Koivuniemi, Paul J.; Tolbert, N. E.; Carlson, Peter S.

1980-01-01

Ribulose-1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39) was crystallized from a heterozygous tobacco (Nicotiana tabacum L.) aurea mutant (Su/su), its wild-type sibling (su/su), and green revertant plants regenerated from green spots found on leaves of haploid Su plants. No differences were found in the specific activity or kinetic parameters of this enzyme, when comparing Su/su and su/su plants of the same age, which had been grown under identical conditions. The enzyme crystallized from revertant plants was also identical to the enzyme from wild-type plants with the exception of one clone, designated R2. R2 has a chromosome number approximately double that of the wild-type (87.0 ± 11.1 versus 48). The enzyme from R2 had a lower Vmax for CO2, although the Km values were identical to those for the enzyme from the wild-type plant. The enzyme from all mutant plants had identical isoelectric points, identical molecular weight as demonstrated by migration on native and sodium dodecyl sulfate (SDS)-polyacrylamide gels, and the same ratio of large to small subunits as the enzyme from the wild-type. The large subunit of the enzyme from tobacco leaves exhibited a different electrophoretic pattern than did the large subunit from spinach; there were two to three bands on SDS-polyacrylamide gels for the tobacco enzyme whereas the enzyme from spinach had only one species of large subunit. Total polyphenol oxidase activity was the same in leaves from the heterozygous mutant (Su/su) and wild-type (su/su) plants when correlated with developmental age as represented by morphology rather than by the chronological age of the plants. There was a marked increase in the soluble activity of this enzyme with increasing age of both plant types and also as a result of varying environmental conditions. Ribulose-1,5-bisphosphate carboxylase/oxygenase activity correlated inversely with increases in the soluble activity of polyphenol oxidase in crude homogenates from which the carboxylase/oxygenase was crystallized over a generation of Su/su and su/su plants. Criteria are outlined for determining if differences in activity of ribulose-1,5-bisphosphate carboxylase/oxygenase are caused by an effect of polyphenol oxidase activity and/or by some other extrinsic parameter. PMID:16661290

Ectomycorrhizal Fungal Communities and Enzymatic Activities Vary across an Ecotone between a Forest and Field.

PubMed

Rúa, Megan A; Moore, Becky; Hergott, Nicole; Van, Lily; Jackson, Colin R; Hoeksema, Jason D

2015-08-28

Extracellular enzymes degrade macromolecules into soluble substrates and are important for nutrient cycling in soils, where microorganisms, such as ectomycorrhizal (ECM) fungi, produce these enzymes to obtain nutrients. Ecotones between forests and fields represent intriguing arenas for examining the effect of the environment on ECM community structure and enzyme activity because tree maturity, ECM composition, and environmental variables may all be changing simultaneously. We studied the composition and enzymatic activity of ECM associated with loblolly pine (Pinus taeda) across an ecotone between a forest where P. taeda is established and an old field where P. taeda saplings had been growing for <5 years. ECM community and environmental characteristics influenced enzyme activity in the field, indicating that controls on enzyme activity may be intricately linked to the ECM community, but this was not true in the forest. Members of the Russulaceae were associated with increased phenol oxidase activity and decreased peroxidase activity in the field. Members of the Atheliaceae were particularly susceptible to changes in their abiotic environment, but this did not mediate differences in enzyme activity. These results emphasize the complex nature of factors that dictate the distribution of ECM and activity of their enzymes across a habitat boundary.

Biochemical changes in rat liver after 18.5 days of spaceflight (41566)

NASA Technical Reports Server (NTRS)

Abraham, S.; Lin, C.Y.; Volkmann, C. M.; Klein, H. P.

1983-01-01

The effect of weightlessness on liver metabolism was investigated using tissue from rats flown in earth orbit for 18.5 days on the Soviet Cosmos 936 biosatellite and the changes in the activities of 28 carbohydrate and lipid enzymes were determined. The activities of two enzymes, palmitoyl-CoA desaturase and lactate dehydrogenase, increased, while the activities of five, glycogen phosphorylase, 6-phosphogluconate dehydrogenase, both acyltransferases which act on alpha-glycerolphosphate and diglycerides, and and aconitate hydratase decreased. The other enzyme activities were found to be unchanged. In addition, increased levels of liver glycogen and palmitoleate were detected which probably resulted from the lowered glycogen phosphorylase and increased palmitoyl-CoA desaturase activities, respectively, in those animals that experienced weightlessness. All of the changes observed in the rats after 18.5 days of spaceflight disappear by 25 days after the flight.

Phosphate-dependent glutaminase in enterocyte mitochondria and its regulation by ammonium and other ions.

PubMed

Masola, B; Zvinavashe, E

2003-06-01

The effects of ammonium and other ions on phosphate dependent glutaminase (PDG) activity in intact rat enterocyte mitochondria were investigated. Sulphate and bicarbonate activated the enzyme in absence and presence of added phosphate. In presence of 10 mM phosphate, ammonium at concentrations <1 mM inhibited the enzyme. This inhibition was reversed by increased concentration of phosphate or sulphate. The inhibition of PDG by ammonium in presence of 10 mM phosphate was biphasic with respect to glutamine concentration, its effect being through a lowering of V(max) at glutamine concentration of

Alternate-Day High-Fat Diet Induces an Increase in Mitochondrial Enzyme Activities and Protein Content in Rat Skeletal Muscle.

PubMed

Li, Xi; Higashida, Kazuhiko; Kawamura, Takuji; Higuchi, Mitsuru

2016-04-06

Long-term high-fat diet increases muscle mitochondrial enzyme activity and endurance performance. However, excessive calorie intake causes intra-abdominal fat accumulation and metabolic syndrome. The purpose of this study was to investigate the effect of an alternating day high-fat diet on muscle mitochondrial enzyme activities, protein content, and intra-abdominal fat mass in rats. Male Wistar rats were given a standard chow diet (CON), high-fat diet (HFD), or alternate-day high-fat diet (ALT) for 4 weeks. Rats in the ALT group were fed a high-fat diet and standard chow every other day for 4 weeks. After the dietary intervention, mitochondrial enzyme activities and protein content in skeletal muscle were measured. Although body weight did not differ among groups, the epididymal fat mass in the HFD group was higher than those of the CON and ALT groups. Citrate synthase and beta-hydroxyacyl CoA dehydrogenase activities in the plantaris muscle of rats in HFD and ALT were significantly higher than that in CON rats, whereas there was no difference between HFD and ALT groups. No significant difference was observed in muscle glycogen concentration or glucose transporter-4 protein content among the three groups. These results suggest that an alternate-day high-fat diet induces increases in mitochondrial enzyme activities and protein content in rat skeletal muscle without intra-abdominal fat accumulation.

Short-Term Responses of Soil Respiration and C-Cycle Enzyme Activities to Additions of Biochar and Urea in a Calcareous Soil

PubMed Central

Song, Dali; Xi, Xiangyin; Huang, Shaomin; Liang, Guoqing; Sun, Jingwen; Zhou, Wei; Wang, Xiubin

2016-01-01

Biochar (BC) addition to soil is a proposed strategy to enhance soil fertility and crop productivity. However, there is limited knowledge regarding responses of soil respiration and C-cycle enzyme activities to BC and nitrogen (N) additions in a calcareous soil. A 56-day incubation experiment was conducted to investigate the combined effects of BC addition rates (0, 0.5, 1.0, 2.5 and 5.0% by mass) and urea (U) application on soil nutrients, soil respiration and C-cycle enzyme activities in a calcareous soil in the North China Plain. Our results showed soil pH values in both U-only and U plus BC treatments significantly decreased within the first 14 days and then stabilized, and CO2emission rate in all U plus BC soils decreased exponentially, while there was no significant difference in the contents of soil total organic carbon (TOC), dissolved organic carbon (DOC), total nitrogen (TN), and C/N ratio in each treatment over time. At each incubation time, soil pH, electrical conductivity (EC), TOC, TN, C/N ratio, DOC and cumulative CO2 emission significantly increased with increasing BC addition rate, while soil potential activities of the four hydrolytic enzymes increased first and then decreased with increasing BC addition rate, with the largest values in the U + 1.0%BC treatment. However, phenol oxidase activity in all U plus BC soils showed a decreasing trend with the increase of BC addition rate. Our results suggest that U plus BC application at a rate of 1% promotes increases in hydrolytic enzymes, does not highly increase C/N and C mineralization, and can improve in soil fertility. PMID:27589265

Effects of Geroprotectors on Age-Related Changes in Proteolytic Digestive Enzyme Activities at Different Lighting Conditions.

PubMed

Morozov, A V; Khizhkin, E A; Svechkina, E B; Vinogradova, I A; Ilyukha, V A; Anisimov, V N; Khavinson, V Kh

2015-10-01

We studied the effect of melatonin and epithalon on age-related changes in proteolytic digestive enzyme activity in the pancreas and gastric mucosa of rats kept under different lighting conditions. In rats kept under standard illumination, pepsin activity and the total proteolytic activity in the stomach and pancreas increased by the age of 12 months, but then decreased. Constant and natural lighting disturbed the age dynamics of proteolytic digestive enzyme activity. Administration of melatonin and epithalon to animals exposed to constant lighting restored age dynamics of pepsin activity and little affected total proteolytic activity.

Enhanced Delivery and Effects of Acid Sphingomyelinase by ICAM-1-Targeted Nanocarriers in Type B Niemann-Pick Disease Mice.

PubMed

Garnacho, Carmen; Dhami, Rajwinder; Solomon, Melani; Schuchman, Edward H; Muro, Silvia

2017-07-05

Acid sphingomyelinase deficiency in type B Niemann-Pick disease leads to lysosomal sphingomyelin storage, principally affecting lungs, liver, and spleen. Infused recombinant enzyme is beneficial, yet its delivery to the lungs is limited and requires higher dosing than liver and spleen, leading to potentially adverse reactions. Previous studies showed increased enzyme pulmonary uptake by nanocarriers targeted to ICAM-1, a protein overexpressed during inflammation. Here, using polystyrene and poly(lactic-co-glycolic acid) nanocarriers, we optimized lung delivery by varying enzyme dose and nanocarrier concentration, verified endocytosis and lysosomal trafficking in vivo, and evaluated delivered activity and effects. Raising the enzyme load of nanocarriers progressively increased absolute enzyme delivery to all lung, liver, and spleen, over the naked enzyme. Varying nanocarrier concentration inversely impacted lung versus liver and spleen uptake. Mouse intravital and postmortem examination verified endocytosis, transcytosis, and lysosomal trafficking using nanocarriers. Compared to naked enzyme, nanocarriers increased enzyme activity in organs and reduced lung sphingomyelin storage and macrophage infiltration. Although old mice with advanced disease showed reactivity (pulmonary leukocyte infiltration) to injections, including buffer without carriers, antibody, or enzyme, younger mice with mild disease did not. We conclude that anti-ICAM nanocarriers may result in effective lung enzyme therapy using low enzyme doses. Copyright © 2017 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.

A significant increase in both basal and maximal calcineurin activity following fluid percussion injury in the rat.

PubMed

Kurz, Jonathan E; Parsons, J Travis; Rana, Aniruddha; Gibson, Cynthia J; Hamm, Robert J; Churn, Severn B

2005-04-01

Calcineurin, a neuronally enriched, calcium-stimulated phosphatase, is an important modulator of many neuronal processes, including several that are physiologically related to the pathology of traumatic brain injury. This study examined the effects of moderate, central fluid percussion injury on the activity of this important neuronal enzyme. Animals were sacrificed at several time-points postinjury and cortical, hippocampal, and cerebellar homogenates were assayed for calcineurin activity by dephosphorylation of p-nitrophenol phosphate. A significant brain injury-dependent increase was observed in both hippocampal and cortical homogenates under both basal and maximally-stimulated reaction conditions. This increase persisted 2-3 weeks post-injury. Brain injury did not alter substrate affinity, but did induce a significant increase in the apparent maximal dephosphorylation rate. Unlike the other brain regions, no change in calcineurin activity was observed in the cerebellum following brain injury. No brain region tested displayed a significant change in calcineurin enzyme levels as determined by Western blot, demonstrating that increased enzyme synthesis was not responsible for the observed increase in activity. The data support the conclusion that fluid percussion injury results in increased calcineurin activity in the rat forebrain. This increased activity has broad physiological implications, possibly resulting in altered cellular excitability or a greater likelihood of neuronal cell death.

Production of polygalacturonase from Coriolus versicolor grown on tomato pomace and its chromatographic behaviour on immobilized metal chelates.

PubMed

do Rosário Freixo, Maria; Karmali, Amin; Arteiro, José Maria

2008-06-01

Tomato pomace and pectin were used as the sole carbon sources for the production of polygalacturonase from a strain of Coriolus versicolor in submerged culture. The culture of C. versicolor grown on tomato pomace exhibited a peak of polygalacturonase activity (1,427 U/l) on the third day of culture with a specific activity of 14.5 U/mg protein. The production of polygalacturonase by C. versicolor grown on pectin as a sole carbon source increased with the time of cultivation, reaching a maximum activity of 3,207 U/l of fermentation broth with a specific activity of 248 U/mg protein. The levels of different isoenzymes of polygalacturonase produced during the culture growth were analysed by native PAGE. Differential chromatographic behaviour of lignocellulosic enzymes produced by C. versicolor (i.e. polygalacturonase, xylanase and laccase) was studied on immobilized metal chelates. The effect of ligand concentration, pH, the length of spacer arm and the nature of metal ion were studied for enzyme adsorption on immobilized metal affinity chromatography (IMAC). The adsorption of these lignocellulosic enzymes onto immobilized metal chelates was pH-dependent since an increase in protein adsorption was observed as the pH was increased from 6.0 to 8.0. The adsorption of polygalacturonase as well as other enzymes to immobilized metal chelates was due to coordination of histidine residues which are available at the protein surface since the presence of imidazole in the equilibration buffer abolished the adsorption of the enzyme to immobilized metal chelates. A one-step purification of polygalacturonase from C. versicolor was devised by using a column of Sepharose 6B-EPI 30-IDA-Cu(II) and purified enzyme exhibited a specific activity of about 150 U/mg protein, final recovery of enzyme activity of 100% and a purification factor of about 10. The use of short spacer arm and the presence of imidazole in equilibration buffer exhibited a higher selectivity for purification of polygalacturonase on this column with a high purification factor. The purified enzyme preparation was analysed by SDS-PAGE as well as by "in situ" detection of enzyme activity.

Development of Activity-based Cost Functions for Cellulase, Invertase, and Other Enzymes

NASA Astrophysics Data System (ADS)

Stowers, Chris C.; Ferguson, Elizabeth M.; Tanner, Robert D.

As enzyme chemistry plays an increasingly important role in the chemical industry, cost analysis of these enzymes becomes a necessity. In this paper, we examine the aspects that affect the cost of enzymes based upon enzyme activity. The basis for this study stems from a previously developed objective function that quantifies the tradeoffs in enzyme purification via the foam fractionation process (Cherry et al., Braz J Chem Eng 17:233-238, 2000). A generalized cost function is developed from our results that could be used to aid in both industrial and lab scale chemical processing. The generalized cost function shows several nonobvious results that could lead to significant savings. Additionally, the parameters involved in the operation and scaling up of enzyme processing could be optimized to minimize costs. We show that there are typically three regimes in the enzyme cost analysis function: the low activity prelinear region, the moderate activity linear region, and high activity power-law region. The overall form of the cost analysis function appears to robustly fit the power law form.

Abeta-degrading enzymes in Alzheimer's disease.

PubMed

Miners, James Scott; Baig, Shabnam; Palmer, Jennifer; Palmer, Laura E; Kehoe, Patrick G; Love, Seth

2008-04-01

In Alzheimer's disease (AD) Abeta accumulates because of imbalance between the production of Abeta and its removal from the brain. There is increasing evidence that in most sporadic forms of AD, the accumulation of Abeta is partly, if not in some cases solely, because of defects in its removal--mediated through a combination of diffusion along perivascular extracellular matrix, transport across vessel walls into the blood stream and enzymatic degradation. Multiple enzymes within the central nervous system (CNS) are capable of degrading Abeta. Most are produced by neurons or glia, but some are expressed in the cerebral vasculature, where reduced Abeta-degrading activity may contribute to the development of cerebral amyloid angiopathy (CAA). Neprilysin and insulin-degrading enzyme (IDE), which have been most extensively studied, are expressed both neuronally and within the vasculature. The levels of both of these enzymes are reduced in AD although the correlation with enzyme activity is still not entirely clear. Other enzymes shown capable of degrading Abetain vitro or in animal studies include plasmin; endothelin-converting enzymes ECE-1 and -2; matrix metalloproteinases MMP-2, -3 and -9; and angiotensin-converting enzyme (ACE). The levels of plasmin and plasminogen activators (uPA and tPA) and ECE-2 are reported to be reduced in AD. Reductions in neprilysin, IDE and plasmin in AD have been associated with possession of APOEepsilon4. We found no change in the level or activity of MMP-2, -3 or -9 in AD. The level and activity of ACE are increased, the level being directly related to Abeta plaque load. Up-regulation of some Abeta-degrading enzymes may initially compensate for declining activity of others, but as age, genetic factors and diseases such as hypertension and diabetes diminish the effectiveness of other Abeta-clearance pathways, reductions in the activity of particular Abeta-degrading enzymes may become critical, leading to the development of AD and CAA.

Vitamin K2 (menaquinone) biosynthesis in Escherichia coli: evidence for the presence of an essential histidine residue in o-succinylbenzoyl coenzyme A synthetase.

PubMed Central

Bhattacharyya, D K; Kwon, O; Meganathan, R

1997-01-01

o-Succinylbenzoyl coenzyme A (OSB-CoA) synthetase, when treated with diethylpyrocarbonate (DEP), showed a time-dependent loss of enzyme activity. The inactivation follows pseudo-first-order kinetics with a second-order rate constant of 9.2 x 10(-4) +/- 1.4 x 10(-4) microM(-1) min(-1). The difference spectrum of the modified enzyme versus the native enzyme showed an increase in A242 that is characteristic of N-carbethoxyhistidine and was reversed by treatment with hydroxylamine. Inactivation due to nonspecific secondary structural changes in the protein and modification of tyrosine, lysine, or cysteine residues was ruled out. Kinetics of enzyme inactivation and the stoichiometry of histidine modification indicate that of the eight histidine residues modified per subunit of the enzyme, a single residue is responsible for the enzyme activity. A plot of the log reciprocal of the half-time of inactivation against the log DEP concentration further suggests that one histidine residue is involved in the catalysis. Further, the enzyme was partially protected from inactivation by either o-succinylbenzoic acid (OSB), ATP, or ATP plus Mg2+ while inactivation was completely prevented by the presence of the combination of OSB, ATP, and Mg2+. Thus, it appears that a histidine residue located at or near the active site of the enzyme is essential for activity. When His341 present in the previously identified ATP binding motif was mutated to Ala, the enzyme lost 65% of its activity and the Km for ATP increased 5.4-fold. Thus, His341 of OSB-CoA synthetase plays an important role in catalysis since it is probably involved in the binding of ATP to the enzyme. PMID:9324253

Regulation of synthesis and activity of NAD(+)-dependent 15-hydroxy-prostaglandin dehydrogenase (15-PGDH) by dexamethasone and phorbol ester in human erythroleukemia (HEL) cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Xun, C.Q.; Ensor, C.M.; Tai, H.H.

1991-06-28

Dexamethasone stimulated 15-PGDH activity in HEL cells in a time and concentration dependent manner. Increase in 15-PGDH activity by dexamethasone was found to be accompanied by an increase in enzyme synthesis as revealed by Western blot and (35S)methionine labeling studies. In addition to dexamethasone, other anti-inflammatory steroids also increased 15-PGDH activity in the order of their glucocorticoid activity. Among sex steroids only progesterone increased significantly 15-PGDH activity. 12-0-Tetradecanoylphorbol-13-acetate (TPA) also induced the synthesis of 15-PGDH but inhibited the enzyme activity. It appears that TPA caused a time dependent inactivation of 15-PGDH by a protein kinase C mediated mechanism.

The molecular basis of the effect of temperature on enzyme activity.

PubMed

Daniel, Roy M; Peterson, Michelle E; Danson, Michael J; Price, Nicholas C; Kelly, Sharon M; Monk, Colin R; Weinberg, Cristina S; Oudshoorn, Matthew L; Lee, Charles K

2009-12-23

Experimental data show that the effect of temperature on enzymes cannot be adequately explained in terms of a two-state model based on increases in activity and denaturation. The Equilibrium Model provides a quantitative explanation of enzyme thermal behaviour under reaction conditions by introducing an inactive (but not denatured) intermediate in rapid equilibrium with the active form. The temperature midpoint (Teq) of the rapid equilibration between the two forms is related to the growth temperature of the organism, and the enthalpy of the equilibrium (DeltaHeq) to its ability to function over various temperature ranges. In the present study, we show that the difference between the active and inactive forms is at the enzyme active site. The results reveal an apparently universal mechanism, independent of enzyme reaction or structure, based at or near the active site, by which enzymes lose activity as temperature rises, as opposed to denaturation which is global. Results show that activity losses below Teq may lead to significant errors in the determination of DeltaG*cat made on the basis of the two-state ('Classical') model, and the measured kcat will then not be a true indication of an enzyme's catalytic power. Overall, the results provide a molecular rationale for observations that the active site tends to be more flexible than the enzyme as a whole, and that activity losses precede denaturation, and provide a general explanation in molecular terms for the effect of temperature on enzyme activity.

Enhanced biomethane production rate and yield from lignocellulosic ensiled forage ley by in situ anaerobic digestion treatment with endogenous cellulolytic enzymes.

PubMed

Speda, Jutta; Johansson, Mikaela A; Odnell, Anna; Karlsson, Martin

2017-01-01

Enzymatic treatment of lignocellulosic material for increased biogas production has so far focused on pretreatment methods. However, often combinations of enzymes and different physicochemical treatments are necessary to achieve a desired effect. This need for additional energy and chemicals compromises the rationale of using enzymes for low energy treatment to promote biogas production. Therefore, simpler and less energy intensive in situ anaerobic digester treatment with enzymes is desirable. However, investigations in which exogenous enzymes are added to treat the material in situ have shown mixed success, possibly because the enzymes used originated from organisms not evolutionarily adapted to the environment of anaerobic digesters. In this study, to examine the effect of enzymes endogenous to methanogenic microbial communities, cellulolytic enzymes were instead overproduced and collected from a dedicated methanogenic microbial community. By this approach, a solution with very high endogenous microbial cellulolytic activity was produced and tested for the effect on biogas production from lignocellulose by in situ anaerobic digester treatment. Addition of enzymes, endogenous to the environment of a mixed methanogenic microbial community, to the anaerobic digestion of ensiled forage ley resulted in significantly increased rate and yield of biomethane production. The enzyme solution had an instant effect on more readily available cellulosic material. More importantly, the induced enzyme solution also affected the biogas production rate from less accessible cellulosic material in a second slower phase of lignocellulose digestion. Notably, this effect was maintained throughout the experiment to completely digested lignocellulosic substrate. The induced enzyme solution collected from a microbial methanogenic community contained enzymes that were apparently active and stable in the environment of anaerobic digestion. The enzymatic activity had a profound effect on the biogas production rate and yield, comparable with the results of many pretreatment methods. Thus, application of such enzymes could enable efficient low energy in situ anaerobic digester treatment for increased biomethane production from lignocellulosic material.

Scaling of oxidative and glycolytic enzymes in mammals.

PubMed

Emmett, B; Hochachka, P W

1981-09-01

The catalytic activities of several oxidative and glycolytic enzymes were determined in the gastrocnemius muscle of 10 mammalian species differing in body weight by nearly 6 orders of magnitude. When expressed in terms of units gm-1, the activities of enzymes functioning in oxidative metabolism (citrate synthase, beta-hydroxybutyrylCoA dehydrogenase, and malate dehydrogenase) decrease as body weight increases. Log-log plots (activity gm-1 vs body mass) yield straight lines with negative slopes that are less than the allometric exponent (-0.25) typically observed for basal metabolic rates. Since the amount of power a muscle can generate depends upon the catalytic potential of its enzyme machinery (the higher the catalytic potential the higher the maximum rate of energy generation), these data predict that the scope for aerobic activity in large mammals should be greater than in small mammals if nothing else becomes limiting, a result in fact recently obtained by Taylor et al. (Respir. Physiol., 1981). In contrast to the scaling of oxidative enzymes, the activities of enzymes functioning in anaerobic glycogenolysis (glycogen phosphorylase, pyruvate kinase, and lactate dehydrogenase) increase as body size increases. Log-log plots (activity gm-1 vs body mass) display a positive slope indicating that the larger the animal the higher the glycolytic potential of its skeletal muscles. This unexpected result may indicate higher relative power costs for burst type locomotion in larger mammals, which is in fact observed in within-species studies of man. However, the scaling of anaerobic muscle power has not been closely assessed in between-species comparisons of mammals varying greatly in body size.

Enhancement in multiple lignolytic enzymes production for optimized lignin degradation and selectivity in fungal pretreatment of sweet sorghum bagasse.

PubMed

Mishra, Vartika; Jana, Asim K; Jana, Mithu Maiti; Gupta, Antriksh

2017-07-01

The objective of this work was to study the increase in multiple lignolytic enzyme productions through the use of supplements in combination in pretreatment of sweet sorghum bagasse (SSB) by Coriolus versicolor such that enzymes act synergistically to maximize the lignin degradation and selectivity. Enzyme activities were enhanced by metallic salts and phenolic compound supplements in SSF. Supplement of syringic acid increased the activities of LiP, AAO and laccase; gallic acid increased MnP; CuSO 4 increased laccase and PPO to improve the lignin degradations and selectivity individually, higher than control. Combination of supplements optimized by RSM increased the production of laccase, LiP, MnP, PPO and AAO by 17.2, 45.5, 3.5, 2.4 and 3.6 folds respectively for synergistic action leading to highest lignin degradation (2.3 folds) and selectivity (7.1 folds). Enzymatic hydrolysis of pretreated SSB yielded ∼2.43 times fermentable sugar. This technique could be widely applied for pretreatment and enzyme productions. Copyright © 2017 Elsevier Ltd. All rights reserved.

Characteristics of maize biochar with different pyrolysis temperatures and its effects on organic carbon, nitrogen and enzymatic activities after addition to fluvo-aquic soil.

PubMed

Wang, Xiubin; Zhou, Wei; Liang, Guoqing; Song, Dali; Zhang, Xiaoya

2015-12-15

In this study, the characteristics of maize biochar produced at different pyrolysis temperatures (300, 450 and 600°C) and its effects on organic carbon, nitrogen and enzymatic activities after addition to fluvo-aquic soil were investigated. As pyrolysis temperature increased, ash content, pH, electrical conductivity, surface area, pore volume and aromatic carbon content of biochar increased while yield, ratios of oxygen:carbon and hydrogen: carbon and alkyl carbon content decreased. During incubation, SOC, total N, and ammonium-N contents increased in all biochar-amended treatments compared with the urea treatment; however, soil nitrate-N content first increased and then decreased with increasing pyrolysis temperature of the applied biochar. Extracellular enzyme activities associated with carbon transformation first increased and then decreased with biochars pyrolyzed at 450 and 600°C. Protease activity markedly increased with increased pyrolysis temperatures, whereas pyrolysis temperature had limited effect on soil urease activity. The results indicated that the responses of extracellular enzymes to biochar were dependent on the pyrolysis temperature, the enzyme itself and incubation time as well. Copyright © 2015. Published by Elsevier B.V.

Degradation of ureidoglycolate in French bean (Phaseolus vulgaris) is catalysed by a ubiquitous ureidoglycolate urea-lyase.

PubMed

Muñoz, Alfonso; Raso, María José; Pineda, Manuel; Piedras, Pedro

2006-06-01

A ureidoglycolate-degrading activity was analysed in different tissues of French bean (Phaseolus vulgaris L.) plants during development. Activity was detected in all the tissues analysed, although values were very low in seeds before germination and in cotyledons. After radicle emergence, the activity increased due to high activity present in the axes. The highest levels of specific activity were found in developing fruits, from which the enzyme was purified and characterised. This is the first ureidoglycolate-degrading activity that has been purified to homogeneity from a ureide legume. The enzyme was purified 280 fold, and the specific activity for the pure enzyme was 4.4 units mg(-1), which corresponds to a turnover number of 1,055 min(-1). The native enzyme has a molecular mass of 240 kDa and consists of six identical or similar-sized subunits each of 38 kDa. The activity of the purified enzyme was completely dependent on manganese and asparagine. The enzyme exhibited hyperbolic, Michaelian kinetics for ureidoglycolate with a K(m) value of 3.9 mM. This enzyme has been characterised as a ureidoglycolate urea-lyase (EC 4.3.2.3).

[Effects of root-knot nematodes on cucumber leaf N and P contents, soil pH, and soil enzyme activities].

PubMed

Xu, Hua; Ruan, Wei-Bin; Gao, Yu-Bao; Song, Xiao-Yan; Wei, Yu-Kun

2010-08-01

A pot experiment was conducted to study the effects of inoculation with root-knot nematodes on the cucumber leaf N and P contents, and the rhizospheric and non-rhizospheric soil pH and enzyme activities. The rhizospheric soil pH didn't have a significant decrease until the inoculation rate reached 6000 eggs per plant. With the increase of inoculation rate, the leaf N and P contents, rhizospheric soil peroxidase activity, and rhizospheric and non-rhizospheric soil polyphenol oxidase activity all decreased gradually, rhizospheric soil catalase activity was in adverse, non-rhizospheric soil pH decreased after an initial increase, and non-rhizospheric soil catalase activity had no regular change. After inoculation, rhizospheric soil urease activity decreased significantly, but rhizospheric and non-rhizospheric soil phosphatase activity and non-rhizospheric soil peroxidase activity only had a significant decrease under high inoculation rate. In most cases, there existed significant correlations between rhizospheric soil pH, enzyme activities, and leaf N and P contents; and in some cases, there existed significant correlations between non-rhizospheric soil pH, enzyme activities, and leaf N and P contents.

Effect of enzyme induction on nephrotoxicity of halothane-related compounds.

PubMed Central

Hitt, B A; Mazze, R I

1977-01-01

Nephrotoxicity following administration of methoxyflurane has been shown to be directly related to anesthetic metabolism to inorganic fluoride. Enzyme induction should increase metabolic rate and the amount of inorganic fluoride that is released. In vivo studies in Fischer 344 rats show that enzyme induction with phenobarbital or phenytoin increases defluorination following methoxyflurane anesthesia but not after enflurane or isoflurane. In vitro, methoxyflurane defluorinase activity was increased far more than that of any of the other anesthetics. These data suggest that treatment with enzyme inducing drugs increases the risk of nephrotoxocity only if methoxyflurane is the anesthetic agent. PMID:612443

Structure-activity relationships of 4-hydroxyalkenals in the conjugation catalysed by mammalian glutathione transferases.

PubMed Central

Danielson, U H; Esterbauer, H; Mannervik, B

1987-01-01

The substrate specificities of 15 cytosolic glutathione transferases from rat, mouse and man have been explored by use of a homologous series of 4-hydroxyalkenals, extending from 4-hydroxypentenal to 4-hydroxypentadecenal. Rat glutathione transferase 8-8 is exceptionally active with the whole range of 4-hydroxyalkenals, from C5 to C15. Rat transferase 1-1, although more than 10-fold less efficient than transferase 8-8, is the second most active transferase with the longest chain length substrates. Other enzyme forms showing high activities with these substrates are rat transferase 4-4 and human transferase mu. The specificity constants, kcat./Km, for the various enzymes have been determined with the 4-hydroxyalkenals. From these constants the incremental Gibbs free energy of binding to the enzyme has been calculated for the homologous substrates. The enzymes responded differently to changes in the length of the hydrocarbon side chain and could be divided into three groups. All glutathione transferases displayed increased binding energy in response to increased hydrophobicity of the substrate. For some of the enzymes, steric limitations of the active site appear to counteract the increase in binding strength afforded by increased chain length of the substrate. Comparison of the activities with 4-hydroxyalkenals and other activated alkenes provides information about the active-site properties of certain glutathione transferases. The results show that the ensemble of glutathione transferases in a given species may serve an important physiological role in the conjugation of the whole range of 4-hydroxyalkenals. In view of its high catalytic efficiency with all the homologues, rat glutathione transferase 8-8 appears to have evolved specifically to serve in the detoxication of these reactive compounds of oxidative metabolism. PMID:3426557

Function and biotechnology of extremophilic enzymes in low water activity

PubMed Central

2012-01-01

Enzymes from extremophilic microorganisms usually catalyze chemical reactions in non-standard conditions. Such conditions promote aggregation, precipitation, and denaturation, reducing the activity of most non-extremophilic enzymes, frequently due to the absence of sufficient hydration. Some extremophilic enzymes maintain a tight hydration shell and remain active in solution even when liquid water is limiting, e.g. in the presence of high ionic concentrations, or at cold temperature when water is close to the freezing point. Extremophilic enzymes are able to compete for hydration via alterations especially to their surface through greater surface charges and increased molecular motion. These properties have enabled some extremophilic enzymes to function in the presence of non-aqueous organic solvents, with potential for design of useful catalysts. In this review, we summarize the current state of knowledge of extremophilic enzymes functioning in high salinity and cold temperatures, focusing on their strategy for function at low water activity. We discuss how the understanding of extremophilic enzyme function is leading to the design of a new generation of enzyme catalysts and their applications to biotechnology. PMID:22480329

Lignocellulolytic enzyme production of Pleurotus ostreatus growth in agroindustrial wastes

PubMed Central

da Luz, José Maria Rodrigues; Nunes, Mateus Dias; Paes, Sirlaine Albino; Torres, Denise Pereira; de Cássia Soares da Silva, Marliane; Kasuya, Maria Catarina Megumi

2012-01-01

The mushroom Pleurotus ostreatus has nutritional and medicinal characteristics that depend on the growth substrate. In nature, this fungus grows on dead wood, but it can be artificially cultivated on agricultural wastes (coffee husks, eucalyptus sawdust, corncobs and sugar cane bagasse). The degradation of agricultural wastes involves some enzyme complexes made up of oxidative (laccase, manganese peroxidase and lignin peroxidase) and hydrolytic enzymes (cellulases, xylanases and tanases). Understanding how these enzymes work will help to improve the productivity of mushroom cultures and decrease the potential pollution that can be caused by inadequate discharge of the agroindustrial residues. The objective of this work was to assess the activity of the lignocellulolytic enzymes produced by two P. ostreatus strains (PLO 2 and PLO 6). These strains were used to inoculate samples of coffee husks, eucalyptus sawdust or eucalyptus bark add with or without 20 % rice bran. Every five days after substrate inoculation, the enzyme activity and soluble protein concentration were evaluated. The maximum activity of oxidative enzymes was observed at day 10 after inoculation, and the activity of the hydrolytic enzymes increased during the entire period of the experiment. The results show that substrate composition and colonization time influenced the activity of the lignocellulolytic enzymes. PMID:24031982

[Effects of altitudes on soil microbial biomass and enzyme activity in alpine-gorge regions.

PubMed

Cao, Rui; Wu, Fu Zhong; Yang, Wan Qin; Xu, Zhen Feng; Tani, Bo; Wang, Bin; Li, Jun; Chang, Chen Hui

2016-04-22

In order to understand the variations of soil microbial biomass and soil enzyme activities with the change of altitude, a field incubation was conducted in dry valley, ecotone between dry valley and mountain forest, subalpine coniferous forest, alpine forest and alpine meadow from 1563 m to 3994 m of altitude in the alpine-gorge region of western Sichuan. The microbial biomass carbon and nitrogen, and the activities of invertase, urease and acid phosphorus were measured in both soil organic layer and mineral soil layer. Both the soil microbial biomass and soil enzyme activities showed the similar tendency in soil organic layer. They increased from 2158 m to 3028 m, then decreased to the lowest value at 3593 m, and thereafter increased until 3994 m in the alpine-gorge region. In contrast, the soil microbial biomass and soil enzyme activities in mineral soil layer showed the trends as, the subalpine forest at 3028 m > alpine meadow at 3994 m > montane forest ecotone at 2158 m > alpine forest at 3593 m > dry valley at 1563 m. Regardless of altitudes, soil microbial biomass and soil enzyme activities were significantly higher in soil organic layer than in mineral soil layer. The soil microbial biomass was significantly positively correlated with the activities of the measured soil enzymes. Moreover, both the soil microbial biomass and soil enzyme activities were significantly positively correlated with soil water content, organic carbon, and total nitrogen. The activity of soil invertase was significantly positively correlated with soil phosphorus content, and the soil acid phosphatase was so with soil phosphorus content and soil temperature. In brief, changes in vegetation and other environmental factors resulting from altitude change might have strong effects on soil biochemical properties in the alpine-gorge region.

Characterization of Purified Staphylococcal Lipase1

PubMed Central

Vadehra, D. V.; Harmon, L. G.

1967-01-01

Purified staphylococcal lipase had an optimal pH of 8.3 for activity at 37 C, and an optimal temperature of 45 C at pH 8.0. During storage, the enzyme lost less than 10% of the activity over a period of 21 days at 4 and -23 C. The enzyme retained 93% of the activity when heated for 30 min at 50 C and was 95% destroyed in 30 min at 70 C. The purified lipase was capable of hydrolyzing a variety of natural fats and oils. However, the enzyme was three times more active on nonhydrogenated soybean oil than on hydrogenated soybean oil with an iodine value of <3.0. The enzyme was also capable of hydrolyzing fatty acids on the α, β, and α′ positions of a synthetic mixed triglyceride. In general, the presence of oxidizing agents increased the activity and the presence of reducing agents decreased the activity of the lipase enzyme. PMID:6035042

Elevated temperature and PCO2 shift metabolic pathways in differentially oxidative tissues of Notothenia rossii.

PubMed

Strobel, Anneli; Leo, Elettra; Pörtner, Hans O; Mark, Felix C

2013-09-01

Mitochondrial plasticity plays a central role in setting the capacity for acclimation of aerobic metabolism in ectotherms in response to environmental changes. We still lack a clear picture if and to what extent the energy metabolism and mitochondrial enzymes of Antarctic fish can compensate for changing temperatures or PCO2 and whether capacities for compensation differ between tissues. We therefore measured activities of key mitochondrial enzymes (citrate synthase (CS), cytochrome c oxidase (COX)) from heart, red muscle, white muscle and liver in the Antarctic fish Notothenia rossii after warm- (7°C) and hypercapnia- (0.2kPa CO2) acclimation vs. control conditions (1°C, 0.04kPa CO2). In heart, enzymes showed elevated activities after cold-hypercapnia acclimation, and a warm-acclimation-induced upward shift in thermal optima. The strongest increase in enzyme activities in response to hypercapnia occurred in red muscle. In white muscle, enzyme activities were temperature-compensated. CS activity in liver decreased after warm-normocapnia acclimation (temperature-compensation), while COX activities were lower after cold- and warm-hypercapnia exposure, but increased after warm-normocapnia acclimation. In conclusion, warm-acclimated N. rossii display low thermal compensation in response to rising energy demand in highly aerobic tissues, such as heart and red muscle. Chronic environmental hypercapnia elicits increased enzyme activities in these tissues, possibly to compensate for an elevated energy demand for acid-base regulation or a compromised mitochondrial metabolism, that is predicted to occur in response to hypercapnia exposure. This might be supported by enhanced metabolisation of liver energy stores. These patterns reflect a limited capacity of N. rossii to reorganise energy metabolism in response to rising temperature and PCO2. © 2013.

The influence of gemfibrozil on malondialdehyde level and paraoxonase 1 activity in wistar and fisher rats.

PubMed

Macan, Marija; Marija, Macan; Konjevoda, Paško; Paško, Konjevoda; Lovric, Jasna; Jasna, Lovrić; Koprivanac, Marijan; Marijan, Koprivanac; Kelava, Marta; Marta, Kelava; Vrkic, Nada; Nada, Vrkić; Bradamante, Vlasta; Vlasta, Bradamante

2011-06-01

There are diverse experimental data about the influence of gemfibrozil (GEM) on the production of hydrogen peroxide (H(2)O(2)) and antioxidant enzymes. We investigated the influence of GEM treatment on the production of malondialdehyde (MDA) level in tissues of normolipidaemic Wistar and Fisher rats which is an index of lipid peroxidation. Because serum paraoxonase 1 (PON1) is an important enzyme with specific protective function on metabolism of lipid peroxides, we examined the influence of GEM on PON1 activity in liver and serum. MDA level and enzyme activities were also determined 10 days after withdrawal of GEM treatment. The significantly increased levels of MDA in liver, kidney and heart of both rat strains were obtained after 3 weeks of GEM treatment. We propose two possibilities for the increase of MDA levels caused by GEM, induction of peroxisome proliferation and activities of enzymes that participated in occurrence of H(2)O(2) and possible reduction of enzyme activities including in H(2)O(2) metabolism. Ten days after withdrawal of GEM treatment, MDA levels in all tissue levels of both rat strains were less in comparison with GEM treatment. GEM caused a significant drop of PON1 activity in serum and liver of Fisher rats, and in liver of Wistar rats. We suggest that GEM, through induction of lipid peroxidation, caused the damage of hepatocytes with consequent reduction of PON1 synthesis. The increase in PON1 activity in serum and tissues of both rat strains 10 days after withdrawal of GEM treatment shows the fast recovery of enzyme synthesis. © 2011 The Authors. Basic & Clinical Pharmacology & Toxicology © 2011 Nordic Pharmacological Society.

Mutations that Allow SIR2 Orthologs to Function in a NAD+-Depleted Environment.

PubMed

Ondracek, Caitlin R; Frappier, Vincent; Ringel, Alison E; Wolberger, Cynthia; Guarente, Leonard

2017-03-07

Sirtuin enzymes depend on NAD + to catalyze protein deacetylation. Therefore, the lowering of NAD + during aging leads to decreased sirtuin activity and may speed up aging processes in laboratory animals and humans. In this study, we used a genetic screen to identify two mutations in the catalytic domain of yeast Sir2 that allow the enzyme to function in an NAD + -depleted environment. These mutant enzymes give rise to a significant increase of yeast replicative lifespan and increase deacetylation by the Sir2 ortholog, SIRT1, in mammalian cells. Our data suggest that these mutations increase the stability of the conserved catalytic sirtuin domain, thereby increasing the catalytic efficiency of the mutant enzymes. Our approach to identifying sirtuin mutants that permit function in NAD + -limited environments may inform the design of small molecules that can maintain sirtuin activity in aging organisms. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Solution Behavior and Activity of a Halophilic Esterase under High Salt Concentration

PubMed Central

Rao, Lang; Zhao, Xiubo; Pan, Fang; Li, Yin; Xue, Yanfen; Ma, Yanhe; Lu, Jian R.

2009-01-01

Background Halophiles are extremophiles that thrive in environments with very high concentrations of salt. Although the salt reliance and physiology of these extremophiles have been widely investigated, the molecular working mechanisms of their enzymes under salty conditions have been little explored. Methodology/Principal Findings A halophilic esterolytic enzyme LipC derived from archeaon Haloarcula marismortui was overexpressed from Escherichia coli BL21. The purified enzyme showed a range of hydrolytic activity towards the substrates of p-nitrophenyl esters with different alkyl chains (n = 2−16), with the highest activity being observed for p-nitrophenyl acetate, consistent with the basic character of an esterase. The optimal esterase activities were found to be at pH 9.5 and [NaCl] = 3.4 M or [KCl] = 3.0 M and at around 45°C. Interestingly, the hydrolysis activity showed a clear reversibility against changes in salt concentration. At the ambient temperature of 22°C, enzyme systems working under the optimal salt concentrations were very stable against time. Increase in temperature increased the activity but reduced its stability. Circular dichroism (CD), dynamic light scattering (DLS) and small angle neutron scattering (SANS) were deployed to determine the physical states of LipC in solution. As the salt concentration increased, DLS revealed substantial increase in aggregate sizes, but CD measurements revealed the maximal retention of the α-helical structure at the salt concentration matching the optimal activity. These observations were supported by SANS analysis that revealed the highest proportion of unimers and dimers around the optimal salt concentration, although the coexistent larger aggregates showed a trend of increasing size with salt concentration, consistent with the DLS data. Conclusions/Significance The solution α-helical structure and activity relation also matched the highest proportion of enzyme unimers and dimers. Given that all the solutions studied were structurally inhomogeneous, it is important for future work to understand how the LipC's solution aggregation affected its activity. PMID:19759821

[Intensity of lipid peroxidation and antioxidant enzyme activity in arterial and venous walls during hypervitaminosis D].

PubMed

Harbuzova, V Iu

2002-01-01

The intensity of the lipid peroxydation (LPO) and the antioxidant enzyme activity (superoxide dismutase, glutathione peroxydase and catalase) on injecting vitamin D in high doses (10,000 U/kg) was examined in the arterial and venous walls of rabbits. The increase in the amount of the intermediate and final LPO products has been found in the vessels of all types. The lowest intensity of LPO was noted in the vena cava. The decrease in the antioxidant activity has been revealed. But vena cava inferior was the exception because the activity of all studied antioxidant enzymes grew in its wall. This increase is likely to be one of the reasons for vena resistance to the action of damaging factors.

Enzymatic oxidation of ethanol in the gaseous phase.

PubMed

Barzana, E; Karel, M; Klibanov, A M

1989-11-01

The enzymatic conversion of gaseous substrates represents a novel concept in bioprocessing. A critical parameter in such systems is the water activity, A(w) The present article reports the effect of A(w) on the catalytic performance of alcohol oxidase acting on ethanol vapors. Enzyme activity in the gas-phase reaction increases several orders of magnitude, whereas the thermostability decreases drastically when A(w) is increased from 0.11 to 0.97. The enzyme is active on gaseous substrates even at hydration levels below the monolayer coverage. Enhanced thermostability at lower hydrations results in an increase in the optimum temperature of the gas-phase reaction catalyzed by alcohol oxidase. The apparent activation energy decreases as A(w) increases, approaching the value obtained for the enzyme in aqueous solution. The formation of a pread-sorbed ethanol phase on the surface of the support is not a prerequisite for the reaction, suggesting that the reaction occurs by direct interaction of the gaseous substrate with the enzyme. The gas-phase reaction follows Michaelis-Menten kinetics, with a K(m) value almost 100 times lower than that in aqueous solution. Based on vapor-liquid equilibrium data and observed K(m) values, it is postulated that during the gas-phase reaction the ethanol on the enzyme establishes an equilibrium with the ethanol vapor similar to that between ethanol in water and ethanol in the gas phase.

Temperature and UV light affect the activity of marine cell-free enzymes

NASA Astrophysics Data System (ADS)

Thomson, Blair; Hepburn, Christopher David; Lamare, Miles; Baltar, Federico

2017-09-01

Microbial extracellular enzymatic activity (EEA) is the rate-limiting step in the degradation of organic matter in the oceans. These extracellular enzymes exist in two forms: cell-bound, which are attached to the microbial cell wall, and cell-free, which are completely free of the cell. Contrary to previous understanding, cell-free extracellular enzymes make up a substantial proportion of the total marine EEA. Little is known about these abundant cell-free enzymes, including what factors control their activity once they are away from their sites (cells). Experiments were run to assess how cell-free enzymes (excluding microbes) respond to ultraviolet radiation (UVR) and temperature manipulations, previously suggested as potential control factors for these enzymes. The experiments were done with New Zealand coastal waters and the enzymes studied were alkaline phosphatase (APase), β-glucosidase, (BGase), and leucine aminopeptidase (LAPase). Environmentally relevant UVR (i.e. in situ UVR levels measured at our site) reduced cell-free enzyme activities by up to 87 % when compared to controls, likely a consequence of photodegradation. This effect of UVR on cell-free enzymes differed depending on the UVR fraction. Ambient levels of UV radiation were shown to reduce the activity of cell-free enzymes for the first time. Elevated temperatures (15 °C) increased the activity of cell-free enzymes by up to 53 % when compared to controls (10 °C), likely by enhancing the catalytic activity of the enzymes. Our results suggest the importance of both UVR and temperature as control mechanisms for cell-free enzymes. Given the projected warming ocean environment and the variable UVR light regime, it is possible that there could be major changes in the cell-free EEA and in the enzymes contribution to organic matter remineralization in the future.

A novel serine alkaline protease from Bacillus altitudinis GVC11 and its application as a dehairing agent.

PubMed

Vijay Kumar, E; Srijana, M; Kiran Kumar, K; Harikrishna, N; Reddy, Gopal

2011-05-01

A serine alkaline protease from a newly isolated alkaliphilic Bacillus altitudinis GVC11 was purified and characterized. The enzyme was purified to homogeneity by acetone precipitation, DEAE-cellulose anion exchange chromatography with 7.03-fold increase in specific activity and 15.25% recovery. The molecular weight of alkaline protease was estimated to be 28 kDa by SDS PAGE and activity was further assessed by zymogram analysis. The enzyme was highly active over a wide range of pH 8.5 to 12.5 with an optimum pH of 9.5. The optimum temperature of purified enzyme was 45 °C and Ca(2+) further increased the thermal stability of the enzyme. The enzyme activity was enhanced by Ca(2+) and Mg(2+) and inhibited by Hg(2+). The present study is the first report to examine and describe production of highly alkaline protease from Bacillus altitudinis and also its remarkable dehairing ability of goat hide in 18 h without disturbing the collagen and hair integrity.

Competition between Anion Binding and Dimerization Modulates Staphylococcus aureus Phosphatidylinositol-specific Phospholipase C Enzymatic Activity*

PubMed Central

Cheng, Jiongjia; Goldstein, Rebecca; Stec, Boguslaw; Gershenson, Anne; Roberts, Mary F.

2012-01-01

Staphylococcus aureus phosphatidylinositol-specific phospholipase C (PI-PLC) is a secreted virulence factor for this pathogenic bacterium. A novel crystal structure shows that this PI-PLC can form a dimer via helix B, a structural feature present in all secreted, bacterial PI-PLCs that is important for membrane binding. Despite the small size of this interface, it is critical for optimal enzyme activity. Kinetic evidence, increased enzyme specific activity with increasing enzyme concentration, supports a mechanism where the PI-PLC dimerization is enhanced in membranes containing phosphatidylcholine (PC). Mutagenesis of key residues confirm that the zwitterionic phospholipid acts not by specific binding to the protein, but rather by reducing anionic lipid interactions with a cationic pocket on the surface of the S. aureus enzyme that stabilizes monomeric protein. Despite its structural and sequence similarity to PI-PLCs from other Gram-positive pathogenic bacteria, S. aureus PI-PLC appears to have a unique mechanism where enzyme activity is modulated by competition between binding of soluble anions or anionic lipids to the cationic sensor and transient dimerization on the membrane. PMID:23038258

[Spectroscopic analysis of the interaction of ethanol and acid phosphatase from wheat germ].

PubMed

Xu, Dong-mei; Liu, Guang-shen; Wang, Li-ming; Liu, Wei-ping

2004-11-01

Conformational and activity changes of acid phosphatase from wheat germ in ethanol solutions of different concentrations were measured by fluorescence spectra and differential UV-absorption spectra. The effect of ethanol on kinetics of acid phosphatase was determined by using the double reciprocal plot. The results indicate the ethanol has a significant effect on the activity and conformation of acid phosphatase. The activity of acid phosphatase decreased linearly with increasing the concentration of ethanol. Differential UV-absorption spectra of the enzyme denatured in ethanol solutions showed two positive peaks at 213 and 234 nm, respectively. The peaks on the differential UV-absorption spectra suggested that the conformation of enzyme molecule changed from orderly structure to out-of-order crispation. The fluorescence emission peak intensity of the enzyme gradually strengthened with increasing ethanol concentration, which is in concordance with the conformational change of the microenvironments of tyrosine and tryptophan residues. The results indicate that the expression of the enzyme activity correlates with the stability and integrity of the enzyme conformation to a great degree. Ethanol is uncompetitive inhibitor of acid phosphatase.

Ethanol increases affinity of protein kinase C for phosphatidylserine

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chin, J.H.

1986-03-01

Protein kinase C is a calcium-dependent enzyme that requires phospholipid for its activation. It is present in relatively high concentration in the brain and may be involved in neuronal function. The present experiments test whether the membrane disorder induced by ethanol affects the activity of kinase C by changing its interaction with membrane lipid. Fractions rich in kinase C were purified from rat brain cytosol by DEAE-cellulose chromatography and Sephadex G-200 gel filtration. Enzyme activity was assayed by measuring the phosphorylation of histone H1. As expected, phosphatidylserine activated the enzyme, and the stimulation was further increased by the addition ofmore » calcium and/or diacylglycerol. At low concentration of free calcium (0.5-1..mu..M), ethanol (800 mM0 enhanced kinase C activity if the presence of phospholipid. similar results were observed in the absence of calcium. Double reciprocal plots of the data showed that ethanol increased the affinity of the enzyme for phosphatidylserine without affecting the V/sub max. The stimulation of kinase C activity by ethanol was not observed at high calcium concentrations. These experiments suggest that ethanol may activated protein kinase C at physiological levels of calcium by facilitating its transfer into the hydrophobic membrane environment.« less

On the different roles of anions and cations in the solvation of enzymes in ionic liquids.

PubMed

Klähn, Marco; Lim, Geraldine S; Seduraman, Abirami; Wu, Ping

2011-01-28

The solvation of the enzyme Candida antarctica lipase B (CAL-B) was studied in eight different ionic liquids (ILs). The influence of enzyme-ion interactions on the solvation of CAL-B and the structure of the enzyme-IL interface are analyzed. CAL-B and ILs are described with molecular dynamics (MD) simulations in combination with an atomistic empirical force field. The considered cations are based on imidazolium or guanidinium that are paired with nitrate, tetrafluoroborate or hexafluorophosphate anions. The interactions of CAL-B with ILs are dominated by Coulomb interactions with anions, while the second largest contribution stems from van der Waals interactions with cations. The enzyme-ion interaction strength is determined by the ion size and the magnitude of the ion surface charge. The solvation of CAL-B in ILs is unfavorable compared to water because of large formation energies for the CAL-B solute cages in ILs. The internal energy in the IL and of CAL-B increases linearly with the enzyme-ion interaction strength. The average electrostatic potential on the surface of CAL-B is larger in ILs than in water, due to a weaker screening of charged enzyme residues. Ion densities increased moderately in the vicinity of charged residues and decreased close to non-polar residues. An aggregation of long alkyl chains close to non-polar regions and the active site entrance of CAL-B are observed in one IL that involved long non-polar decyl groups. In ILs that contain 1-butyl-3-methylimidazolium cations, the diffusion of one or two cations into the active site of CAL-B occurs during MD simulations. This suggests a possible obstruction of the active site in these ILs. Overall, the results indicate that small ions lead to a stronger electrostatic screening within the solvent and stronger interactions with the enzyme. Also a large ion surface charge, when more hydrophilic ions are used, increases enzyme-IL interactions. An increase of these interactions destabilizes the enzyme and impedes enzyme solvation due to an increase in solute cage formation energies.

Protective effect of dextrans on glucose oxidase denaturation and inactivation.

PubMed

Altikatoglu, Melda; Basaran-Elalmis, Yeliz

2012-08-01

In the present study, the stabilizing effect of dextrans as additives on the denaturation and inactivation of glucose oxidase (GOD) was investigated. Three different molecular weighted dextrans (M(w) 17.5, 75, 188 kD) were used with different concentrations. Dramatically increased enzyme activities were measured after one hour of incubation of enzyme with additives between 25-40°C in water bath. Highest activity value was measured with 75 kDa molecular weighted dextran (in concentration 30% w/v) at pH 5. Dextran as an additive supplied a long shelf-life to the enzyme at 4°C. In the presence of the 75 kDa dextran, the enzyme was more stable and its activity was increased 2.7-fold at 30°C. In addition, dextran protected GOD against inactivation by a n-heptane/aqueous buffer-stirred system.

Novel epoxy activated hydrogels for solving lactose intolerance.

PubMed

Elnashar, Magdy M M; Hassan, Mohamed E

2014-01-01

"Lactose intolerance" is a medical problem for almost 70% of the world population. Milk and dairy products contain 5-10% w/v lactose. Hydrolysis of lactose by immobilized lactase is an industrial solution. In this work, we succeeded to increase the lactase loading capacity to more than 3-fold to 36.3 U/g gel using epoxy activated hydrogels compared to 11 U/g gel using aldehyde activated carrageenan. The hydrogel's mode of interaction was proven by FTIR, DSC, and TGA. The high activity of the epoxy group was regarded to its ability to attach to the enzyme's -SH, -NH, and -OH groups, whereas the aldehyde group could only bind to the enzyme's -NH2 group. The optimum conditions for immobilization such as epoxy chain length and enzyme concentration have been studied. Furthermore, the optimum enzyme conditions were also deliberated and showed better stability for the immobilized enzyme and the Michaelis constants, K m and V max, were doubled. Results revealed also that both free and immobilized enzymes reached their maximum rate of lactose conversion after 2 h, albeit, the aldehyde activated hydrogel could only reach 63% of the free enzyme. In brief, the epoxy activated hydrogels are more efficient in immobilizing more enzymes than the aldehyde activated hydrogel.

Microbial Activity and Silica Degradation in Rice Straw

NASA Astrophysics Data System (ADS)

Kim, Esther Jin-kyung

Abundantly available agricultural residues like rice straw have the potential to be feedstocks for bioethanol production. Developing optimized conditions for rice straw deconstruction is a key step toward utilizing the biomass to its full potential. One challenge associated with conversion of rice straw to bioenergy is its high silica content as high silica erodes machinery. Another obstacle is the availability of enzymes that hydrolyze polymers in rice straw under industrially relevant conditions. Microbial communities that colonize compost may be a source of enzymes for bioconversion of lignocellulose to products because composting systems operate under thermophilic and high solids conditions that have been shown to be commercially relevant. Compost microbial communities enriched on rice straw could provide insight into a more targeted source of enzymes for the breakdown of rice straw polysaccharides and silica. Because rice straw is low in nitrogen it is important to understand the impact of nitrogen concentrations on the production of enzyme activity by the microbial community. This study aims to address this issue by developing a method to measure microbial silica-degrading activity and measure the effect of nitrogen amendment to rice straw on microbial activity and extracted enzyme activity during a high-solids, thermophilic incubation. An assay was developed to measure silica-degrading enzyme or silicase activity. This process included identifying methods of enzyme extraction from rice straw, identifying a model substrate for the assay, and optimizing measurement techniques. Rice straw incubations were conducted with five different levels of nitrogen added to the biomass. Microbial activity was measured by respiration and enzyme activity. A microbial community analysis was performed to understand the shift in community structure with different treatments. With increased levels of nitrogen, respiration and cellulose and hemicellulose degrading activity increased. Silicase activity did not change across nitrogen treatments despite a shift in microbial community with varied nitrogen concentration. Samples treated with different nitrogen concentrations had similar levels of diversity, however the microbial community composition differed with added nitrogen. The results demonstrated that adding nitrogen to rice straw during thermophilic decomposition nurtured a more active microbial community and promoted enzyme secretion thus improving the ability to discover enzymes for rice straw deconstruction. These results can inform future experiments for cultivating a unique, thriving compost-derived microbial community that can successfully decompose rice straw. Understanding the silicase activity of microorganisms may alleviate the challenges associated with silica in various feedstocks.

Complex enzyme hydrolysis releases antioxidative phenolics from rice bran.

PubMed

Liu, Lei; Wen, Wei; Zhang, Ruifen; Wei, Zhencheng; Deng, Yuanyuan; Xiao, Juan; Zhang, Mingwei

2017-01-01

In this study, phenolic profiles and antioxidant activity of rice bran were analyzed following successive treatment by gelatinization, liquefaction and complex enzyme hydrolysis. Compared with gelatinization alone, liquefaction slightly increased the total amount of phenolics and antioxidant activity as measured by ferric reducing antioxidant power (FRAP) and oxygen radical absorbance capacity (ORAC) assays. Complex enzyme hydrolysis significantly increased the total phenolics, flavonoids, FRAP and ORAC by 46.24%, 79.13%, 159.14% and 41.98%, respectively, compared to gelatinization alone. Furthermore, ten individual phenolics present in free or soluble conjugate forms were also analyzed following enzymatic processing. Ferulic acid experienced the largest release, followed by protocatechuic acid and then quercetin. Interestingly, a major proportion of phenolics existed as soluble conjugates, rather than free form. Overall, complex enzyme hydrolysis releases phenolics, thus increasing the antioxidant activity of rice bran extract. This study provides useful information for processing rice bran into functional beverage rich in phenolics. Copyright © 2016 Elsevier Ltd. All rights reserved.

Dissecting the functional significance of non-catalytic carbohydrate binding modules in the deconstruction of plant cell walls

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hahn, Michael G.

The project seeks to investigate the mechanism by which CBMs potentiate the activity of glycoside hydrolases against complete plant cell walls. The project is based on the hypothesis that the wide range of CBMs present in bacterial enzymes maximize the potential target substrates by directing the cognate enzymes not only to different regions of a specific plant cell wall, but also increases the range of plant cell walls that can be degraded. In addition to maximizing substrate access, it was also proposed that CBMs can target specific subsets of hydrolases with complementary activities to the same region of the plantmore » cell wall, thereby maximizing the synergistic interactions between these enzymes. This synergy is based on the premise that the hydrolysis of a specific polysaccharide will increase the access of closely associated polymers to enzyme attack. In addition, it is unclear whether the catalytic module and appended CBM of modular enzymes have evolved unique complementary activities.« less

Regulation of Hydrolytic Enzyme Activity in Aquatic Microbial Communities Hosted by Carnivorous Pitcher Plants.

PubMed

Young, Erica B; Sielicki, Jessica; Grothjan, Jacob J

2018-04-20

Carnivorous pitcher plants Sarracenia purpurea host diverse eukaryotic and bacterial communities which aid in insect prey digestion, but little is known about the functional processes mediated by the microbial communities. This study aimed to connect pitcher community diversity with functional nutrient transformation processes, identifying bacterial taxa, and measuring regulation of hydrolytic enzyme activity in response to prey and alternative nutrient sources. Genetic analysis identified diverse bacterial taxa known to produce hydrolytic enzyme activities. Chitinase, protease, and phosphatase activities were measured using fluorometric assays. Enzyme activity in field pitchers was positively correlated with bacterial abundance, and activity was suppressed by antibiotics suggesting predominantly bacterial sources of chitinase and protease activity. Fungi, algae, and rotifers observed could also contribute enzyme activity, but fresh insect prey released minimal chitinase activity. Activity of chitinase and proteases was upregulated in response to insect additions, and phosphatase activity was suppressed by phosphate additions. Particulate organic P in prey was broken down, appearing as increasing dissolved organic and inorganic P pools within 14 days. Chitinase and protease were not significantly suppressed by availability of dissolved organic substrates, though organic C and N stimulated bacterial growth, resulting in elevated enzyme activity. This comprehensive field and experimental study show that pitcher plant microbial communities dynamically regulate hydrolytic enzyme activity, to digest prey nutrients to simpler forms, mediating biogeochemical nutrient transformations and release of nutrients for microbial and host plant uptake.

Reversal of Acetylcholinesterase Inhibitor Toxicity In Vivo by Inhibitors of Choline Transport.

DTIC Science & Technology

1983-10-31

the increased interaction of acetylcholine with the receptor resulting from the inhibition of the enzyme acetylcholinesterase. . Acetylcholinesterase...competitive inhibitors of acetylcholine at the enzyme receptor. The second category, "reversible" cholinesterase inhibitors, form covalent bonds with the...method of Ellman et al. (46) was used to determine the acetyicholinesterase activity in mouse brain homogenates. Briefly, the enzyme activity was

Markers of oxidative stress and erythrocyte antioxidant enzyme activity in older men and women with differing physical activity.

PubMed

Rowiński, Rafał; Kozakiewicz, Mariusz; Kędziora-Kornatowska, Kornelia; Hübner-Woźniak, Elżbieta; Kędziora, Józef

2013-11-01

The aim of the present study was to examine the relationship between markers of oxidative stress and erythrocyte antioxidant enzyme activity and physical activity in older men and women. The present study included 481 participants (233 men and 248 women) in the age group 65-69 years (127 men and 125 women) and in the age group 90 years and over (106 men and 123 women). The classification of respondents by physical activity was based on answers to the question if, in the past 12 months, they engaged in any pastimes which require physical activity. The systemic oxidative stress status was assessed by measuring plasma iso-PGF2α and protein carbonyl concentration as well as erythrocyte antioxidant enzymes activity, i.e., superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), and glutathione reductase (GR). The concentration of plasma iso-PGF2α and protein carbonyls (CP) was lower in groups of younger men and women compared to the respective older groups. In all examined groups, physical activity resulted in decrease of these oxidative stress markers and simultaneously caused adaptive increase in the erythrocyte SOD activity. Additionally, in active younger men CAT, GPx, and GR activities were higher than in sedentary ones. In conclusion, oxidative stress increase is age-related, but physical activity can reduce oxidative stress markers and induce adaptive increase in the erythrocyte antioxidant enzyme activity, especially SOD, even in old and very old men and women. © 2013.

Long-term fertilization, but not warming, shifts rates of ectomycorrhizal nutrient cycling in Arctic tussock tundra.

NASA Astrophysics Data System (ADS)

Dunleavy, H.; Mack, M. C.

2017-12-01

The role of ectomycorrhizae (ECM) in Arctic nutrient cycling may be changing as temperature, nutrient availability, and ECM shrub abundance and size increase. A shift in ECM function has been proposed as a possible mechanism for shrub expansion. While several studies demonstrate a higher abundance of ECM as well as community compositional shifts in response to long-term experimental warming and fertilization, direct measurements of functional responses are missing. To understand the potential role of ECM in soil biogeochemical processes of the changing Arctic, we investigated the functional response of ECM to 30 years of summer warming and increased nutrient availability by measuring potential activities of extracellular enzymes associated with nitrogen (N) and phosphorous (P) acquisition on ECM root tips. We hypothesize ECM enzyme activities will be higher with warmer temperatures. Conversely, fertilization will lower ECM enzyme activities as N and P become less limiting to host plants. Preliminary results strongly support our latter hypothesis, but not the first. Warming decreased hydrolytic P-associated and labile N-associated enzyme activities on individual root tips (pmol/min/mm2 root tip) by 30% and 83%, respectively. However, warming increased ECM abundance and did not alter community-level activities (pmol/min/cm3 soil). Fertilization decreased hydrolytic and oxidative enzymatic activities on individual root tips by 34 to 80% as well as on a community level by 67 to 93%, even though ECM shrubs were almost monodominant. The combined effect of warming and fertilization decreased labile N-associated enzyme activity by 82%, but had little effect on oxidative and other hydrolytic enzyme activities. Although both warming and fertilization decreased root tip activities, reflecting a potential reduction in plant allocation to mycorrhizal nutrient acquisition, only fertilization lowered rates of ECM nutrient cycling. The indirect relationship between ECM abundance and individual root tip activity highlights the importance of measuring ECM function to assess the role of this symbiosis in nutrient cycling.

The effect of exogenous calcium on mitochondria, respiratory metabolism enzymes and ion transport in cucumber roots under hypoxia.

PubMed

He, Lizhong; Li, Bin; Lu, Xiaomin; Yuan, Lingyun; Yang, Yanjuan; Yuan, Yinghui; Du, Jing; Guo, Shirong

2015-08-25

Hypoxia induces plant stress, particularly in cucumber plants under hydroponic culture. In plants, calcium is involved in stress signal transmission and growth. The ultimate goal of this study was to shed light on the mechanisms underlying the effects of exogenous calcium on the mitochondrial antioxidant system, the activity of respiratory metabolism enzymes, and ion transport in cucumber (Cucumis sativus L. cv. Jinchun No. 2) roots under hypoxic conditions. Our experiments revealed that exogenous calcium reduces the level of reactive oxygen species (ROS) and increases the activity of antioxidant enzymes in mitochondria under hypoxia. Exogenous calcium also enhances the accumulation of enzymes involved in glycolysis and the tricarboxylic acid (TCA) cycle. We utilized fluorescence and ultrastructural cytochemistry methods to observe that exogenous calcium increases the concentrations of Ca(2+) and K(+) in root cells by increasing the activity of plasma membrane (PM) H(+)-ATPase and tonoplast H(+)-ATPase and H(+)-PPase. Overall, our results suggest that hypoxic stress has an immediate and substantial effect on roots. Exogenous calcium improves metabolism and ion transport in cucumber roots, thereby increasing hypoxia tolerance in cucumber.

The synergistic effects of insecticidal essential oils and piperonyl butoxide on biotransformational enzyme activities in Aedes aegypti (Diptera: Culicidae).

PubMed

Waliwitiya, Ranil; Nicholson, Russell A; Kennedy, Christopher J; Lowenberger, Carl A

2012-05-01

The biochemical mechanisms underlying the increased toxicity of several plant essential oils (thymol, eugenol, pulegone, terpineol, and citronellal) against fourth instar of Aedes aegypti L. when exposed simultaneously with piperonyl butoxide (PBO) were examined. Whole body biotransformational enzyme activities including cytochrome P450-mediated oxidation (ethoxyresorufin O-dethylase [EROD]), glutathione S-transferase (GST), and beta-esterase activity were measured in control, essential oil-exposed only (single chemical), and essential oil + PBO (10 mg/liter) exposed larvae. At high concentrations, thymol, eugenol, pulegone, and citronellal alone reduced EROD activity by 5-25% 16 h postexposure. Terpineol at 10 mg/liter increased EROD activity by 5 +/- 1.8% over controls. The essential oils alone reduced GST activity by 3-20% but PBO exposure alone did not significantly affect the activity of any of the measured enzymes. All essential oils in combination with PBO reduced EROD activity by 58-76% and reduced GST activity by 3-85% at 16 h postexposure. This study indicates a synergistic interaction between essential oils and PBO in inhibiting the cytochrome P450 and GST detoxification enzymes in Ae. aegypti.

Effects of acoustic wave resonance oscillation on immobilized enzyme

NASA Astrophysics Data System (ADS)

Nishiyama, Hiroshi; Watanabe, Tomoya; Inoue, Yasunobu

2014-03-01

In aiming at developing a new method to artificially activate enzyme catalysts immobilized on surface, the effects of resonance oscillation of bulk acoustic waves were studied. Glucose oxidase (GOD) was immobilized by a covalent coupling method on a ferroelectric lead zirconate titanate (PZT) device that was able to generate thickness-extensional resonance oscillation (TERO). Glucose oxidation by the GOD enzyme was studied in a microreactor. The generation of TERO immediately increased the catalytic activity of immobilized GOD by a factor of 2-3. With turn-off of TERO, no significant activity decrease occurred, and 80-90% of the enhanced activity was maintained while the reaction proceeded. The almost complete reversion of the activity to the original low level before TERO generation was observed when the immobilized GOD was exposed to a glucose substrate-free solution. These results indicated that the presence of glucose substrate was essential for TERO-induced GOD activation and preservation of the increased activity level. The influences of reaction temperature, glucose concentration, pH, and rf electric power on the TERO activation showed that TERO strengthened the interactions of the immobilized enzyme with glucose substrate and hence promoted the formation of an activation complex.

Small heat shock protein AgsA: an effective stabilizer of enzyme activities.

PubMed

Tomoyasu, Toshifumi; Tabata, Atsushi; Ishikawa, Yoko; Whiley, Robert A; Nagamune, Hideaki

2013-01-01

A small heat shock protein, AgsA, possesses chaperone activity that can reduce the amount of heat-aggregated protein in vivo, and suppress the aggregation of chemical- and heat-denatured proteins in vitro. Therefore, we examined the ability of AgsA to stabilize the activity of several enzymes by using this chaperone activity. We observed that AgsA can stabilize the enzymatic activities of Renilla (Renilla reniformis) luciferase, firefly (Photinus pyralis) luciferase, and β-galactosidase, and showed comparable or greater stabilization of these enzymes than bovine serum albumin (BSA), a well-known stabilizer of enzyme activities. In particular, AgsA revealed better stabilization of Renilla luciferase and β-galactosidase than BSA under disulfide bond-reducing conditions with dithiothreitol. In addition, AgsA also increased the enzymatic performance of β-galactosidase and various restriction enzymes to a comparable or greater extent than BSA. These data indicate that AgsA may be useful as a general stabilizer of enzyme activities. Copyright © 2012 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

[Protein fractions and their enzyme activity in the rat myocardium in a Kosmos-936 biosatellite experiment].

PubMed

Tigranian, R A; Nosova, E A; Kolchina, E V; Veresotskaia, N A; Kurkina, L M

1981-01-01

The effect of artificial gravity on protein fractions and their enzyme activity in the myocardium of rats flown on board Cosmos-936 was studied. In weightless rats the content of sarcoplasmic proteins increased at R + O and that of T fraction proteins decreased at R + 25. In centrifuged rats such changes were not seen. In centrifuged rats the enzyme activity of sarcoplasmic proteins did not alter. In weightless rats ATPase activity of myosin decreased significantly, and in centrifuged rats it remained almost unchanged.

Immobilization of β-galactosidase from Kluyveromyces lactis onto polymeric membrane surfaces: effect of surface characteristics.

PubMed

Güleç, Hacı Ali

2013-04-01

The aim of this study was to investigate the effects of surface characteristics of plain and plasma modified cellulose acetate (CA) membranes on the immobilization yield of β-galactosidases from Kluyveromyces lactis (KLG) and its galacto-oligosaccharide (GOS) yield, respectively. Low pressure plasma treatments involving oxygen plasma activation, plasma polymerization (PlsP) of ethylenediamine (EDA) and PlsP of 2-mercaptoethanol were used to modify plain CA membrane surfaces. KLG enzyme was immobilized onto plain and oxygen plasma treated membrane surfaces by simple adsorption. Oxygen plasma activation increased the hydrophylicity of CA membrane surfaces and it improved the immobilization yield of the enzyme by 42%. KLG enzyme was also immobilized onto CA membrane surfaces through amino groups created by PlsP of EDA via covalent binding. Plasma action at 60W plasma power and 15 min. exposure time improved the amount of membrane bounded enzyme by 3.5-fold. The enrichment of the amount of amino groups via polyethyleneimine (PEI) addition enhanced this increase from 3.5-fold to 4.5-fold. Although high enzyme loading was achived (65-83%), both of the methods dramatically decreased the enzyme activity (11-12%) and GOS yield due to probably negative effects of active amino groups. KLG enzyme was more effectively immobilized onto thiolated CA membrane surface created by PlsP of 2-mercaptoethanol with high immobilization yield (70%) and especially high enzyme activity (46%). Immobilized enzymes on the CA membranes treated by PlsP were successively reutilized for 5-8 cycles at 25°C and enzymatic derivatives retained approximately 75-80% of their initial activites at the end of the reactions. Copyright © 2012 Elsevier B.V. All rights reserved.

Ectomycorrhizal Fungal Communities and Enzymatic Activities Vary across an Ecotone between a Forest and Field

PubMed Central

Rúa, Megan A.; Moore, Becky; Hergott, Nicole; Van, Lily; Jackson, Colin R.; Hoeksema, Jason D.

2015-01-01

Extracellular enzymes degrade macromolecules into soluble substrates and are important for nutrient cycling in soils, where microorganisms, such as ectomycorrhizal (ECM) fungi, produce these enzymes to obtain nutrients. Ecotones between forests and fields represent intriguing arenas for examining the effect of the environment on ECM community structure and enzyme activity because tree maturity, ECM composition, and environmental variables may all be changing simultaneously. We studied the composition and enzymatic activity of ECM associated with loblolly pine (Pinus taeda) across an ecotone between a forest where P. taeda is established and an old field where P. taeda saplings had been growing for <5 years. ECM community and environmental characteristics influenced enzyme activity in the field, indicating that controls on enzyme activity may be intricately linked to the ECM community, but this was not true in the forest. Members of the Russulaceae were associated with increased phenol oxidase activity and decreased peroxidase activity in the field. Members of the Atheliaceae were particularly susceptible to changes in their abiotic environment, but this did not mediate differences in enzyme activity. These results emphasize the complex nature of factors that dictate the distribution of ECM and activity of their enzymes across a habitat boundary. PMID:29376908

Purification and Characterization of Thermostable and Detergent-Stable α-Amylase from Anoxybacillus sp. AH1

PubMed Central

Bekler, Fatma Matpan; Pirinççioğlu, Hemşe; Güven, Reyhan Gül; Güven, Kemal

2016-01-01

Summary A thermostable and detergent-stable α-amylase from a newly isolated Anoxybacillus sp. AH1 was purified and characterized. Maximum enzyme production (1874.8 U/mL) was obtained at 24 h of incubation. The amylase was purified by using Sephadex G-75 gel filtration, after which an 18-fold increase in specific activity and a yield of 9% were achieved. The molecular mass of the purified enzyme was estimated at 85 kDa by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The optimum pH and temperature values of the enzyme were 7.0 and 60 °C, respectively. The enzyme was highly stable in the presence of 30% glycerol, retaining 85% of its original activity at 60 °C within 120 min. Km and vmax values were 0.102 µmol and 0.929 µmol/min, respectively, using Lineweaver-Burk plot. The enzyme activity was increased by various detergents, but it was significantly inhibited in the presence of urea. Mg2+ and Ca2+ also significantly activated α-amylase, while Zn2+, Cu2+ and metal ion chelators ethylenediaminetetraacetic acid (EDTA) and 1,10-phenanthroline (phen) greatly inhibited the enzyme activity. α-Amylase activity was enhanced by β-mercaptoethanol (β-ME) and dithiothreitol (DTT) to a great extent, but inhibited by p-chloromercuribenzoic acid (PCMB). Iodoacetamide (IAA) and N-ethylmaleimide (NEM) had a slight, whereas phenylmethylsulfonyl fluoride (PMSF) had a strong inhibitory effect on the amylase activity. PMID:27904395

Stabilizing effect of biochar on soil extracellular enzymes after a denaturing stress.

PubMed

Elzobair, Khalid A; Stromberger, Mary E; Ippolito, James A

2016-01-01

Stabilizing extracellular enzymes may maintain enzymatic activity while protecting enzymes from proteolysis and denaturation. A study determined whether a fast pyrolysis hardwood biochar (CQuest™) would reduce evaporative losses, subsequently stabilizing soil extracellular enzymes and prohibiting potential enzymatic activity loss following a denaturing stress (microwaving). Soil was incubated in the presence of biochar (0%, 1%, 2%, 5%, or 10% by wt.) for 36 days and then exposed to microwave energies (0, 400, 800, 1600, or 3200 J g(-1) soil). Soil enzymes (β-glucosidase, β-d-cellobiosidase, N-acetyl-β-glucosaminidase, phosphatase, leucine aminopeptidase, β-xylosidase) were analyzed by fluorescence-based assays. Biochar amendment reduced leucine aminopeptidase and β-xylosidase potential activity after the incubation period and prior to stress exposure. The 10% biochar rate reduced soil water loss at the lowest stress level (400 J microwave energy g(-1) soil). Enzyme stabilization was demonstrated for β-xylosidase; intermediate biochar application rates prevented a complete loss of this enzyme's potential activity after soil was exposed to 400 (1% biochar treatment) or 1600 (5% biochar treatment) J microwave energy g(-1) soil. Remaining enzyme potential activities were not affected by biochar, and activities decreased with increasing stress levels. We concluded that biochar has the potential to reduce evaporative soil water losses and stabilize certain extracellular enzymes where activity is maintained after a denaturing stress; this effect was biochar rate and enzyme dependent. While biochar may reduce the potential activity of certain soil extracellular enzymes, this phenomenon was not universal as the majority of enzymes assayed in this study were unaffected by exposure to biochar. Copyright © 2015 Elsevier Ltd. All rights reserved.

Thermal inactivation kinetics of β-galactosidase during bread baking.

PubMed

Zhang, Lu; Chen, Xiao Dong; Boom, Remko M; Schutyser, Maarten A I

2017-06-15

In this study, β-galactosidase was utilized as a model enzyme to investigate the mechanism of enzyme inactivation during bread baking. Thermal inactivation of β-galactosidase was investigated in a wheat flour/water system at varying temperature-moisture content combinations, and in bread during baking at 175 or 205°C. In the wheat flour/water system, the thermostability of β-galactosidase increased with decreased moisture content, and a kinetic model was accurately fitted to the corresponding inactivation data (R 2 =0.99). Interestingly, the residual enzyme activity in the bread crust (about 30%) was hundredfold higher than that in the crumb (about 0.3%) after baking, despite the higher temperature in the crust throughout baking. This result suggested that the reduced moisture content in the crust increased the thermostability of the enzyme. Subsequently, the kinetic model reasonably predicted the enzyme inactivation in the crumb using the same parameters derived from the wheat flour/water system. However, the model predicted a lower residual enzyme activity in the crust compared with the experimental result, which indicated that the structure of the crust may influence the enzyme inactivation mechanism during baking. The results reported can provide a quantitative understanding of the thermal inactivation kinetics of enzyme during baking, which is essential to better retain enzymatic activity in bakery products supplemented with heat-sensitive enzymes. Copyright © 2017 Elsevier Ltd. All rights reserved.

Antioxidant enzyme activities are affected by salt content and temperature and influence muscle lipid oxidation during dry-salted bacon processing.

PubMed

Jin, Guofeng; He, Lichao; Yu, Xiang; Zhang, Jianhao; Ma, Meihu

2013-12-01

Fresh pork bacon belly was used as material and manufactured into dry-salted bacon through salting and drying-ripening. During processing both oxidative stability and antioxidant enzyme stability were evaluated by assessing peroxide value (PV), thiobarbituric acid reactive substances (TBARS) and activities of catalase, glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD), and their correlations were also analysed. The results showed that all antioxidant enzyme activities decreased (p<0.05) until the end of process; GSH-Px was the most unstable one followed by catalase. Antioxidant enzyme activities were negatively correlated with TBARS (p<0.05), but the correlations were decreased with increasing process temperature. Salt showed inhibitory effect on all antioxidant enzyme activities and was concentration dependent. These results indicated that when process temperature and salt content were low at the same time during dry-salted bacon processing, antioxidant enzymes could effectively control lipid oxidation. Copyright © 2013 Elsevier Ltd. All rights reserved.

DOE Office of Scientific and Technical Information (OSTI.GOV)

THOMAS, PAUL; JANAVE, M. T.

Mangoes were gamma-irradiated at a dose rate of 4 Krad per min in doses of 15 to 200 Krad. Methods are described for extraction of the enzyme, assay of enzyme activity, and estimation of total phenolic constituents, ascorbic acid, and pH. Above doses of 75 Krad discoloration increased with dose and longer storage periods. An increase in activity of polyphenol oxidase was found with increasing radiation doses; a several-fold increase was observed at 200 Krad. This increase was correlated with external manifestations of radiation injury. Possible ways in which the activation of polyphenol oxidase in mango fruits is brought aboutmore » by irradiation are discussed. (HLW)« less

Enhancement of hepatic detoxification enzyme activity by dietary mercuric acetate

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wagstaff, D.J.

1973-01-01

This report deals with stimulation of liver microsomal enzymes by dietary mercuric acetate (HgAc) and interactions of HgAc with phenobarbital sodium (PB). There is a diphasic response of microsomal enzymes in rats exposed to mercurials. Detoxication activity increased as the dietary dose of HgAc was increased. Liver weight was unaffected by ingestion of HgAc . Toxicity of HgAc increased with dosage. There were no deaths among animals fed diets of 2000 ppM HgAc or less but all five animals fed the diet of 5000 ppM died after five but before ten days on the experiment. The mercury-phenobarbital interactions support speculationmore » that mercury in combination with other chemicals in the environment may have enzyme stimulatory capacity at low exposure levels. 25 references, 1 figure, 1 table.« less

Cholinesterase assay for monitoring the kinetics of the JD6. 5 organophosphorus acid anhydrase in detoxification of diisopropylfluorophosphate. Final report, January-June 1988

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yeh, H.R.; Cheng, T.C.; DeFrank, J.J.

1992-06-01

In the present studies, cholinesterase was used for monitoring the enzymatic activities of the JD6.5 organophosphorus acid anhydrase. The kinetic data indicated that: (1) the first order of kinetic constants (k) and Vmax values of the enzymatic reactions increased as the concentrations of the enzyme increased; (2) while the half-life (tl/2) of diisopropylfluorophosphate (DFP) hydrolysis decreased as the enzyme concentrations increased; (3) the minimum time required for hydrolysis of 9mM of DFP was 3 min at the concentrations of the enzyme present; Km values of DFP were found to be in range of 5mM; and (4) both MnCl2 and NaClmore » were found to be required for the optimal activity of the enzyme.« less

Immobilization and stabilization of pectinase by multipoint attachment onto an activated agar-gel support.

PubMed

Li, Tuoping; Li, Suhong; Wang, Na; Tain, Lirui

2008-08-15

Pectinase was immobilized on an activated agar-gel support by multipoint attachment. The maximal activity of immobilized pectinase was obtained at 5°C, pH 3.6, with a 24h reaction time at an enzyme dose of 0.52mg protein/g gel, and the gel was activated with 1.0M glycidol. These conditions increased the thermal stability of the immobilized pectinase 19-fold compared with the free enzyme at 65°C. The optimal temperature for pectinase activity changed from 40 to 50°C after immobilization; however, the optimal pH remained unchanged. The immobilized enzyme also exhibited great operational stability, and an 81% residual activity was observed in the immobilized enzyme after 10 batch reactions. Copyright © 2008 Elsevier Ltd. All rights reserved.

Enzyme Sorption onto Soil and Biocarbon Amendments Alters Catalytic Capacity and Depends on the Specific Protein and pH

NASA Astrophysics Data System (ADS)

Foster, E.; Fogle, E. J.; Cotrufo, M. F.

2017-12-01

Enzymes catalyze biogeochemical reactions in soils and play a key role in nutrient cycling in agricultural systems. Often, to increase soil nutrients, agricultural managers add organic amendments and have recently experimented with charcoal-like biocarbon products. These amendments can enhance soil water and nutrient holding capacity through increasing porosity. However, the large surface area of the biocarbon has the potential to sorb nutrients and other organic molecules. Does the biocarbon decrease nutrient cycling through sorption of enzymes? In a laboratory setting, we compared the interaction of two purified enzymes β-glucosidase and acid phosphatase with a sandy clay loam and two biocarbons. We quantified the sorbed enzymes at three different pHs using a Bradford protein assay and then measured the activity of the sorbed enzyme via high-throughput fluorometric analysis. Both sorption and activity depended upon the solid phase, pH, and specific enzyme. Overall the high surface area biocarbon impacted the catalytic capacity of the enzymes more than the loam soil, which may have implications for soil nutrient management with these organic amendments.

The role of electrostatic interactions in protease surface diffusion and the consequence for interfacial biocatalysis.

PubMed

Feller, Bob E; Kellis, James T; Cascão-Pereira, Luis G; Robertson, Channing R; Frank, Curtis W

2010-12-21

This study examines the influence of electrostatic interactions on enzyme surface diffusion and the contribution of diffusion to interfacial biocatalysis. Surface diffusion, adsorption, and reaction were investigated on an immobilized bovine serum albumin (BSA) multilayer substrate over a range of solution ionic strength values. Interfacial charge of the enzyme and substrate surface was maintained by performing the measurements at a fixed pH; therefore, electrostatic interactions were manipulated by changing the ionic strength. The interfacial processes were investigated using a combination of techniques: fluorescence recovery after photobleaching, surface plasmon resonance, and surface plasmon fluorescence spectroscopy. We used an enzyme charge ladder with a net charge ranging from -2 to +4 with respect to the parent to systematically probe the contribution of electrostatics in interfacial enzyme biocatalysis on a charged substrate. The correlation between reaction rate and adsorption was determined for each charge variant within the ladder, each of which displayed a maximum rate at an intermediate surface concentration. Both the maximum reaction rate and adsorption value at which this maximum rate occurs increased in magnitude for the more positive variants. In addition, the specific enzyme activity increased as the level of adsorption decreased, and for the lowest adsorption values, the specific enzyme activity was enhanced compared to the trend at higher surface concentrations. At a fixed level of adsorption, the specific enzyme activity increased with positive enzyme charge; however, this effect offers diminishing returns as the enzyme becomes more highly charged. We examined the effect of electrostatic interactions on surface diffusion. As the binding affinity was reduced by increasing the solution ionic strength, thus weakening electrostatic interaction, the rate of surface diffusion increased considerably. The enhancement in specific activity achieved at the lowest adsorption values is explained by the substantial rise in surface diffusion at high ionic strength due to decreased interactions with the surface. Overall, knowledge of the electrostatic interactions can be used to control surface parameters such as surface concentration and surface diffusion, which intimately correlate with surface biocatalysis. We propose that the maximum reaction rate results from a balance between adsorption and surface diffusion. The above finding suggests enzyme engineering and process design strategies for improving interfacial biocatalysis in industrial, pharmaceutical, and food applications.

Saccharification of ozonated sugarcane bagasse using enzymes from Myceliophthora thermophila JCP 1-4 for sugars release and ethanol production.

PubMed

de Cassia Pereira, Josiani; Travaini, Rodolfo; Paganini Marques, Natalia; Bolado-Rodríguez, Silvia; Bocchini Martins, Daniela Alonso

2016-03-01

The saccharification of ozonated sugarcane bagasse (SCB) by enzymes from Myceliophthora thermophila JCP 1-4 was studied. Fungal enzymes provided slightly higher sugar release than commercial enzymes, working at 50°C. Sugar release increased with temperature increase. Kinetic studies showed remarkable glucose release (4.99 g/L, 3%w/w dry matter) at 60°C, 8 h of hydrolysis, using an enzyme load of 10 FPU (filter paper unit). FPase and β-glucosidase activities increased during saccharification (284% and 270%, respectively). No further significant improvement on glucose release was observed increasing the enzyme load above 7.5 FPU per g of cellulose. Higher dry matter contents increased sugars release, but not yields. The fermentation of hydrolysates by Saccharomyces cerevisiae provided glucose-to-ethanol conversions around to 63%. Copyright © 2015 Elsevier Ltd. All rights reserved.

Mutant α-galactosidase A enzymes identified in Fabry disease patients with residual enzyme activity: biochemical characterization and restoration of normal intracellular processing by 1-deoxygalactonojirimycin

PubMed Central

Ishii, Satoshi; Chang, Hui-Hwa; Kawasaki, Kunito; Yasuda, Kayo; Wu, Hui-Li; Garman, Scott C.; Fan, Jian-Qiang

2007-01-01

Fabry disease is a lysosomal storage disorder caused by the deficiency of α-Gal A (α-galactosidase A) activity. In order to understand the molecular mechanism underlying α-Gal A deficiency in Fabry disease patients with residual enzyme activity, enzymes with different missense mutations were purified from transfected COS-7 cells and the biochemical properties were characterized. The mutant enzymes detected in variant patients (A20P, E66Q, M72V, I91T, R112H, F113L, N215S, Q279E, M296I, M296V and R301Q), and those found mostly in mild classic patients (A97V, A156V, L166V and R356W) appeared to have normal Km and Vmax values. The degradation of all mutants (except E59K) was partially inhibited by treatment with kifunensine, a selective inhibitor of ER (endoplasmic reticulum) α-mannosidase I. Metabolic labelling and subcellular fractionation studies in COS-7 cells expressing the L166V and R301Q α-Gal A mutants indicated that the mutant protein was retained in the ER and degraded without processing. Addition of DGJ (1-deoxygalactonojirimycin) to the culture medium of COS-7 cells transfected with a large set of missense mutant α-Gal A cDNAs effectively increased both enzyme activity and protein yield. DGJ was capable of normalizing intracellular processing of mutant α-Gal A found in both classic (L166V) and variant (R301Q) Fabry disease patients. In addition, the residual enzyme activity in fibroblasts or lymphoblasts from both classic and variant hemizygous Fabry disease patients carrying a variety of missense mutations could be substantially increased by cultivation of the cells with DGJ. These results indicate that a large proportion of mutant enzymes in patients with residual enzyme activity are kinetically active. Excessive degradation in the ER could be responsible for the deficiency of enzyme activity in vivo, and the DGJ approach may be broadly applicable to Fabry disease patients with missense mutations. PMID:17555407

Lead nitrate-induced development of hypercholesterolemia in rats: sterol-independent gene regulation of hepatic enzymes responsible for cholesterol homeostasis.

PubMed

Kojima, Misaki; Masui, Toshimitsu; Nemoto, Kiyomitsu; Degawa, Masakuni

2004-12-01

Changes in the gene expressions of hepatic enzymes responsible for cholesterol homeostasis were examined during the process of lead nitrate (LN)-induced development of hypercholesterolemia in male rats. Total cholesterol levels in the liver and serum were significantly increased at 3-72 h and 12-72 h, respectively, after LN-treatment (100 micromol/kg, i.v.). Despite the development of hypercholesterolemia, the genes for hepatic 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) and other enzymes (FPPS, farnesyl diphosphate synthase; SQS, squalene synthase; CYP51, lanosterol 14alpha-demethylase) responsible for cholesterol biosynthesis were activated at 3-24 h and 12-18 h, respectively. On the other hand, the gene expression of cholesterol 7alpha-hydroxylase (CYP7A1), a catabolic enzyme of cholesterol, was remarkably suppressed at 3-72 h. The gene expression levels of cytokines interleukin-1beta (IL-1beta) and TNF-alpha, which activate the HMGR gene and suppress the CYP7A1 gene, were significantly increased at 1-3 h and 3-24 h, respectively. Furthermore, gene activation of SREBP-2, a gene activator of several cholesterogenic enzymes, occurred before the gene activations of FPPS, SQS and CYP51. This is the first report demonstrating sterol-independent gene regulation of hepatic enzymes responsible for cholesterol homeostasis in LN-treated male rats. The mechanisms for the altered-gene expressions of hepatic enzymes in LN-treated rats are discussed.

Dose and time-dependent effects of cyanide on thiosulfate sulfurtransferase, 3-mercaptopyruvate sulfurtransferase, and cystathionine λ-lyase activities.

PubMed

Singh, Poonam; Rao, Pooja; Bhattacharya, Rahul

2013-12-01

We assessed the dose-dependent effect of potassium cyanide (KCN) on thiosulfate sulfurtransferase (TST), 3-mercaptopyruvate sulfurtransferase (3-MPST), and cystathionine λ-lyase (CST) activities in mice. The time-dependent effect of 0.5 LD50 KCN on cyanide level and cytochrome c oxidase (CCO), TST, 3-MPST, and CST activities was also examined. Furthermore, TST, 3-MPST, and CST activities were measured in stored mice cadavers. Hepatic and renal TST activity increased by 0.5 LD50 KCN but diminished by ≥2.0 LD50. After 0.5 LD50 KCN, the elevated hepatic cyanide level was accompanied by increased TST, 3-MPST, and CST activities, and CCO inhibition. Elevated renal cyanide level was only accompanied by increased 3-MPST activity. No appreciable change in enzyme activities was observed in mice cadavers. The study concludes that high doses of cyanide exert saturating effects on its detoxification enzymes, indicating their exogenous use during cyanide poisoning. Also, these enzymes are not reliable markers of cyanide poisoning in autopsied samples. © 2013 Wiley Periodicals, Inc.

Involvement of Reactive Oxygen Species and Mitochondrial Proteins in Biophoton Emission in Roots of Soybean Plants under Flooding Stress.

PubMed

Kamal, Abu Hena Mostafa; Komatsu, Setsuko

2015-05-01

To understand the mechanism of biophoton emission, ROS and mitochondrial proteins were analyzed in soybean plants under flooding stress. Enzyme activity and biophoton emission were increased in the flooding stress samples when assayed in reaction mixes specific for antioxidant enzymes and reactive oxygen species; although the level of the hydroxyl radicals was increased at day 4 (2 days of flooding) compared to nonflooding at day 4, the emission of biophotons did not change. Mitochondria were isolated and purified from the roots of soybean plants grown under flooding stress by using a Percoll gradient, and proteins were analyzed by a gel-free proteomic technique. Out of the 98 mitochondrial proteins that significantly changed abundance under flooding stress, 47 increased and 51 decreased at day 4. The mitochondrial enzymes fumarase, glutathione-S-transferase, and aldehyde dehydrogenase increased at day 4 in protein abundance and enzyme activity. Enzyme activity and biophoton emission decreased at day 4 by the assay of lipoxygenase under stress. Aconitase, acyl CoA oxidase, succinate dehydrogenase, and NADH ubiquinone dehydrogenase were up-regulated at the transcription level. These results indicate that oxidation and peroxide scavenging might lead to biophoton emission and oxidative damage in the roots of soybean plants under flooding stress.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gavva, S.R.; Harris, B.G.; Cook, P.F.

A thiol group at the malate-binding site of the NAD-malic enzyme from Ascaris suum has been modified to thiocyanate. The modified enzyme generally exhibits slight increases in K{sub NAD} and K{sub i metal} and decreases in V{sub max} as the metal size increases from Mg{sup 2+} to Mn{sup 2+} to Cd{sup 2+}, indicative of crowding in the site. The K{sub malate} value increases 10- to 30-fold, suggesting that malate does not bind optimally to the modified enzyme. Deuterium isotope effects on V and V/K{sub malate} increase with all three metal ions compared to the native enzyme concomitant with a decreasemore » in the {sup 13}C isotope effect, suggesting a switch in the rate limitation of the hydride transfer and decarboxylation steps with hydride transfer becoming more rate limiting. The {sup 13}C effect decreases only slightly when obtained with deuterated malate, suggestive of the presence of a secondary {sup 13}C effect in the hydride transfer step, similar to data obtained with non-nicotinamide-containing dinucleotide substrates for the native enzyme (see the preceding paper in this issue). The native enzyme is inactivated in a time-dependent manner by Cd{sup 2+}. This inactivation occurs whether the enzyme alone is present or whether the enzyme is turning over with Cd{sup 2+} as the divalent metal activator. Upon inactivation, only Cd{sup 2+} ions are bound at high stoichiometry to the enzyme, which eventually becomes denatured. Conversion of the active-site thiol to thiocyanate makes it more difficult to inactivate the enzyme by treatment with Cd{sup 2+}.« less

Developmental and hormone-induced changes of mitochondrial electron transport chain enzyme activities during the last instar larval development of maize stem borer, Chilo partellus (Lepidoptera: Crambidae).

PubMed

VenkatRao, V; Chaitanya, R K; Naresh Kumar, D; Bramhaiah, M; Dutta-Gupta, A

2016-12-01

The energy demand for structural remodelling in holometabolous insects is met by cellular mitochondria. Developmental and hormone-induced changes in the mitochondrial respiratory activity during insect metamorphosis are not well documented. The present study investigates activities of enzymes of mitochondrial electron transport chain (ETC) namely, NADH:ubiquinone oxidoreductase or complex I, Succinate: ubiquinone oxidoreductase or complex II, Ubiquinol:ferricytochrome c oxidoreductase or complex III, cytochrome c oxidase or complex IV and F 1 F 0 ATPase (ATPase), during Chilo partellus development. Further, the effect of juvenile hormone (JH) analog, methoprene, and brain and corpora-allata-corpora-cardiaca (CC-CA) homogenates that represent neurohormones, on the ETC enzyme activities was monitored. The enzymatic activities increased from penultimate to last larval stage and thereafter declined during pupal development with an exception of ATPase which showed high enzyme activity during last larval and pupal stages compared to the penultimate stage. JH analog, methoprene differentially modulated ETC enzyme activities. It stimulated complex I and IV enzyme activities, but did not alter the activities of complex II, III and ATPase. On the other hand, brain homogenate declined the ATPase activity while the injected CC-CA homogenate stimulated complex I and IV enzyme activities. Cumulatively, the present study is the first to show that mitochondrial ETC enzyme system is under hormone control, particularly of JH and neurohormones during insect development. Copyright © 2015 Elsevier Inc. All rights reserved.

Binding affinity and adhesion force of organophosphate hydrolase enzyme with soil particles related to the isoelectric point of the enzyme.

PubMed

Islam, Shah Md Asraful; Yeasmin, Shabina; Islam, Md Saiful; Islam, Md Shariful

2017-07-01

The binding affinity of organophosphate hydrolase enzyme (OphB) with soil particles in relation to the isoelectric point (pI) was studied. Immobilization of OphB with soil particles was observed by confocal microscopy, Fourier transform infrared spectroscopy (FT-IR), and Atomic force microscopy (AFM). The calculated pI of OphB enzyme was increased from 8.69 to 8.89, 9.04 and 9.16 by the single, double and triple mutant of OphB enzyme, respectively through the replacement of negatively charged aspartate with positively charged histidine. Practically, the binding affinity was increased to 5.30%, 11.50%, and 16.80% for single, double and triple mutants, respectively. In contrast, enzyme activity of OphB did not change by the mutation of the enzyme. On the other hand, adhesion forces were gradually increased for wild type OphB enzyme (90 pN) to 96, 100 and 104 pN for single, double and triple mutants of OphB enzyme, respectively. There was an increasing trend of binding affinity and adhesion force by the increase of isoelectric point (pI) of OphB enzyme. Copyright © 2017 Elsevier Inc. All rights reserved.

Changes in selected physical property and enzyme activity of rice and barley koji during fermentation and storage.

PubMed

Bechman, Allison; Phillips, Robert D; Chen, Jinru

2012-06-01

Koji are solid-state fermentation products made by inoculating steamed grains with the spores of fungi, particularly Aspergillus spp. This research was undertaken to identify the fermentation and storage conditions optimal for the production and maintenance of selected hydrolytic enzymes, such as α-amlyase and protease, in koji. Steamed rice and barley were inoculated with 2 × 10 ¹¹ Aspergillus oryzae spores per kilogram of grains and fermented for 118 h in a growth chamber at 28 to 32 °C with controlled relative humidities. Samples were drawn periodically during fermentation and storage at -20, 4, or 32 °C, and α-amylase and protease activity, mold counts, a(w), moisture contents, and pH of collected samples were determined. It was observed that the a(w), moisture contents, and pH of the koji were influenced by the duration of fermentation and temperature of storage. The α-amylase activity of both koji increased as the populations of A. oryzae increased during the exponential growth phase. The enzyme activity of barley koji was significantly higher than that of rice koji, reaching a peak activity of 211.87 or 116.57 U at 46 and 58 h, respectively, into the fermentation process. The enzyme activity in both products started to decrease once the mold culture entered the stationary growth phase. The protease activities of both koji were low and remained relatively stable during fermentation and storage. These results suggest that rice and barley koji can be used as sources of α-amylase and desired enzyme activity can be achieved by controlling the fermentation and storage conditions. Amylases and proteases are 2 important hydrolytic enzymes. In the food industry, these enzymes are used to break down starches and proteins while reducing the viscosity of foods. Although amylases and proteases are found in plants and animals, commercial enzymes are often produced using bacteria or molds through solid state fermentation, which is designed to use natural microbial process to produce enzymes in a controlled environment. A properly produced and maintained koji with a high hydrolytic enzyme activity can serve as an important source of the enzymes for the food industry. © 2012 Institute of Food Technologists®

Grape seed proanthocyanidins ameliorates isoproterenol-induced myocardial injury in rats by stabilizing mitochondrial and lysosomal enzymes: an in vivo study.

PubMed

Karthikeyan, K; Sarala Bai, B R; Niranjali Devaraj, S

2007-11-30

This study was designed to examine the effects of grape seed proanthocyanidins (GSP) against myocardial injury (MI) induced by isoproterenol (ISO), in a rat model. Induction of rats with ISO (85 mg/kg body weight, subcutaneously) for 2 days resulted in a significant decrease in the activities of heart mitochondrial enzymes (isocitrate dehydrogenase, succinate dehydrogenase, malate dehydrogenase and alpha-ketoglutarate dehydrogenase) and respiratory chain enzymes (NADH dehydrogenase and cytochrome c oxidase). The activities of lysosomal enzymes (alpha-d-glucuronidase, alpha-d-N-acetylglucosaminidase, cathepsin-D, acid phosphatases and alpha-d-galactosidase) were increased significantly in the heart and serum of ISO-induced rats. The prior administration of GSP for 6 days a week for 5 weeks significantly increased the activities of mitochondrial and respiratory chain enzymes and significantly decreased the activities of lysosomal enzymes in the heart tissues of ISO-induced rats, which proves the stress stabilizing action of GSP. Oral administration of grape seed proanthocyanidins alone (50, 100 and 150 mg/kg) to normal rats did not show any significant effect in all the parameters studied. These biochemical functional alterations were supported by the macroscopic enzyme mapping assay of ischemic myocardium. Thus, this study shows that 100 and 150 mg/kg of GSP gives protection against ISO-induced MI and demonstrates that GSP has a significant effect in the protection of heart.

Changes in Biochemical Characteristics and Activities of Ripening Associated Enzymes in Mango Fruit during the Storage at Different Temperatures

PubMed Central

Kimura, Yoshinobu

2014-01-01

As a part of the study to explore the possible strategy for enhancing the shelf life of mango fruits, we investigated the changes in biochemical parameters and activities of ripening associated enzymes of Ashwina hybrid mangoes at 4-day regular intervals during storage at −10°C, 4°C, and 30 ± 1°C. Titratable acidity, vitamin C, starch content, and reducing sugar were higher at unripe state and gradually decreased with the increasing of storage time at all storage temperatures while phenol content, total soluble solid, total sugar, and nonreducing sugar contents gradually increased. The activities of amylase, α-mannosidase, α-glucosidase, and invertase increased sharply within first few days and decreased significantly in the later stage of ripening at 30 ± 1°C. Meanwhile polyphenol oxidase, β-galactosidase, and β-hexosaminidase predominantly increased significantly with the increasing days of storage till later stage of ripening. At −10°C and 4°C, the enzymes as well as carbohydrate contents of storage mango changed slightly up to 4 days and thereafter the enzyme became fully dormant. The results indicated that increase in storage temperature and time correlated with changes in biochemical parameters and activities of glycosidases suggested the suppression of β-galactosidase and β-hexosaminidase might enhance the shelf life of mango fruits. PMID:25136564

Bacterial quorum sensing and nitrogen cycling in rhizosphere soil

DOE Office of Scientific and Technical Information (OSTI.GOV)

DeAngelis, K.M.; Lindow, S.E.; Firestone, M.K.

2008-10-01

Plant photosynthate fuels carbon-limited microbial growth and activity, resulting in increased rhizosphere nitrogen (N)-mineralization. Most soil organic N is macromolecular (chitin, protein, nucleotides); enzymatic depolymerization is likely rate-limiting for plant N accumulation. Analyzing Avena (wild oat) planted in microcosms containing sieved field soil, we observed increased rhizosphere chitinase and protease specific activities, bacterial cell densities, and dissolved organic nitrogen (DON) compared to bulk soil. Low-molecular weight DON (<3000 Da) was undetectable in bulk soil but comprised 15% of rhizosphere DON. Extracellular enzyme production in many bacteria requires quorum sensing (QS), cell-density dependent group behavior. Because proteobacteria are considered major rhizospheremore » colonizers, we assayed the proteobacterial QS signals acyl-homoserine lactones (AHLs), which were significantly increased in the rhizosphere. To investigate the linkage between soil signaling and N cycling, we characterized 533 bacterial isolates from Avena rhizosphere: 24% had chitinase or protease activity and AHL production; disruption of QS in 7 of 8 eight isolates disrupted enzyme activity. Many {alpha}-Proteobacteria were newly found with QS-controlled extracellular enzyme activity. Enhanced specific activities of N-cycling enzymes accompanied by bacterial density-dependent behaviors in rhizosphere soil gives rise to the hypothesis that QS could be a control point in the complex process of rhizosphere N-mineralization.« less

The effect of enzymes upon metabolism, storage, and release of carbohydrates in normal and abnormal endometria.

PubMed

Hughes, E C

1976-07-01

This paper presents preliminary data concerning the relationship of various components of glandular epithelium and effect of enzymes on metabolism, storage, and release of certain substances in normal and abnormal endometria. Activity of these endometrial enzymes has been compared between two groups: 252 patients with normal menstrual histories and 156 patients, all over the age of 40, with abnormal uterine bleeding. Material was obtained by endometrial biopsy or curettage. In the pathologic classification of the group of 156, 30 patients had secretory endometria, 88 patients had endometria classified as proliferative, 24 were classified as endometrial hyperplasia, and 14 were classified as adenocarcinoma. All tissue was studied by histologic, histochemical, and biochemical methods. Glycogen synthetase activity caused synthesis of glucose to glycogen, increasing in amount until midcycle, when glycogen phosphorylase activity caused the breakdown to glucose during the regressive stage of endometrial activity. This normal cyclic activity did not occur in the abnormal endometria, where activity of both enzymes continued at low constant tempo. Only the I form of glycogen synthetase increased as the tissue became more hyperplastic. With the constant glycogen content and the increased activity of both the TPN isocitric dehydrogenase and glucose-6-phosphate dehydrogenase in the hyperplastic and cancerous endometria, tissue energy was created, resulting in abnormal cell proliferation. These altered biochemical and cellular activities may be the basis for malignant cell growth.

Release of enzymes from cells: transport and distribution within the extracellular space.

PubMed

Mattenheimer, H; Friedel, R

1977-01-01

The distribution in the extracellular space of enzymes released from organ cells was investigated using three models: (1) comparison of enzyme activities in blood plasma and lymph of the ductus thoracicus (dog) and plasma and intestinal lymph (rat); (2) i.v. injection of heterologous, homologous and autologous enzymes in order to increase acutely the activities and to measure the rate constants for the distribution and elimination of the enzymes (rat); or (3) plasmapheresis in order to create an enzyme activity gradient from the interstitial space and to determine the rate constants for the reestablishment of the equilibrium between the extra and intravascular compartments (rat). The results suggest that the enzymes are mainly released into the interstitial fluid and transported via the lymph into the intravascular compartment. From there the enzymes diffuse back into the interstitial compartment and are eliminated by a yet unknown mechanism. Transport of enzymes across the capillary membranes in both directions depends on (1) the permeability of the capillary membranes, which varies from region to region and (2) the molecular seizes of the enzymes.

Redox regulation of antioxidant enzymes: post-translational modulation of catalase and glutathione peroxidase activity by resveratrol in diabetic rat liver.

PubMed

Sadi, Gökhan; Bozan, Davut; Yildiz, Huseyin Bekir

2014-08-01

Resveratrol is a strong antioxidant that exhibits blood glucose-lowering effects, which might contribute to its usefulness in preventing complications associated with diabetes. The present study aimed to investigate resveratrol effects on catalase (CAT) and glutathione peroxidase (GPx) gene and protein expression, their phosphorylation states and activities in rat liver of STZ-induced diabetes. Diabetes increased the levels of total protein phosphorylation and p-CAT, while mRNA expression, protein levels, and activity were reduced. Although diabetes induced transcriptional repression over GPx, it did not affect the protein levels and activity. When resveratrol was administered to diabetic rats, an increase in activity was associated with an increase in p-GPx levels. Decrease in Sirtuin1 (SIRT1) and nuclear factor erythroid 2-related factor (Nrf2) and increase in nuclear factor kappa B (NFκB) gene expression in diabetes were associated with a decrease in CAT and GPx mRNA expression. A possible compensatory mechanism for reduced gene expression of antioxidant enzymes is proved to be nuclear translocation of redox-sensitive Nrf2 and NFκB in diabetes which is confirmed by the increase in nuclear and decrease in cytoplasmic protein levels of Nrf2 and NFκB. Taken together, these findings revealed that an increase in the oxidized state in diabetes intricately modified the cellular phosphorylation status and regulation of antioxidant enzymes. Gene regulation of antioxidant enzymes was accompanied by nuclear translocation of Nrf2 and NFκB. Resveratrol administration also activated a coordinated cytoprotective response against diabetes-induced changes in liver tissues.

Time- and dose-dependent differential regulation of copper-zinc superoxide dismutase and manganese superoxide dismutase enzymatic activity and mRNA level by vitamin E in rat blood cells.

PubMed

Hajiani, Maliheh; Razi, Farideh; Golestani, Aboualfazl; Frouzandeh, Mehdi; Owji, Ali Akbar; Khaghani, Shahnaz; Ghannadian, Naghmeh; Shariftabrizi, Ahmad; Pasalar, Parvin

2012-01-01

Vitamin E is the most important lipid-soluble antioxidant. Recently, it has been proposed as a gene regulator, and its gene modulation effects have been observed at different levels of gene expression and cell signaling. This study was performed to investigate the effects of vitamin E on the activity and expression of the most important endogenous antioxidant enzyme, superoxide dismutase (SOD), in rat plasma. Twenty-eight male Sprauge-Dawley rats were divided into four groups: control group and three dosing groups. The control group received the vehicle (liquid paraffin), and the dosing groups received twice-weekly intraperitoneal injections of 10, 30, and 100 mg/kg of vitamin E ((±)-α-Tocopherol) for 6 weeks. Quantitative real-time reverse transcription-polymerase chain reaction and enzyme assays were used to assess the levels of Cu/Zn-SOD and Mn-SOD mRNA and enzyme activity levels in blood cells at 0, 2, 4, and 6 weeks following vitamin E administration. Catalase enzyme activity and total antioxidant capacity were also assessed in plasma at the same time intervals. Mn-SOD activity was significantly increased in the 100 and 30 mg/kg dosing groups after 4 and 6 weeks, with corresponding significant increase in their mRNA levels. Cu/Zn-SOD activity was not significantly changed in response to vitamin E administration at any time points, whereas Cu/Zn-SOD mRNA levels were significantly increased after longer time points with high doses (30 and 100 mg/kg) of vitamin E. Catalase enzyme activity was transiently but significantly increased after 4 weeks of vitamin E treatment in 30 and 100 mg/kg dosing groups. Total antioxidant status was significantly increased after 4 and 6 weeks in the 100 mg/kg dosing group. Only the chronic administration of higher doses of alpha-tocopherol is associated with the increased activity and expression of Mn-SOD in rats. Cu/Zn-SOD activity and expression does not dramatically change in response to vitamin E.

Development of in vivo biotransformation enzyme assays for ecotoxicity screening: In vivo measurement of phases I and II enzyme activities in freshwater planarians.

PubMed

Li, Mei-Hui

2016-08-01

The development of a high-throughput tool is required for screening of environmental pollutants and assessing their impacts on aquatic animals. Freshwater planarians can be used in rapid and sensitive toxicity bioassays. Planarians are known for their remarkable regeneration ability but much less known for their metabolic and xenobiotic biotransformation abilities. In this study, the activities of different phase I and II enzymes were determined in vivo by directly measuring fluorescent enzyme substrate disappearance or fluorescent enzyme metabolite production in planarian culture media. For phase I enzyme activity, O-deethylation activities with alkoxyresorufin could not be detected in planarian culture media. By contrast, O-deethylation activities with alkoxycoumarin were detected in planarian culture media. Increases in 7-ethoxycoumarin O-deethylase (ECOD) activities was only observed in planarians exposed to 1μM, but not 10μM, β-naphthoflavone for 24h. ECOD activity was inhibited in planarians exposed to 10 and 100μM rifampicin or carbamazepine for 24h. For phase II enzyme activity, DT-diaphorase, arylsulfatases, uridine 5'-diphospho (UDP)-glucuronosyltransferase or catechol-O-methyltransferase activity was determined in culture media containing planarians. The results of this study indicate that freshwater planarians are a promising model organism to monitor exposure to environmental pollutants or assess their impacts through the in vivo measurement of phase I and II enzyme activities. Copyright © 2016. Published by Elsevier Inc.

Alterations in the skin of Labeo rohita exposed to an azo dye, Eriochrome black T: a histopathological and enzyme biochemical investigation.

PubMed

Srivastava, Ayan; Verma, Neeraj; Mistri, Arup; Ranjan, Brijesh; Nigam, Ashwini Kumar; Kumari, Usha; Mittal, Swati; Mittal, Ajay Kumar

2017-03-01

Histopathological changes and alterations in the activity of certain metabolic and antioxidant enzymes were analyzed in the head skin of Labeo rohita, exposed to sublethal test concentrations of the azo dye, Eriochrome black T for 4 days, using 24 h renewal bioassay method. Hypertrophied epithelial cells, increased density of mucous goblet cells, and profuse mucous secretion at the surface were considered to protect the skin from toxic impact of the azo dye. Degenerative changes including vacuolization, shrinkage, decrease in dimension, and density of club cells with simultaneous release of their contents in the intercellular spaces were associated to plug them, preventing indiscriminate entry of foreign matter. On exposure of fish to the dye, significant decline in the activity of enzymes-alkaline phosphatase, acid phosphatase, carboxylesterase, succinate dehydrogenase, catalase, and peroxidase-was associated with the binding of dye to the enzymes. Gradual increase in the activity of lactate dehydrogenase was considered to reflect a shift from aerobic to anaerobic metabolism. On transfer of azo dye exposed fish to freshwater, skin gradually recovers and, by 8 days, density and area of mucous goblet cells, club cells, and activity of the enzymes appear similar to that of controls. Alteration in histopathology and enzyme activity could be considered beneficial tool in monitoring environmental toxicity, valuable in the sustenance of fish populations.

Regulation of SIRT 1 mediated NAD dependent deacetylation: A novel role for the multifunctional enzyme CD38

DOE Office of Scientific and Technical Information (OSTI.GOV)

Aksoy, Pinar; Escande, Carlos; Seccion Biologia Celular, Facultad de Ciencias, Universidad de la Republica, Igua 4225, Montevideo

2006-10-13

The SIRT 1 enzyme is a NAD dependent deacetylase implicated in ageing, cell protection, and energy metabolism in mammalian cells. How the endogenous activity of SIRT 1 is modulated is not known. The enzyme CD38 is a multifunctional enzyme capable of synthesis of the second messenger, cADPR, NAADP, and ADPR. However, the major enzymatic activity of CD38 is the hydrolysis of NAD. Of particular interest is the fact that CD38 is present on the inner nuclear membrane. Here, we investigate the modulation of the SIRT 1 activity by CD38. We propose that by modulating availability of NAD to the SIRT1more » enzyme, CD38 may regulate SIRT1 enzymatic activity. We observed that in CD38 knockout mice, tissue levels of NAD are significantly increased. We also observed that incubation of purified recombinant SIRT1 enzyme with CD38 or nuclear extracts of wild-type mice led to a significant inhibition of its activity. In contrast, incubation of SIRT1 with cellular extract from CD38 knockout mice was without effect. Furthermore, the endogenous activity of SIRT1 was several time higher in nuclear extracts from CD38 knockout mice when compared to wild-type nuclear extracts. Finally, the in vivo deacetylation of the SIRT1 substrate P53 is increased in CD38 knockout mice tissue. Our data support the novel concept that nuclear CD38 is a major regulator of cellular/nuclear NAD level, and SIRT1 activity. These findings have strong implications for understanding the basic mechanisms that modulate intracellular NAD levels, energy homeostasis, as well as ageing and cellular protection modulated by the SIRT enzymes.« less

The early ontogeny of digestive and metabolic enzyme activities in two commercial strains of arctic charr (Salvelinus alpinus L.).

PubMed

Lemieux, Hélène; Le François, Nathalie R; Blier, Pierre U

2003-10-01

The extent to which growth performance is linked to digestive or energetic capacities in the early life stages of a salmonid species was investigated. We compared two strains of Arctic charr known to have different growth potentials during their early development (Fraser and Yukon gold). Trypsin, lipase, and amylase activities of whole alevins were measured at regular intervals from hatching through 65 days of development. To assess catabolic ability, we also measured five enzymes representing the following metabolic pathways: amino acid oxidation (amino aspartate transferase), fatty acid oxidation (beta-hydroxy acyl CoA-dehydrogenase), tricarboxylic acid cycle (citrate synthase), glycolysis (pyruvate kinase), and anaerobic glycolysis (lactate dehydrogenase). The measurement of these enzyme activities in individual fish allowed a clear evaluation of digestive capacity in relation to energetic demand. We also compared triploid and diploid individuals within the Yukon gold strain. For the whole experimental period, diploid Yukon gold fish exhibited the highest growth rate (1.08+/-0.18% length/day) followed by triploid Yukon gold fish (1.00+/-0.28% length/day) and finally Fraser strain fish (0.84+/-0.28% length/day). When differences in enzyme activities were observed, the Fraser strain showed higher enzyme activities at a given length than the Yukon gold strain (diploid and triploid). Higher growth performance appears to be linked to lower metabolic capacity. Our results suggest that fish may have to reach an important increase in the ratio of digestive to catabolic enzyme activities or a leveling off of metabolic enzyme activities before the onset of large increases in mass. Copyright 2003 Wiley-Liss, Inc.

Effect of high-intensity intermittent swimming training on fatty acid oxidation enzyme activity in rat skeletal muscle.

PubMed

Terada, Shin; Tabata, Izumi; Higuchi, Mitsuru

2004-02-01

We previously reported that high-intensity exercise training significantly increased citrate synthase (CS) activity, a marker of oxidative enzyme, in rat skeletal muscle to a level equaling that attained after low-intensity prolonged exercise training (Terada et al., J Appl Physiol 90: 2019-2024, 2001). Since mitochondrial oxidative enzymes and fatty acid oxidation (FAO) enzymes are often increased simultaneously, we assessed the effect of high-intensity intermittent swimming training on FAO enzyme activity in rat skeletal muscle. Male Sprague-Dawley rats (3 to 4 weeks old) were assigned to a 10-day period of high-intensity intermittent exercise training (HIT), low-intensity prolonged exercise training (LIT), or sedentary control conditions. In the HIT group, the rats repeated fourteen 20 s swimming sessions with a weight equivalent to 14-16% of their body weight. Between the exercise sessions, a 10 s pause was allowed. Rats in the LIT group swam 6 h/day in two 3 h sessions separated by 45 min of rest. CS activity in the triceps muscle of rats in the HIT and LIT groups was significantly higher than that in the control rats by 36 and 39%, respectively. Furthermore, 3-beta hydroxyacyl-CoA dehydrogenase (HAD) activity, an important enzyme in the FAO pathway in skeletal muscle, was higher in the two training groups than in the control rats (HIT: 100%, LIT: 88%). No significant difference in HAD activity was observed between the two training groups. In conclusion, the present investigation demonstrated that high-intensity intermittent swimming training elevated FAO enzyme activity in rat skeletal muscle to a level similar to that attained after 6 h of low-intensity prolonged swimming exercise training.

Comparison of short- and long-term exposure effects of cruciferous and apiaceous vegetables on carcinogen metabolizing enzymes in Wistar rats.

PubMed

Kim, Jae Kyeom; Strapazzon, Noemia; Gallaher, Cynthia M; Stoll, Dwight R; Thomas, William; Gallaher, Daniel D; Trudo, Sabrina P

2017-10-01

Cruciferous and apiaceous vegetables may be chemopreventive due to their ability to modulate carcinogen-metabolizing enzymes but whether the effects on such enzymes are sustained over time is unknown. To examine the short- and long-term effects of the vegetables, rats were fed one of four diets for 7, 30, or 60 d: AIN-93G, CRU (21% cruciferous vegetables-fresh broccoli, green cabbage, watercress), API (9% apiaceous vegetables - fresh parsnips, celery), or API + CRU (10.5% CRU + 4.5% API). Although CRU increased activity and protein expression of cytochrome P450 (CYP) 1A1 and CYP1A2 after 7 d, only activity was sustained after 30 and 60 d. There was a trend towards an interaction between the length of feeding period and CRU for CYP1A1 activity; activity increased with greater time of feeding. API increased CYP1A2 activity but decreased sulfotransferase 1A1 activity after 7 d, although not at later times. Altogether, increased CYP1A activity by CRU was maintained with long term feeding while protein amount decreased, suggesting influence by mechanisms other than, or in addition to, transcriptional regulation. Thus, response patterns and interactions with length of feeding may differ, depending upon the types of vegetables and enzymes, requiring caution when interpreting the results of short-term feeding studies. Copyright © 2017 Elsevier Ltd. All rights reserved.

Effect of immobile isolated enzymes from rumen liquid by using alginate matrices on the bay leaf extraction

NASA Astrophysics Data System (ADS)

Paramita, Vita; Yulianto, Mohammad Endy; Yohana, Eflita; Arifan, Fahmi; Hanifah, Amjad, Muhammad Taqiyuddin

2015-12-01

This research aims to develop the enzymatically of bay leaves phytochemical extraction process. The novelty and the main innovations of this research is the development of extraction process by using enzymatic extractor and isolate the enzymes from rumen liquid to shift the equilibrium phase, increase the extraction rate and increase the extraction yield. The activity of rumen liquid enzyme was represented by the activity of cellulase and protease. The analyze of total flavonoid content was performed by using UV-Vis Spectrofometry. The activity of immobilized enzyme of cellulase (0.08±0.00 U/ml) was lower than the un-immobilized one (0.23±0.00 U/ml). However, there was no difference activity of the immobilized (0.75±0.00 U/ml) and un-immobilized (0.76±0.01 U/ml) of protease. The model of mass transfer of un-immobilized enzyme can be fitted on the experimental data, however the model of mass transfer of immobilized enzyme did not match with the experimental data. The mass transfer coefficient of enzymatic extraction flavonoids bay leaf without immobilization was 0.17167 s-1 which greater than the reported value of obtained KLa from extraction by using electric heating.

Cytochrome P450-Mediated Phytoremediation using Transgenic Plants: A Need for Engineered Cytochrome P450 Enzymes

PubMed Central

Kumar, Santosh; Jin, Mengyao; Weemhoff, James L

2013-01-01

There is an increasing demand for versatile and ubiquitous Cytochrome P450 (CYP) biocatalysts for biotechnology, medicine, and bioremediation. In the last decade there has been an increase in realization of the power of CYP biocatalysts for detoxification of soil and water contaminants using transgenic plants. However, the major limitations of mammalian CYP enzymes are that they require CYP reductase (CPR) for their activity, and they show relatively low activity, stability, and expression. On the other hand, bacterial CYP enzymes show limited substrate diversity and usually do not metabolize herbicides and industrial contaminants. Therefore, there has been a considerable interest for biotechnological industries and the scientific community to design CYP enzymes to improve their catalytic efficiency, stability, expression, substrate diversity, and the suitability of P450-CPR fusion enzymes. Engineered CYP enzymes have potential for transgenic plants-mediated phytoremediation of herbicides and environmental contaminants. In this review we discuss: 1) the role of CYP enzymes in phytoremediation using transgenic plants, 2) problems associated with wild-type CYP enzymes in phytoremediation, and 3) examples of engineered CYP enzymes and their potential role in transgenic plant-mediated phytoremediation. PMID:25298920

Induction of human spermine oxidase SMO(PAOh1) is regulated at the levels of new mRNA synthesis, mRNA stabilization and newly synthesized protein

PubMed Central

2004-01-01

The oxidation of polyamines induced by antitumour polyamine analogues has been associated with tumour response to specific agents. The human spermine oxidase, SMO(PAOh1), is one enzyme that may play a direct role in the cellular response to the antitumour polyamine analogues. In the present study, the induction of SMO(PAOh1) enzyme activity by CPENSpm [N1-ethyl-N11-(cyclopropyl)methyl-4,8,diazaundecane] is demonstrated to be a result of newly synthesized mRNA and protein. Inhibition of new RNA synthesis by actinomycin D inhibits both the appearance of SMO(PAOh1) mRNA and enzyme activity. Similarly, inhibition of newly synthesized protein with cycloheximide prevents analogue-induced enzyme activity. Half-life determinations indicate that stabilization of SMO(PAOh1) protein does not play a significant role in analogue-induced activity. However, half-life experiments using actinomycin D indicate that CPENSpm treatment not only increases mRNA expression, but also leads to a significant increase in mRNA half-life (17.1 and 8.8 h for CPENSpm-treated cells and control respectively). Using reporter constructs encompassing the SMO(PAOh1) promoter region, a 30–90% increase in transcription is observed after exposure to CPENSpm. The present results are consistent with the hypothesis that analogue-induced expression of SMO(PAOh1) is a result of increased transcription and stabilization of SMO(PAOh1) mRNA, leading to increased protein production and enzyme activity. These data indicate that the major level of control of SMO(PAOh1) expression in response to polyamine analogues exposure is at the level of mRNA. PMID:15496143

DOE Office of Scientific and Technical Information (OSTI.GOV)

Alhenc-Gelas, F.; Weare, J.A.; Johnson, R.L. Jr.

CE was purified from human lung, and antisera were raised in rabbits. Antisera inhibited the activity of the purified enzyme from lung and kidney and the plasma CE of normal persons and sarcoid patients. With antisera at a titer of 1:100,000, a sensitive, direct RIA was developed. CE purified from lung or kidney and CE present in normal and in sarcoid plasma gave parallel logit-log displacement lines, suggesting immunological identity. The level of CE in normal human plasma was 400 +/- 131 ng/ml. In untreated sarcoid patients, the enzyme level and activity increased in parallel. There was a negative correlationmore » (r . -0.81) between enzyme level and diffusing capacity of the lung for CO in sarcoid patients. Synthetic inhibitors such as captopril or MK 421 did not interfere with the RIA, permitting enzyme levels to be monitored in patients undergoing acute inhibitor therapy. During administration of MK 421, CE activity was negligible and plasma levels of CE did not change. In contrast, renin activity increased eightfold during the inhibitor therapy.« less

Effect of Sodium Fluoride Ingestion on Malondialdehyde Concentration and the Activity of Antioxidant Enzymes in Rat Erythrocytes

PubMed Central

Morales-González, José A.; Gutiérrez-Salinas, José; García-Ortiz, Liliana; del Carmen Chima-Galán, María; Madrigal-Santillán, Eduardo; Esquivel-Soto, Jaime; Esquivel-Chirino, César; González-Rubio, Manuel García-Luna y

2010-01-01

Fluoride intoxication has been shown to produce diverse deleterious metabolic alterations within the cell. To determine the effects of sodium fluoride (NaF) treatment on malondialdehyde (MDA) levels and on the activity of antioxidant enzymes in rat erythrocytes, Male Wistar rats were treated with 50 ppm of NaF or were untreated as controls. Erythrocytes were obtained from rats sacrificed weekly for up to eight weeks and the concentration of MDA in erythrocyte membrane was determined. In addition, the activity of the enzymes superoxide, dismutase, catalase, and glutathione peroxidase were determined. Treatment with NaF produces an increase in the concentration of malondialdehyde in the erythrocyte membrane only after the eight weeks of treatment. On the other hand, antioxidant enzyme activity was observed to increase after the fourth week of NaF treatment. In conclusion, intake of NaF produces alterations in the erythrocyte of the male rat, which indicates induction of oxidative stress. PMID:20640162

Dose of Phenobarbital and Age of Treatment at Early Life are Two Key Factors for the Persistent Induction of Cytochrome P450 Enzymes in Adult Mouse Liver.

PubMed

Tien, Yun-Chen; Liu, Ke; Pope, Chad; Wang, Pengcheng; Ma, Xiaochao; Zhong, Xiao-bo

2015-12-01

Drug treatment of neonates and infants and its long-term consequences on drug responses have emerged in recent years as a major challenge for health care professionals. In the current study, we use phenobarbital as a model drug and mouse as an in vivo model to demonstrate that the dose of phenobarbital and age of treatment are two key factors for the persistent induction of gene expression and consequential increases of enzyme activities of Cyp2b, Cyp2c, and Cyp3a in adult livers. We show that phenobarbital treatment at early life of day 5 after birth with a low dose (<100 mg/kg) does not change expression and enzyme activities of Cyp2b, Cyp2c, and Cyp3a in adult mouse liver, whereas phenobarbital treatment with a high dose (>200 mg/kg) significantly increases expression and enzyme activities of these P450s in adult liver. We also demonstrate that phenobarbital treatment before day 10 after birth, but not at later ages, significantly increases mRNAs, proteins, and enzyme activities of the tested P450s. Such persistent induction of P450 gene expression and enzyme activities in adult livers by phenobarbital treatment only occurs within a sensitive age window early in life. The persistent induction in gene expression and enzyme activities is higher in female mice than in male mice for Cyp2b10 but not for Cyp2c29 and Cyp3a11. These results will stimulate studies to evaluate the long-term impacts of drug treatment with different doses at neonatal and infant ages on drug metabolism, therapeutic efficacy, and drug-induced toxicity throughout the rest of life. Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics.

Effect of various factors on the activity of trehalase from the larvae of Sesamia inferens Walker (Insect).

PubMed

Agarwal, A K

1976-12-15

Trehalase from the salivary glands and the midgut of Sesamia inferens showed optimum activity at pH 5.8, and at temperatures of 50 and 60 degrees C respectively. The increase in the incubation period, enzyme concentration, and substrate concentration respectively increased the end-product, the hydrolysis, and the rate of hydrolysis of the substrate. Dialysis did not affect, tryptophan accelerated, and other amino acids and end-product inhibited the enzyme activity.

Metabolic enzyme activities of abyssal and hadal fishes: pressure effects and a re-evaluation of depth-related changes

NASA Astrophysics Data System (ADS)

Gerringer, M. E.; Drazen, J. C.; Yancey, P. H.

2017-07-01

Metabolic enzyme activities of muscle tissue have been useful and widely-applied indicators of whole animal metabolic capacity, particularly in inaccessible systems such as the deep sea. Previous studies have been conducted at atmospheric pressure, regardless of organism habitat depth. However, maximum reaction rates of some of these enzymes are pressure dependent, complicating the use of metabolic enzyme activities as proxies of metabolic rates. Here, we show pressure-related rate changes in lactate and malate dehydrogenase (LDH, MDH) and pyruvate kinase (PK) in six fish species (2 hadal, 2 abyssal, 2 shallow). LDH maximal reaction rates decreased with pressure for the two shallow species, but, in contrast to previous findings, it increased for the four deep species, suggesting evolutionary changes in LDH reaction volumes. MDH maximal reaction rates increased with pressure in all species (up to 51±10% at 60 MPa), including the tide pool snailfish, Liparis florae (activity increase at 60 MPa 44±9%), suggesting an inherent negative volume change of the reaction. PK was inhibited by pressure in all species tested, including the hadal liparids (up to 34±3% at 60 MPa), suggesting a positive volume change during the reaction. The addition of 400 mM TMAO counteracted this inhibition at both 0.5 and 2.0 mM ADP concentrations for the hadal liparid, Notoliparis kermadecensis. We revisit depth-related trends in metabolic enzyme activities according to these pressure-related rate changes and new data from seven abyssal and hadal species from the Kermadec and Mariana trenches. Results show that, with abyssal and hadal species, pressure-related rate changes are another variable to be considered in the use of enzyme activities as proxies for metabolic rate, in addition to factors such as temperature and body mass. Intraspecific increases in tricarboxylic acid cycle enzymes with depth of capture, independent of body mass, in two hadal snailfishes suggest improved nutritional condition for individuals deeper in the hadal zone, likely related to food availability. These new data inform the discussion of factors controlling metabolism in the deep sea, including the visual interactions hypothesis and extend published trends to the planet's deepest-living fishes.

Engineering acidic Streptomyces rubiginosus D-xylose isomerase by rational enzyme design.

PubMed

Waltman, Mary Jo; Yang, Zamin Koo; Langan, Paul; Graham, David E; Kovalevsky, Andrey

2014-02-01

To maximize bioethanol production from lignocellulosic biomass, all sugars must be utilized. Yeast fermentation can be improved by introducing the d-xylose isomerase enzyme to convert the pentose sugar d-xylose, which cannot be fermented by Saccharomyces cerevisiae, into the fermentable ketose d-xylulose. The low activity of d-xylose isomerase, especially at the low pH required for optimal fermentation, limits its use. A rational enzyme engineering approach was undertaken, and seven amino acid positions were replaced to improve the activity of Streptomyces rubiginosus d-xylose isomerase towards its physiological substrate at pH values below 6. The active-site design was guided by mechanistic insights and the knowledge of amino acid protonation states at low pH obtained from previous joint X-ray/neutron crystallographic experiments. Tagging the enzyme with 6 or 12 histidine residues at the N-terminus resulted in a significant increase in the active-site affinity towards substrate at pH 5.8. Substituting an asparagine at position 215, which hydrogen bonded to the metal-bound Glu181 and Asp245, with an aspartate gave a variant with almost an order of magnitude lower KM than measured for the native enzyme, with a 4-fold increase in activity. Other studied variants showed similar (Asp57Asn, Glu186Gln/Asn215Asp), lower (Asp57His, Asn247Asp, Lys289His, Lys289Glu) or no (Gln256Asp, Asp287Asn, ΔAsp287) activity in acidic conditions relative to the native enzyme.

Soil microbial communities and enzyme activities in sea-buckthorn (Hippophae rhamnoides) plantation at different ages.

PubMed

Yang, Miao; Yang, Dan; Yu, Xuan

2018-01-01

The aim of this study was to assess the impact of forest age and season on the soil microbial community and enzyme activities in sea-buckthorn plantation system and to determine the relative contributions to soil microbial properties. Soil sampling was carried out in the dry season (April) and wet season (September) in four areas, including: abandoned farmland (NH), an 8-year- old plantation (young plantation, 8Y), a 13-year-old plantation (middle-aged plantation, 13Y), and an 18-year-old plantation (mature plantation, 18Y). The results showed that forest age and season have a significant effect on soil microbial community structure and enzyme activities. The total, bacterial, fungal, Gram-negative (G+), and Gram-positive (G-) PLFAs increased gradually with forest age, with the highest values detected in 18Y. All the detected enzyme activities showed the trend as a consequence of forest age. The microbial PLFAs and soil enzyme activities were higher in the wet season than the dry season. However, there were no significant interactions between forest age and season. A Correlation analysis suggested that soil microbial communities and enzyme activities were significantly and positively correlated with pH, total nitrogen (TN) and available phosphorus (AP). Season had a stronger influence on soil microbial communities than forest age. In general, sea-buckthorn plantations establishment might be a potential tool for maintaining and increasing soil fertility in arid and semi-arid regions.

Soil microbial communities and enzyme activities in sea-buckthorn (Hippophae rhamnoides) plantation at different ages

PubMed Central

Yang, Miao; Yang, Dan

2018-01-01

The aim of this study was to assess the impact of forest age and season on the soil microbial community and enzyme activities in sea-buckthorn plantation system and to determine the relative contributions to soil microbial properties. Soil sampling was carried out in the dry season (April) and wet season (September) in four areas, including: abandoned farmland (NH), an 8-year- old plantation (young plantation, 8Y), a 13-year-old plantation (middle-aged plantation, 13Y), and an 18-year-old plantation (mature plantation, 18Y). The results showed that forest age and season have a significant effect on soil microbial community structure and enzyme activities. The total, bacterial, fungal, Gram-negative (G+), and Gram-positive (G-) PLFAs increased gradually with forest age, with the highest values detected in 18Y. All the detected enzyme activities showed the trend as a consequence of forest age. The microbial PLFAs and soil enzyme activities were higher in the wet season than the dry season. However, there were no significant interactions between forest age and season. A Correlation analysis suggested that soil microbial communities and enzyme activities were significantly and positively correlated with pH, total nitrogen (TN) and available phosphorus (AP). Season had a stronger influence on soil microbial communities than forest age. In general, sea-buckthorn plantations establishment might be a potential tool for maintaining and increasing soil fertility in arid and semi-arid regions. PMID:29324845

Profiling the orphan enzymes.

PubMed

Sorokina, Maria; Stam, Mark; Médigue, Claudine; Lespinet, Olivier; Vallenet, David

2014-06-06

The emergence of Next Generation Sequencing generates an incredible amount of sequence and great potential for new enzyme discovery. Despite this huge amount of data and the profusion of bioinformatic methods for function prediction, a large part of known enzyme activities is still lacking an associated protein sequence. These particular activities are called "orphan enzymes". The present review proposes an update of previous surveys on orphan enzymes by mining the current content of public databases. While the percentage of orphan enzyme activities has decreased from 38% to 22% in ten years, there are still more than 1,000 orphans among the 5,000 entries of the Enzyme Commission (EC) classification. Taking into account all the reactions present in metabolic databases, this proportion dramatically increases to reach nearly 50% of orphans and many of them are not associated to a known pathway. We extended our survey to "local orphan enzymes" that are activities which have no representative sequence in a given clade, but have at least one in organisms belonging to other clades. We observe an important bias in Archaea and find that in general more than 30% of the EC activities have incomplete sequence information in at least one superkingdom. To estimate if candidate proteins for local orphans could be retrieved by homology search, we applied a simple strategy based on the PRIAM software and noticed that candidates may be proposed for an important fraction of local orphan enzymes. Finally, by studying relation between protein domains and catalyzed activities, it appears that newly discovered enzymes are mostly associated with already known enzyme domains. Thus, the exploration of the promiscuity and the multifunctional aspect of known enzyme families may solve part of the orphan enzyme issue. We conclude this review with a presentation of recent initiatives in finding proteins for orphan enzymes and in extending the enzyme world by the discovery of new activities.

Altered fibre types in gastrocnemius muscle of high wheel-running selected mice with mini-muscle phenotypes.

PubMed

Guderley, Helga; Joanisse, Denis R; Mokas, Sophie; Bilodeau, Geneviève M; Garland, Theodore

2008-03-01

Selective breeding of mice for high voluntary wheel running has favoured characteristics that facilitate sustained, aerobically supported activity, including a "mini-muscle" phenotype with markedly reduced hind limb muscle mass, increased mass-specific activities of oxidative enzymes, decreased % myosin heavy chain IIb, and, in the medial gastrocnemius, reduced twitch speed, reduced mass-specific isotonic power, and increased fatigue resistance. To evaluate whether selection has altered fibre type expression in mice with either "mini" or normal muscle phenotypes, we examined fibre types of red and white gastrocnemius. In both the medial and lateral gastrocnemius, the mini-phenotype increased activities of oxidative enzymes and decreased activities of glycolytic enzymes. In red muscle samples, the mini-phenotype markedly changed fibre types, with the % type I and type IIA fibres and the surface area of type IIA fibres increasing; in addition, mice from selected lines in general had an increased % type IIA fibres and larger type I fibres as compared with mice from control lines. White muscle samples from mini-mice showed dramatic structural alterations, with an atypical distribution of extremely small, unidentifiable fibres surrounded by larger, more oxidative fibres than normally present in white muscle. The increased proportion of oxidative fibres and these atypical small fibres together may explain the reduced mass and increased mitochondrial enzyme activities in mini-muscles. These and previous results demonstrate that extension of selective breeding beyond the time when the response of the selected trait (i.e. distance run) has levelled off can still modify the mechanistic underpinnings of this behaviour.

A possible role for CD26/DPPIV enzyme activity in the regulation of psoriatic pruritus.

PubMed

Komiya, Eriko; Hatano, Ryo; Otsuka, Haruna; Itoh, Takumi; Yamazaki, Hiroto; Yamada, Taketo; Dang, Nam H; Tominaga, Mitsutoshi; Suga, Yasushi; Kimura, Utako; Takamori, Kenji; Morimoto, Chikao; Ohnuma, Kei

2017-06-01

Psoriasis (PSO) is one of the most common chronic inflammatory skin diseases, and pruritus affects approximately 60-90% of patients with PSO. However, the pathogenesis of pruritus in PSO remains unclear. Dipeptidyl peptidase IV (DPPIV) enzyme activity is involved in the regulation of peptide hormones, chemokines and neurotransmitters. Our aim is to evaluate for a potential association between DPPIV and an increased risk of pruritus, and to identify possible underlying treatment targets in affected patients. Utilizing clinical serum samples of PSO patients and in vivo experimental pruritus models, we evaluated for a potential association between DPPIV and an increased risk for pruritus, and attempted to identify possible underlying treatment targets in pruritus of PSO. We first showed that levels of DPPIV enzyme activity in sera of patients with PSO were significantly increased compared to those of healthy controls. We next evaluated levels of substance-P (SP), which is a neurotransmitter for pruritus and a substrate for DPPIV enzyme. Truncated form SP cleaved by DPPIV was significantly increased in sera of PSO. In an in vivo pruritus model induced by SP, scratching was decreased by treatment with a DPPIV inhibitor. Moreover, DPPIV-knockout mice showed attenuation of scratching induced by SP. Finally, scratching was decreased following the administration of a DPPIV inhibitor in an imiquimod-induced PSO model. On the other hand, scratching induced by imiquimod was increased in DPPIV overexpressing-mice. These results suggest that inhibition of DPPIV enzyme activity regulates pruritus in PSO. Copyright © 2017 Japanese Society for Investigative Dermatology. Published by Elsevier B.V. All rights reserved.

Expression and chromatin structures of cellulolytic enzyme gene regulated by heterochromatin protein 1.

PubMed

Zhang, Xiujun; Qu, Yinbo; Qin, Yuqi

2016-01-01

Heterochromatin protein 1 (HP1, homologue HepA in Penicillium oxalicum ) binding is associated with a highly compact chromatin state accompanied by gene silencing or repression. HP1 loss leads to the derepression of gene expression. We investigated HepA roles in regulating cellulolytic enzyme gene expression, as an increasingly number of studies have suggested that cellulolytic enzyme gene expression is not only regulated by transcription factors, but is also affected by the chromatin status. Among the genes that exhibited significant differences between the hepA deletion strain (Δ hepA ) and the wild type (WT), most (95.0 %) were upregulated in Δ hepA compared with WT. The expression of the key transcription factor for cellulolytic enzyme gene (e.g., repressor CreA and activator ClrB) increased significantly. However, the deletion of hepA led to downregulation of prominent extracellular cellulolytic enzyme genes. Among the top 10 extracellular glycoside hydrolases (Amy15A, Amy13A, Cel7A/CBHI, Cel61A, Chi18A, Cel3A/BGLI, Xyn10A, Cel7B/EGI, Cel5B/EGII, and Cel6A/CBHII), in which secretion amount is from the highest to the tenth in P . oxalicum secretome, eight genes, including two amylase genes ( amy15A and amy13A ), all five cellulase genes ( cel7A / cbh1 , cel6A / cbh2 , cel7B / eg1 , cel5B / eg2 , and cel3A / bgl1 ), and the cellulose-active LPMO gene ( cel61A ) expression were downregulated. Results of chromatin accessibility real-time PCR (CHART-PCR) showed that the chromatin of all three tested upstream regions opened specifically because of the deletion of hepA in the case of two prominent cellulase genes cel7A/cbh1 and cel7B/eg1 . However, the open chromatin status did not occur along with the activation of cellulolytic enzyme gene expression. The overexpression of hepA upregulated the cellulolytic enzyme gene expression without chromatin modification. The overexpression of hepA remarkably activated the cellulolytic enzyme synthesis, not only in WT (~150 % filter paper activity (FPA) increase), but also in the industry strain RE-10 (~20-30 % FPA increase). HepA is required for chromatin condensation of prominent cellulase genes. However, the opening of chromatin mediated by the deletion of hepA was not positively correlated with cellulolytic enzyme gene activation. HepA is actually a positive regulator for cellulolytic enzyme gene expression and could be a promising target for genetic modification to improve cellulolytic enzyme synthesis.

A meta-analysis of soil exoenzyme responses to simulated climate change

NASA Astrophysics Data System (ADS)

Gebhardt, M.; Espinosa, N. J.; Blankinship, J. C.; Gallery, R. E.

2017-12-01

Microorganisms produce extracellular enzymes to decompose plant matter and drive biogeochemical transformations in soils. Climate change factors, such as warming and altered precipitation patterns, can impact enzyme activity through both direct and indirect mechanisms. Although many individual studies have examined how soil exoenzyme activities respond to climate change manipulations, there is disagreement surrounding the direction of these responses. We performed a synthesis of published studies to examine the influence of warming and altered precipitation on microbial exoenzyme activity. We found that warming increased enzyme activity with a more pronounced effect for oxidative relative to hydrolytic enzymes. Reduced precipitation consistently decreased exoenzyme activity. These responses, however, varied by season, biome, and enzyme type. The majority of studies fitting our criteria (e.g., experiments lasting a minimum of one growing season, paired treatments and controls) were located in North America and Europe. Inferences from this analysis therefore exclude many important ecosystems such as hyper-arid, wetlands, and artic systems. Carbon degrading enzyme activities were less sensitive to climate change manipulations when compared to phosphorus and nitrogen degrading enzyme activities. Linking enzyme activity to biogeochemical processes requires concomitant measurements of organic and inorganic carbon pools, mineralogy, nutrients, microbial biomass and community structure, and heterotrophic respiration within individual studies. Furthermore, linking these parameters to climate and environmental factors will require a comprehensive and consistent inclusion of biotic and abiotic variables among researchers and experiments. Globally, soils contain the largest carbon pools. Understanding the impacts of large-scale perturbations on soil enzyme activity will help to constrain predictions on the fate of biogeochemical transformations and improve model projections.

Increased brain lysosomal pepstatin-insensitive proteinase activity in patients with neurodegenerative diseases.

PubMed

Junaid, M A; Pullarkat, R K

1999-04-02

A recent study has shown mutations in CLN2 gene, that encodes a novel lysosomal pepstatin-insensitive proteinase (LPIP), in the pathophysiology of late-infantile neuronal ceroid lipofuscinosis (LINCL). We have measured the LPIP activities in brains from various forms of human neuronal ceroid lipofuscinoses (NCL), canine ceroid lipofuscinosis and other neurodegenerative disorders with a highly sensitive assay using a tetrapeptide Gly-Phe-Phe-Leu-amino-trifluoromethyl coumarin (AFC) as substrate. Brain LPIP has a pH optimum of 3.5 and an apparent km of 100 microM for the crude enzyme. The enzyme activity is totally absent in LINCL patients. Pronounced increase in the LPIP activity was seen in patients suffering from infantile (INCL), juvenile (JNCL) and adult (ANCL) forms of neuronal ceroid lipofuscinoses. LPIP activity was also found to be increased about two-fold in Alzheimer's disease when compared with normal or age-matched controls, while in globoidal-cell leukodystrophy (Krabbe's disease) it was similar to the normal controls. Although mannose-6-phosphorylated LPIP is increased 13-fold in brains of patients with JNCL, this form of LPIP did not have any enzyme activity. The mechanism by which LPIP activities are increased in a wide range of neurodegenerative diseases is unknown, although neuronal loss, followed by gliosis are common characteristics of these diseases.

Soil properties and enzyme activities as affected by biogas slurry irrigation in the Three Gorges Reservoir areas of China.

PubMed

Chen, Shiling; Yu, Weiwei; Zhang, Zhi; Luo, Surong

2015-03-01

Biogas slurry, as a quality organic fertilizer, is widely used on large scale livestock farmland in Southwest China. In the present study, slurry collected from anaerobic tank of dairy farm was used to irrigate farmland having typical purple soil in Chongquing, China. The study revealed that irrigation with biogasslurry increased soil ammonium nitrogen and soil nitrate by 47.8 and 19% respectively as compared to control check. The average soil available phosphorus and soil phosphorus absorption co-efficient changed slightly. Relative enzyme activities of N and P transformation were indicated by catalase, urease, invertase and phosphatase activity. Irrigation period and irrigation quantity were selected as variable factor Catalase, invertase and urease activity was highest when irrigation period and irrigation quantitiy was 4 days and 500 ml; whereas highest phosphatase activity increased significantly in purple irrigated by biogas slurry. The result of the present study is helpful in finding optimum irrigation conditions required for enzyme activity within defined range. It further reveals that biogas slurry enriches soil with various nutrients by enhancing N, P content and enzyme activities as well as it also deals with large number of biogas slurry for protecting the environment.

Climate and root proximity as dominant drivers of enzyme activity and C and N isotopic signature in soil

NASA Astrophysics Data System (ADS)

Stock, Svenja; Köster, Moritz; Dippold, Michaela; Boy, Jens; Matus, Francisco; Merino, Carolina; Nájera, Francisco; Spielvogel, Sandra; Gorbushina, Anna; Kuzyakov, Yakov

2017-04-01

The Chilean ecosystems provide a unique study area to investigate biotic controls on soil organic matter (SOM) decomposition and mineral weathering depending on climate (from hyper arid to temperate humid). Microorganisms play a crucial role in the SOM decomposition, nutrient release and cycling. By means of extracellular enzymes microorganisms break down organic compounds and provide nutrients for plants. Soil moisture (abiotic factor) and root carbon (biotic factor providing easily available energy source for microorganisms), are important factors for microbial decomposition of SOM and show strong gradients along the investigated climatic gradient. A high input of root carbon increases microbial activity and enzyme production, and facilitates SOM breakdown and nutrient release The aim of this study was to determine the potential enzymatic SOM decomposition and nutrient release depending on root proximity and precipitation. C and N contents, δ13C and δ15N values, and kinetics (Vmax, Km) of six extracellular enzymes, responsible for C, N, and P cycles, were quantified in vertical (soil depth) and horizontal (from roots to bulk soil) gradients in two climatic regions: within a humid temperate forest and a semiarid open forest. The greater productivity of the temperate forest was reflected by higher C and N contents compared to the semiarid forest. Regression lines between δ13C and -[ln(%C)] showed a stronger isotopic fractionation from top- to subsoil at the semiarid open forest, indicating a faster SOM turnover compared to the humid temperate forest. This is the result of more favorable soil conditions (esp. temperature and smaller C/N ratios) in the semiarid forest. Depth trends of δ15N values indicated N limitation in both soils, though the limitation at the temperate site was stronger. The activity of enzymes degrading cellulose and hemicellulose increased with C content. Activity of enzymes involved in C, N and P cycles decreased from top- to subsoil and with distance to roots. Chitinase and acid phosphatase activities increased with increasing C contents and indicated a faster substrate turnover in soil under the temperate forest compared to the semiarid forest. In contrast, Tyrosin-aminopeptidase activities indicated a faster substrate turnover under semiarid forest than the temperate forest, and strongly increased with increasing N content. We conclude that the N availability and SOM turnover under semiarid open forest is higher than under humid temperate forest. The enzyme activities are depending on depth only indirectly and are driven mainly by soil C content, which is directly affected by root carbon input.

Cadmium Phytoavailability and Enzyme Activity under Humic Acid Treatment in Fluvo-aquic Soil

NASA Astrophysics Data System (ADS)

Liu, Borui; Huang, Qing; Su, Yuefeng

2018-01-01

A pot experiment was conducted to investigate the cadmium (Cd) availability to pakchois (Brassica chinensis L.) as well as the enzyme activities in fluvo-aquic soil under humic acid treatment. The results showed that the phytoavailability of Cd in soil decreased gradually as humic acid concentration rose (0 to 12 g·kg-1), while the activities of urease (UE), alkaline phosphatase (ALP) and catalase (CAT) kept increasing (P < 0.05). The correlation analysis indicated that humic acid was effective for reducing the devastation to soil enzymes due to the Cd pollution. In conclusion, humic acid is effective for the reduction of both Cd phytoavailability and the damage to enzyme activities due to Cd pollution in fluvo-aquic soil

Differences in activity of cytochrome C oxidase in brain between sleep and wakefulness.

PubMed

Nikonova, Elena V; Vijayasarathy, Camasamudram; Zhang, Lin; Cater, Jacqueline R; Galante, Raymond J; Ward, Stephen E; Avadhani, Narayan G; Pack, Allan I

2005-01-01

Increased mRNA level of subunit 1 cytochrome c oxidase (COXI) during wakefulness and after short-term sleep deprivation has been described in brain. We hypothesized that this might contribute to increased activity of cytochrome oxidase (COX) enzyme during wakefulness, as part of the mechanisms to provide sufficient amounts of adenosine triphosphate to meet increased neuronal energy demands. COX activity was measured in isolated mitochondria from different brain regions in groups of rats with 3 hours of spontaneous sleep, 3 hours of spontaneous wake, and 3 hours of sleep deprivation. The group with 3 hours of spontaneous wake was added to delineate the circadian component of changes in the enzyme activity. Northern blot analysis was performed to examine the mRNA levels of 2 subunits of the enzyme COXI and COXIV, encoded by mitochondrial and nuclear DNA, respectively. Laboratory of Biochemistry, Department of Animal Biology, and Center for Sleep and Respiratory Neurobiology, University of Pennsylvania. 2-month-old male Fischer rats (N = 21) implanted for polygraphic recording. For COX activity, there was a main effect by analysis of variance of experimental group (P < .0001) with significant increases in COX activity in wake and sleep-deprived groups as compared to the sleep group. A main effect of brain region was also significant (P < .001). There was no difference between brain regions in the degree of increase in enzyme activity in wakefulness. Both COXI and COXIV mRNA were increased with wakefulness as compared to sleep. There is an increase in COX activity after both 3 hours of spontaneous wake and 3 hours of sleep deprivation as compared with 3 hours of spontaneous sleep in diverse brain regions, which could be, in part, explained by the increased levels of bigenomic transcripts of the enzyme. This likely contributes to increased adenosine triphosphate production during wakefulness. ADP, adenosine diphosphate; ATP, adenosine triphosphate; COXI, cytochrome c oxidase subunit 1 mRNA; COX, cytochrome c oxidase (protein); CREB, cyclic AMP response element binding protein; DNA, deoxyribonucleic acid; EDTA, ethylenediaminetetraacetic acid; EEG, electroencephalography; EMG, electromyography; GABP, GA binding protein; HEPES, 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid; mRNA, messenger ribonucleic acid; NADH, nicotinamid adenine dinucleotide, reduced; NDII, NADH dehydrogenase subunit 2 mRNA; NRF, nuclear respiratory factor.

[Changes in active cysteine cathepsins in lysosomes from tissues thyroid papillary carcinomas with various biological characteristics].

PubMed

Kalinichenko, O V; Myshunina, T M; Tron'ko, M D

2013-01-01

To clarify possible role of cysteine cathepsin H, B and L in the proteolytic processes that contribute to the progression of tumor growth in the thyroid, we studied their activity in lysosomes isolated from the tissue of papillary carcinomas. It was shown that for these enzymes there is a dependence of the changes in their activity on a number of biological characteristics of the tumors. Thus, the sharp increase in the activity ofcathepsin H observed in lysosomes of tissue carcinomas category T2 and T3, with intra-and ekstrathyroid and lymphatic invasion of tumor cells. An increase in the activity of cathepsin B is set in the lysosomes of tissue heterogeneous follicular structure, especially in the presence of solid areas, in comparison with typical papillary tumors and in the lysosomes of tissue carcinomas in intrathyroid and cathepsin L-at extrathyroid invasion. A common feature of the enzymes is to increase the activity of cathepsins in lysosomes of tissue nonencapsulated papillary carcinomas. These enzymes probably do not take part in the invasion of tumor cells into blood vessels and in the mechanisms of tumor metastasis to regional lymph nodes. The latter shows no changes in the activity of cathepsins in lysosomes of tissue carcinomas category N1. The results indicate the different role of cathepsin H, B and L in thyroid carcinogenesis, where each enzyme has its specific function.

Oxygen requirements for formation and activity of the squalene expoxidase in Saccharomyces cerevisiae

NASA Technical Reports Server (NTRS)

Jahnke, L.; Klein, H. P.

1983-01-01

The effect of oxygen on squalene epoxidase activity in Saccharomyces cerevisiae was investigated. In cells grown in standing cultures, the epoxidase was localized mainly in the 'mitochondrial' fraction. Upon aeration, enzyme activity increased and the newly formed enzyme was associated with the 'microsomal' fraction. At 0.03 percent (vol/vol) oxygen, epoxidase levels doubled, whereas the ergosterol level was only slightly increased. Cycloheximide inhibited the increase in epoxidase under these conditions. An apparent K sub m for oxygen of 0.38 percent (vol/vol) was determined from a crude particulate preparation for the epoxidase.

Studies on the protective effect of dietary fish oil on uranyl-nitrate-induced nephrotoxicity and oxidative damage in rat kidney.

PubMed

Priyamvada, Shubha; Khan, Sara A; Khan, Md Wasim; Khan, Sheeba; Farooq, Neelam; Khan, Farah; Yusufi, A N K

2010-01-01

Human and animal exposure demonstrates that uranium is nephrotoxic. However, attempts to reduce it were not found suitable for clinical use. Dietary fish oil (FO) enriched in omega-3 fatty acids reduces the severity of cardiovascular and renal diseases. Present study investigates the protective effect of FO on uranyl nitrate (UN)-induced renal damage. Rats prefed with experimental diets for 15 days, given single nephrotoxic dose of UN (0.5mg/kg body weight) intraperitoneally. After 5d of UN treatment, serum/urine parameters, enzymes of carbohydrate metabolism, brush border membrane (BBM), oxidative stress and phosphate transport were analyzed in rat kidney. UN nephrotoxicity was characterized by increased serum creatinine and blood urea nitrogen. UN increased the activity of lactate dehydrogenase and NADP-malic enzyme whereas decreased malate, isocitrate and glucose-6-phophate dehydrogenases; glucose-6-phophatase, fructose-1, 6-bisphosphatase and BBM enzyme activities. UN caused oxidant/antioxidant imbalances as reflected by increased lipid peroxidation, activities of superoxide dismutase, glutathione peroxidase and decreased catalase activity. Feeding FO alone increased activities of enzymes of glucose metabolism, BBM, oxidative stress and Pi transport. UN-elicited alterations were prevented by FO feeding. However, corn oil had no such effects and was not similarly effective. In conclusion, FO appears to protect against UN-induced nephrotoxicity by improving energy metabolism and antioxidant defense mechanism. Copyright 2009 Elsevier Ltd. All rights reserved.

[Importance of the 11β-hydroxysteroid dehydrogenase enzyme in clinical disorders].

PubMed

Feldman, Karolina; Likó, István; Nagy, Zsolt; Szappanos, Agnes; Grolmusz, Vince Kornél; Tóth, Miklós; Rácz, Károly; Patócs, Attila

2013-02-24

Glucocorticoids play an important role in the regulation of carbohydrate and amino acid metabolism, they modulate the function of the immune system, and contribute to stress response. Increased and decreased production of glucocorticoids causes specific diseases. In addition to systemic hypo- or hypercortisolism, alteration of local synthesis and metabolism of cortisol may result in tissue-specific hypo- or hypercortisolism. One of the key enzymes participating in the local synthesis and metabolism of cortisol is the 11β-hydroxysteroid dehydrogenase enzyme. Two isoforms, type 1 and type 2 enzymes are located in the endoplasmic reticulum and catalyze the interconversion of hormonally active cortisol and inactive cortisone. The type 1 enzyme mainly works as an activator, and it is responsible for the generation of cortisol from cortisone in liver, adipose tissue, brain and bone. The gene encoding this enzyme is located on chromosome 1. The authors review the physiological and pathophysiological processes related to the function of the type 1 11β-hydroxysteroid dehydrogenase enzyme. They summarize the potential significance of polymorphic variants of the enzyme in clinical diseases as well as knowledge related to inhibitors of enzyme activity. Although further studies are still needed, inhibition of the enzyme activity may prove to be an effective tool for the treatment of several diseases such as obesity, osteoporosis and type 2 diabetes.

[Erythremia: the activity of erythrocyte antioxidant enzymes and the association with iron deficiency].

PubMed

Petukhov, V I; Kumerova, A O; Letse, A G; Silova, A A; Shkesters, A P; Krishchuna, M A; Mironova, N A

1997-01-01

Concentration of malonic dialdehyde (MDA) and activity of antioxidant enzymes G-6-PD, glutation peroxidase (GP), glutation reductase, catalase, superoxide dismutase were measured in red cells of patients with polycythemia vera. Plasmic ions Fe3+ were estimated by means of electron-paramagnetic resonance. MDA concentration and antioxidant enzymes (except GP) in polycythemia red cells were found increased, while the activity of selenium-dependent GP was reduced, the inhibition being greatest in severe iron deficiency. It is suggested that GP activity in red cells depends on both selenium levels in the body and concentrations of non-hematic iron.

Role of conformational dynamics in the evolution of novel enzyme function.

PubMed

Maria-Solano, Miguel A; Serrano-Hervás, Eila; Romero-Rivera, Adrian; Iglesias-Fernández, Javier; Osuna, Sílvia

2018-05-21

The free energy landscape concept that describes enzymes as an ensemble of differently populated conformational sub-states in dynamic equilibrium is key for evaluating enzyme activity, enantioselectivity, and specificity. Mutations introduced in the enzyme sequence can alter the populations of the pre-existing conformational states, thus strongly modifying the enzyme ability to accommodate alternative substrates, revert its enantiopreferences, and even increase the activity for some residual promiscuous reactions. In this feature article, we present an overview of the current experimental and computational strategies to explore the conformational free energy landscape of enzymes. We provide a series of recent publications that highlight the key role of conformational dynamics for the enzyme evolution towards new functions and substrates, and provide some perspectives on how conformational dynamism should be considered in future computational enzyme design protocols.

ORENZA: a web resource for studying ORphan ENZyme activities

PubMed Central

Lespinet, Olivier; Labedan, Bernard

2006-01-01

Background Despite the current availability of several hundreds of thousands of amino acid sequences, more than 36% of the enzyme activities (EC numbers) defined by the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (NC-IUBMB) are not associated with any amino acid sequence in major public databases. This wide gap separating knowledge of biochemical function and sequence information is found for nearly all classes of enzymes. Thus, there is an urgent need to explore these sequence-less EC numbers, in order to progressively close this gap. Description We designed ORENZA, a PostgreSQL database of ORphan ENZyme Activities, to collate information about the EC numbers defined by the NC-IUBMB with specific emphasis on orphan enzyme activities. Complete lists of all EC numbers and of orphan EC numbers are available and will be periodically updated. ORENZA allows one to browse the complete list of EC numbers or the subset associated with orphan enzymes or to query a specific EC number, an enzyme name or a species name for those interested in particular organisms. It is possible to search ORENZA for the different biochemical properties of the defined enzymes, the metabolic pathways in which they participate, the taxonomic data of the organisms whose genomes encode them, and many other features. The association of an enzyme activity with an amino acid sequence is clearly underlined, making it easy to identify at once the orphan enzyme activities. Interactive publishing of suggestions by the community would provide expert evidence for re-annotation of orphan EC numbers in public databases. Conclusion ORENZA is a Web resource designed to progressively bridge the unwanted gap between function (enzyme activities) and sequence (dataset present in public databases). ORENZA should increase interactions between communities of biochemists and of genomicists. This is expected to reduce the number of orphan enzyme activities by allocating gene sequences to the relevant enzymes. PMID:17026747

The steady-state kinetics of the NADH-dependent nitrite reductase from Escherichia coli K 12. Nitrite and hydroxylamine reduction.

PubMed Central

Jackson, R H; Cole, J A; Cornish-Bowden, A

1981-01-01

The reduction of both NO2- and hydroxylamine by the NADH-dependent nitrite reductase of Escherichia coli K 12 (EC 1.6.6.4) appears to follow Michaelis-Menten kinetics over a wide range of NADH concentrations. Substrate inhibition can, however, be detected at low concentrations of the product NAD+. In addition, NAD+ displays mixed product inhibition with respect to NADH and mixed or uncompetitive inhibition with respect to hydroxylamine. These inhibition characteristics are consistent with a mechanism in which hydroxylamine binds during catalysis to a different enzyme form from that generated when NAD+ is released. The apparent maximum velocity with NADH as varied substrate increases as the NAD+ concentration increases from 0.05 to 0.7 mM with 1 mM-NO2- or 100 mM-hydroxylamine as oxidized substrate. This increase is more marked for hydroxylamine reduction than for NO2- reduction. Models incorporating only one binding site for NAD can account for the variation in the Michaelis-Menten parameters for both NADH and hydroxylamine with [NAD+] for hydroxylamine reduction. According to these models, activation of the reaction occurs by reversal of an over-reduction of the enzyme by NADH. If the observed activation of the enzyme by NAD+ derives both from activation of the generation of the enzyme-hydroxylamine complex from the enzyme-NO2- complex during NO2- reduction and from activation of the reduction of the enzyme-hydroxylamine complex to form NH4+, then the variation of Vapp. for NO2- or hydroxylamine with [NAD+] is consistent with the occurrence of the same enzyme-hydroxylamine complex as an intermediate in both reactions. PMID:6279095

Ectomycorrhizal fungi enhance nitrogen and phosphorus nutrition of Nothofagus dombeyi under drought conditions by regulating assimilative enzyme activities.

PubMed

Alvarez, Maricel; Huygens, Dries; Olivares, Erick; Saavedra, Isabel; Alberdi, Miren; Valenzuela, Eduardo

2009-08-01

Drought stress conditions (DC) reduce plant growth and nutrition, restraining the sustainable reestablishment of Nothofagus dombeyi in temperate south Chilean forest ecosystems. Ectomycorrhizal symbioses have been documented to enhance plant nitrogen (N) and phosphorus (P) uptake under drought, but the regulation of involved assimilative enzymes remains unclear. We studied 1-year-old N. dombeyi (Mirb.) Oerst. plants in association with the ectomycorrhizal fungi Pisolithus tinctorius (Pers.) Coker & Couch. and Descolea antartica Sing. In greenhouse experiments, shoot and root dry weights, mycorrhizal colonization, foliar N and P concentrations, and root enzyme activities [glutamate synthase (glutamine oxoglutarate aminotransferase (GOGAT), EC 1.4.1.13-14), glutamine synthetase (GS, EC 6.3.1.2), glutamate dehydrogenase (GDH, EC 1.4.1.2-4), nitrate reductase (NR, EC 1.6.6.1), and acid phosphomonoesterase (PME, EC 3.1.3.1-2)] were determined as a function of soil-water content. Inoculation of N. dombeyi with P. tinctorius and D. antartica significantly stimulated plant growth and increased plant foliar N and P concentrations, especially under DC. Ectomycorrhizal inoculation increased the activity of all studied enzymes relative to non-mycorrhizal plants under drought. We speculate that GDH is a key enzyme involved in the enhancement of ectomycorrhizal carbon (C) availability by fuelling the tricarboxylic acid (TCA) cycle under conditions of drought-induced carbon deficit. All studied assimilative enzymes of the ectomycorrhizal associations, involved in C, N, and P transfers, are closely interlinked and interdependent. The up-regulation of assimilative enzyme activities by ectomycorrhizal fungal root colonizers acts as a functional mechanism to increase seedling endurance to drought. We insist upon incorporating ectomycorrhizal inoculation in existing Chilean afforestation programs.

Oligo-carrageenan kappa increases NADPH, ascorbate and glutathione syntheses and TRR/TRX activities enhancing photosynthesis, basal metabolism, and growth in Eucalyptus trees.

PubMed

González, Alberto; Moenne, Fabiola; Gómez, Melissa; Sáez, Claudio A; Contreras, Rodrigo A; Moenne, Alejandra

2014-01-01

In order to analyze the effect of OC kappa in redox status, photosynthesis, basal metabolism and growth in Eucalyptus globulus, trees were treated with water (control), with OC kappa at 1 mg mL(-1), or treated with inhibitors of NAD(P)H, ascorbate (ASC), and glutathione (GSH) syntheses and thioredoxin reductase (TRR) activity, CHS-828, lycorine, buthionine sulfoximine (BSO), and auranofin, respectively, and with OC kappa, and cultivated for 4 months. Treatment with OC kappa induced an increase in NADPH, ASC, and GSH syntheses, TRR and thioredoxin (TRX) activities, photosynthesis, growth and activities of basal metabolism enzymes such as rubisco, glutamine synthetase (GlnS), adenosine 5'-phosphosulfate reductase (APR), involved in C, N, and S assimilation, respectively, Krebs cycle and purine/pyrimidine synthesis enzymes. Treatment with inhibitors and OC kappa showed that increases in ASC, GSH, and TRR/TRX enhanced NADPH synthesis, increases in NADPH and TRR/TRX enhanced ASC and GSH syntheses, and only the increase in NADPH enhanced TRR/TRX activities. In addition, the increase in NADPH, ASC, GSH, and TRR/TRX enhanced photosynthesis and growth. Moreover, the increase in NADPH, ASC and TRR/TRX enhanced activities of rubisco, Krebs cycle, and purine/pyrimidine synthesis enzymes, the increase in GSH, NADPH, and TRR/TRX enhanced APR activity, and the increase in NADPH and TRR/TRX enhanced GlnS activity. Thus, OC kappa increases NADPH, ASC, and GSH syntheses leading to a more reducing redox status, the increase in NADPH, ASC, GSH syntheses, and TRR/TRX activities are cross-talking events leading to activation of photosynthesis, basal metabolism, and growth in Eucalyptus trees.

Improved activity of α-chymotrypsin on silica particles - A high-pressure stopped-flow study.

PubMed

Schuabb, Vitor; Winter, Roland; Czeslik, Claus

2016-11-01

Pressure is well known to affect the catalytic rate of enzymes dissolved in solution. To better understand enzyme kinetics at aqueous-solid interfaces, we have carried out a high-pressure stopped-flow activity study of α-chymotrypsin (α-CT) that is adsorbed on silica particles and, for comparison, dissolved in solution. The enzyme reaction was modulated using pressures up to 2000bar and recorded using the high-pressure stopped-flow technique. The results indicate an 8-fold enhancement of the turnover number upon α-CT adsorption and a further increase of the catalytic rate in the pressure range up to 1000bar. From the pressure dependence of the catalytic rate, apparent activation volumes have been determined. In the adsorbed state of α-CT, a pronounced change of the activation volume is found with increasing pressure. Furthermore, owing to suppression of its autolysis, a significantly longer storage time of α-CT can be achieved when the enzyme is adsorbed on silica particles. The results obtained are discussed in terms of a surface-induced selection of conformational substates of the enzyme-substrate complex. Copyright © 2016 Elsevier B.V. All rights reserved.

Effect of vilon and epithalon on activity of enzymes in epithelial and subepithelial layers in small intestine of old rats.

PubMed

Khavinson, V Kh; Timofeeva, N M; Malinin, V V; Gordova, L A; Nikitina, A A

2002-12-01

Per os administration of Vilon (Lys-Glu) or Epithalon (Ala-Glu-Asp-Gly) to aged Wistar rats for 1 month significantly increased activity of membrane enzymes maltase and alkaline phosphatase in epithelial layer of the small intestine. In addition, Vilon significantly increased activity of cytosolic glycyl-L-leucine dipeptidase in the stromal and seromuscular layers of the small intestine in comparison with the control rats not treated with this agent. These findings suggest improvement of trophic and barrier functions of the small intestine and corroborate the hypothesis on the existence of not only epithelial, but also subepithelial enzymatic barrier supporting the enzyme system in the small intestine, especially in aged animals.

Phosphatidylserine metabolism modification precedes manganese-induced apoptosis and phosphatidylserine exposure in PC12 cells.

PubMed

Ferrara, G; Gambelunghe, A; Mozzi, R; Marchetti, M C; Migliorati, G; Muzi, G; Buratta, S

2013-12-01

Long-term exposure to high manganese (Mn) levels can lead to Parkinson-like neurological disorders. Molecular mechanisms underlying Mn cytotoxicity have been not defined. It is known that Mn induces apoptosis in PC12 cells and that this involves the activation of some signal transduction pathways. Although the role of phospholipids in apoptosis and signal transduction is well-known, the membrane phospholipid component in Mn-related damage has not yet been investigated. Phosphatidylserine (PS) facilitates protein translocation from cytosol to plasma membrane and PS exposure on the cell surface allows macrophage recognition of apoptotic cells. This study investigates the effects of MnCl2 on PS metabolism in PC12 cells, relating them to those on cell apoptosis. Apoptosis induction decreased PS radioactivity of PC12 cells incubated with radioactive serine. MnCl2 reduced PS radioactivity even under conditions that did not affect cell viability or PS exposure, suggesting that the effects on PS metabolism may represent an early event in cell apoptosis. Thus the latter conditions that also induced a greater PS decarboxylation were utilized for further investigating on the effects on PS synthesis, by measuring the activity and expression of PS-synthesizing enzymes, in cell lysates and in total cellular membranes (TM). Compared with corresponding controls, enzyme activity of MnCl2-treated cells was lower in cell lysates and greater in TM. Evaluating the expression of two isoforms of PS-synthesizing enzyme (PSS), PSSII was increased both in cell lysate and TM, while PSSI was unchanged. MnCl2 addition to control cell lysate reduced enzyme activity. These results suggest Mn plays a dual role on PS synthesis. Once inside the cell, Mn inhibits the enzyme/s, thus accounting for reduced PS synthesis in lysates and intact cells. On the other hand, it increases PSSII expression in cell membranes. The possibility that this occurs to counteract the direct effects of Mn ions on enzyme activity cannot be excluded. The effects on membrane enzyme activity and expression may also participate to PS exposure, observed at longer periods of treatment, by increasing membrane PS content. Copyright © 2013 Elsevier Inc. All rights reserved.

Patterns of functional enzyme activity in fungus farming ambrosia beetles.

PubMed

De Fine Licht, Henrik H; Biedermann, Peter H W

2012-06-06

In wood-dwelling fungus-farming weevils, the so-called ambrosia beetles (Curculionidae: Scolytinae and Platypodinae), wood in the excavated tunnels is used as a medium for cultivating fungi by the combined action of digging larvae (which create more space for the fungi to grow) and of adults sowing and pruning the fungus. The beetles are obligately dependent on the fungus that provides essential vitamins, amino acids and sterols. However, to what extent microbial enzymes support fungus farming in ambrosia beetles is unknown. Here we measure (i) 13 plant cell-wall degrading enzymes in the fungus garden microbial consortium of the ambrosia beetle Xyleborinus saxesenii, including its primary fungal symbionts, in three compartments of laboratory maintained nests, at different time points after gallery foundation and (ii) four specific enzymes that may be either insect or microbially derived in X. saxesenii adult and larval individuals. We discovered that the activity of cellulases in ambrosia fungus gardens is relatively small compared to the activities of other cellulolytic enzymes. Enzyme activity in all compartments of the garden was mainly directed towards hemicellulose carbohydrates such as xylan, glucomannan and callose. Hemicellulolytic enzyme activity within the brood chamber increased with gallery age, whereas irrespective of the age of the gallery, the highest overall enzyme activity were detected in the gallery dump material expelled by the beetles. Interestingly endo-β-1,3(4)-glucanase activity capable of callose degradation was identified in whole-body extracts of both larvae and adult X. saxesenii, whereas endo-β-1,4-xylanase activity was exclusively detected in larvae. Similar to closely related fungi associated with bark beetles in phloem, the microbial symbionts of ambrosia beetles hardly degrade cellulose. Instead, their enzyme activity is directed mainly towards comparatively more easily accessible hemicellulose components of the ray-parenchyma cells in the wood xylem. Furthermore, the detection of xylanolytic enzymes exclusively in larvae (which feed on fungus colonized wood) and not in adults (which feed only on fungi) indicates that only larvae (pre-) digest plant cell wall structures. This implies that in X. saxesenii and likely also in many other ambrosia beetles, adults and larvae do not compete for the same food within their nests - in contrast, larvae increase colony fitness by facilitating enzymatic wood degradation and fungus cultivation.

Kinetic study of an enzymic cycling system coupled to an enzymic step: determination of alkaline phosphatase activity.

PubMed Central

Valero, E; Varón, R; García-Carmona, F

1995-01-01

A kinetic study is made of a system consisting of a specific enzymic cycling assay coupled to an enzymic reaction. A kinetic analysis of this system is presented, and the accumulation of chromophore involved in the cycle is seen to be parabolic, i.e. the rate of the reaction increases continuously with constant acceleration. The system is illustrated by the measurement of alkaline phosphatase activity using beta-NADP+ as substrate. The enzymes alcohol dehydrogenase and diaphorase are used to cycle beta-NAD+ in the presence of ethanol and p-Iodonitrotetrazolium Violet. During each turn of the cycle, one molecule of the tetrazolium salt is reduced to an intensely coloured formazan. A simple procedure for evaluating the kinetic parameters involved in the system and for optimizing this cycling assay is described. The method is applicable to the measurement of any enzyme, and its amplification capacity as well as the simplicity of determining kinetic parameters enable it to be employed in enzyme immunoassays to increase the magnitude of the measured response. PMID:7619054

Loss in photosynthesis during senescence is accompanied by an increase in the activity of β-galactosidase in leaves of Arabidopsis thaliana: modulation of the enzyme activity by water stress.

PubMed

Pandey, Jitendra Kumar; Dash, Sidhartha Kumar; Biswal, Basanti

2017-07-01

The precise nature of the developmental modulation of the activity of cell wall hydrolases that breakdown the wall polysaccharides to maintain cellular sugar homeostasis under sugar starvation environment still remains unclear. In this work, the activity of β-galactosidase (EC 3.2.1.23), a cell-wall-bound enzyme known to degrade the wall polysaccharides, has been demonstrated to remarkably enhance during senescence-induced loss in photosynthesis in Arabidopsis thaliana. The enhancement in the enzyme activity reaches a peak at the terminal phase of senescence when the rate of photosynthesis is at its minimum. Although the precise nature of chemistry of the interface between the decline in photosynthesis and enhancement in the activity of the enzyme could not be fully resolved, the enhancement in its activity in dark and its suppression in light or with exogenous sugars may indicate the involvement of loss of photosynthetic production of sugars as a key factor that initiates and stimulates the activity of the enzyme. The hydrolase possibly participates in the catabolic network of cell wall polysaccharides to produce sugars for execution of energy-dependant senescence program in the background of loss of photosynthesis. Drought stress experienced by the senescing leaves accelerates the decline in photosynthesis with further stimulation in the activity of the enzyme. The stress recovery of photosynthesis and suppression of the enzyme activity on withdrawal of stress support the proposition of photosynthetic modulation of the cell-wall-bound enzyme activity.

Effect of LED photobiomodulation on fluorescent light induced changes in cellular ATPases and Cytochrome c oxidase activity in Wistar rat.

PubMed

A, Ahamed Basha; C, Mathangi D; R, Shyamala

2016-12-01

Fluorescent light exposure at night alters cellular enzyme activities resulting in health defects. Studies have demonstrated that light emitting diode photobiomodulation enhances cellular enzyme activities. The objectives of this study are to evaluate the effects of fluorescent light induced changes in cellular enzymes and to assess the protective role of pre exposure to 670 nm LED in rat model. Male Wistar albino rats were divided into 10 groups of 6 animals each based on duration of exposure (1, 15, and 30 days) and exposure regimen (cage control, exposure to fluorescent light [1800 lx], LED preexposure followed by fluorescent light exposure and only LED exposure). Na + -K + ATPase, Ca 2+ ATPase, and cytochrome c oxidase of the brain, heart, kidney, liver, and skeletal muscle were assayed. Animals of the fluorescent light exposure group showed a significant reduction in Na + -K + ATPase and Ca 2+ ATPase activities in 1 and 15 days and their increase in animals of 30-day group in most of the regions studied. Cytochrome c oxidase showed increase in their level at all the time points assessed in most of the tissues. LED light preexposure showed a significant enhancement in the degree of increase in the enzyme activities in almost all the tissues and at all the time points assessed. This study demonstrates the protective effect of 670 nm LED pre exposure on cellular enzymes against fluorescent light induced change.

Effect of Patulin from Penicillium vulpinum on the Activity of Glutathione-S-Transferase and Selected Antioxidative Enzymes in Maize

PubMed Central

Ismaiel, Ahmed A.

2017-01-01

The mycotoxin patulin (PAT) was purified from Penicillium vulpinum CM1 culture that has been isolated from a soil cultivated with maize. The effect of PAT and of a fungal culture filtrate on the activities of glutathione-S-transferase (GST) and some antioxidant enzymes viz. ascorbate peroxidase (APX), glutathione reductase (GR), dehydroascorbate reductase (DHAR) and monodehydroascorbate reductase (MDHAR) was investigated in roots and shoots of 8-day-old maize seedlings. PAT and culture filtrate caused significant reduction effects in a dose-related manner on the total GST activity. Upon application of the high PAT concentration (25 μg·mL−1) and of the concentrated fungal filtrate (100%, v/v), the reduction in GST activity of roots was 73.8–76.0% and of shoots was 60–61.7%. Conversely, significant increases in the activities of antioxidant enzymes were induced. Application of 25 μg·PAT·mL−1 increased APX, GR, DHAR, and MDHAR activity of root by 2.40-, 2.00-, 1.24-, and 2.16-fold, respectively. In shoots, the enzymatic activity was increased by 1.57-, 1.45-, 1.45-, and 1.61-fold, respectively. Similar induction values of the enzymatic activity were obtained upon application of the concentrated fungal filtrate. This is the first report describing the response of GST and antioxidant enzyme activities of plant cells to PAT toxicity. PMID:28737668

Glucose-6-phosphate dehydrogenase (G-6-PD) deficiency in Switzerland. Demonstration of a new variant (G-6-PD Aarau) with chronic nonsphaerocytic haemolytic anaemia.

PubMed

Gahr, M; Schröter, W; Sturzenegger, M; Bornhalm, D; Marti, H R

1976-08-01

A new variant of erythrocytic glucose-6-phosphate dehydrogenase has been found in a family of Swiss origin. It is associated with chronic nonsphaerocytic haemolytic anaemia. The enzyme from the erythrocytes of a young boy of this family was partially purified 110-fold and characterized. It revealed reduced catalytic activity, increased thermolability and two maxima of the pH activity curve at pH 7.0 and 8.5. The Km value for glucose-6-phosphate was reduced, that for NADP was normal. The enzyme showed an increased inhibitor constant for NADPH with respect to NADP. Electrophoretic mobility was normal (B+). 2-Desoxyglucose-6-phosphate and galactose-6-phosphate were utilized at normal rates, whereas the analogue deamino-NADP gave an increased utilization rate. The mother of the propositus could be identified as heterozygous for this enzyme deficiency. Chronic haemolysis is possibly due to the increased thermolability of the variant enzyme.

Human neutral brush border endopeptidase EC 3.4.24.11 in urine, its isolation, characterisation and activity in renal diseases.

PubMed

Vlaskou, D; Hofmann, W; Guder, W G; Siskos, P A; Dionyssiou-Asteriou, A

2000-07-01

Human neutral brush border endopeptidase (NEP) was purified from the urine of patients suffering from acute toxic tubulointerstitial nephropathy. An enzyme preparation with specific activity of 102 Ug(-1) protein was obtained. The urinary activities of neutral endopeptidase and alanine aminopeptidase were measured in patients with renal disease and in 30 control patients, resulting in a reference range from 0.1 to 0.7 Ug(-1) creatinine and 1.4-14.1 Ug(-1) creatinine, respectively. Urine enzyme activities were highest in patients with acute tubulotoxic renal diseases. Neutral endopeptidase and alanine aminopeptidase activities were found to be 6.5- and 10-fold higher than the upper value of the reference range, respectively. Smaller increases in the rate of excretion of these enzymes (2.5- and 3.5-fold), respectively, were observed in patients suffering from acute tubular insufficiency and even lower increases, 2- and 1.5-fold, respectively, were observed in patients with chronic renal diseases. In diabetics and kidney transplant patients the enzyme excretion rates were within the reference range. Assay of both transmembrane metalloproteinases in urine may prove valuable in serving as markers for renal toxicity. Together with beta-NAG these enzymes could be employed as differentiation markers between acute and chronic tubular insufficiency.

Effect of the electrical currents generated by the intestinal smooth muscle layers on pancreatic enzyme activity: an in vitro study.

PubMed

Dabek, Marta; Podgurniak, Paweł; Piedra, Jose L Valverde; Szymańczyk, Sylwia; Filip, Rafał; Wojtasz-Pajak, Anna; Werpachowska, Eliza; Podgurniak, Malgorzata; Pierzynowski, Stefan G

2007-05-01

Gut enzymes in the small intestine are exposed to extremely low electrical currents (ELEC) generated by the smooth muscle. In the present study, the in vitro tests were undertaken to evaluate the effect of these electric currents on the activity of the proteolytic pancreatic digestive enzymes. A simulator generating the typical electrical activity of pig gut was used for these studies. The electric current emitted by the simulator was transmitted to the samples, containing enzyme and its substrate, using platinum plate electrodes. All samples were incubated at 37 degrees C for 1 h. The changes in optical density, corresponding to enzyme activity, in samples stimulated for 1 h with ELEC was compared with that not exposed to ELEC. The obtained results show that the electrical current with the characteristics of the myoelectrical migrating complex (MMC) has an influence on pancreatic enzyme activity. Increased endopeptidase and reduced exopeptidase activity was noticed in samples treated with ELEC. This observation can be of important as analyzed factors which can alter enzymatic activity of the gut, can thus also affect feed/food digestibility. (c) 2007 Wiley-Liss, Inc.

Heat shock protein 70 (Hsp70)-stimulated deoxycytidine deaminases from a human lymphoma cell but not the activation-induced cytidine deaminase (AID) from Ramos 6.4 human Burkitt's lymphoma cells

PubMed Central

2010-01-01

Deoxycytidine deaminase enzyme activity was reduced in lysates of human leukemic THP1 cells 24 h after transfection with siRNA designed to inhibit cell synthesis of heat shock protein 70 (Hsp70)1a and Hsp701b. The cytidine deaminase enzyme activity from the cell lysates was purified from an affinity column which contained bound single-stranded oligodeoxycytidylic acid. Deficient enzyme activity in certain elution fractions from the siRNA-transfected cells was restored by including recombinant HSP 70 in the assays. Enzyme activity in some other fractions was increased after siRNA transfection. Activation-induced cytidine deaminase (AID) is a central factor in the immune response. A more specific assay for AID was used to study the influence of Hsp70 on AID activity. Unlike Hsp70's ability to stimulate certain enzymes of DNA base excision repair and other cytidine deaminases, it had little effect on AID activity in vitro, or was weakly inhibitory. PMID:20680536

Electrochemical Nanoparticle-Enzyme Sensors for Screening Bacterial Contamination in Drinking Water

PubMed Central

Chen, Juhong; Jiang, Ziwen; Ackerman, Jonathan D.; Yazdani, Mahdieh; Hou, Singyuk

2015-01-01

Traditional plating and culturing methods used to quantify bacteria commonly require hours to days from sampling to results. We present here a simple, sensitive and rapid electrochemical method for bacteria detection in drinking water based on gold nanoparticle-enzyme complexes. The gold nanoparticles were functionalized with positively charged quaternary amine headgroups that could bind to enzymes through electrostatic interactions, resulting in inhibition of enzymatic activity. In the presence of bacteria, the nanoparticles released from the enzymes and preferentially bound to the bacteria, resulting in an increase in enzyme activity, releasing a redox-active phenol from the substrate. We employed this strategy for the electrochemical sensing of Escherichia coli and Staphylococcus aureus, resulting in a rapid detection (<1h) with high sensitivity (102 CFU·mL−1). PMID:26042607

Effect of potential probiotic Rhodotorula benthica D30 on the growth performance, digestive enzyme activity and immunity in juvenile sea cucumber Apostichopus japonicus.

PubMed

Wang, Ji-hui; Zhao, Liu-qun; Liu, Jin-feng; Wang, Han; Xiao, Shan

2015-04-01

The effects of dietary addition of yeast Rhodotorula benthica (R. benthica) D30 which isolated from local sea mud at levels of 0 (control), 10(5), 10(6) and 10(7) CFU/g feed on the growth performance, digestive enzyme activity, immunity and disease resistance of juvenile sea cucumber Apostichopus japonicus were investigated. It was shown that dietary addition of R. benthica D30 significantly increased the growth rates of sea cucumbers (p < 0.05). The amylase activity, cellulase activity and alginase activity were increased for the animals from three probiotics treated groups. And with the supplemented concentration increased, the values of those digestive enzyme activities increased as well. Dietary addition of R. benthica D30 at the level of 10(7) CFU significantly increased the lysozyme, phagocytic and total nitric oxide synthase activity of A. japonicus (p < 0.05). While, the highest values of the phenoloxidase and alkaline phosphatase activity were found in sea cucumbers fed with R. benthica D30 at the level of 10(6) CFU. Whereas adding R. benthica D30 to diet had no significant effects on the total coelomocyte counts and acid phosphatase activity of A. japonicus (p > 0.05). It was observed that adding R. benthica D30 could significantly decrease the cumulative mortality of sea cucumbers. The present study demonstrated that dietary addition of R. benthica D30 could increase growth performance and some digestive enzyme activities, improve immunity and disease resistance of A. japonicus. And the medium (10(6) CFU) and high (10(7) CFU) additional levels showed better effects. It suggests that yeast R. benthica D30 could be a good probiotic for aquaculture. Copyright © 2015 Elsevier Ltd. All rights reserved.

Recombinant deamidated mutants of Erwinia chrysanthemi L-asparaginase have similar or increased activity compared to wild-type enzyme.

PubMed

Gervais, David; Foote, Nicholas

2014-10-01

The enzyme Erwinia chrysanthemi L-asparaginase (ErA) is an important biopharmaceutical product used in the treatment of acute lymphoblastic leukaemia. Like all proteins, certain asparagine (Asn) residues of ErA are susceptible to deamidation to aspartic acid (Asp), which may be a concern with respect to enzyme activity and potentially to pharmaceutical efficacy. Recombinant ErA mutants containing Asn to Asp changes were expressed, purified and characterised. Two mutants with single deamidation sites (N41D and N281D) were found to have approximately the same specific activity (1,062 and 924 U/mg, respectively) as the wild-type (908 U/mg). However, a double mutant (N41D N281D) had an increased specific activity (1261 U/mg). The N41D mutation conferred a slight increase in the catalytic constant (k cat 657 s(-1)) when compared to the WT (k cat 565 s(-1)), which was further increased in the double mutant, with a k cat of 798 s(-1). Structural analyses showed that the slight changes caused by point mutation of Asn41 to Asp may have reduced the number of hydrogen bonds in this α-helical part of the protein structure, resulting in subtle changes in enzyme turnover, both structurally and catalytically. The increased α-helical content observed with the N41D mutation by circular dichroism spectroscopy correlates with the difference in k cat, but not K m. The N281D mutation resulted in a lower glutaminase activity compared with WT and the N41D mutant, however the N281D mutation also imparted less stability to the enzyme at elevated temperatures. Taken as a whole, these data suggest that ErA deamidation at the Asn41 and Asn281 sites does not affect enzyme activity and should not be a concern during processing, storage or clinical use. The production of recombinant deamidated variants has proven an effective and powerful means of studying the effect of these changes and may be a useful strategy for other biopharmaceutical products.

Nitrogen Deposition Reduces Decomposition Rates Through Shifts in Microbial Community Composition and Function

NASA Astrophysics Data System (ADS)

Waldrop, M.; Zak, D.; Sinsabaugh, R.

2002-12-01

Atmospheric nitrogen (N) deposition may alter soil biological activity in northern hardwood forests by repressing phenol oxidase enzyme activity and altering microbial community composition, thereby slowing decomposition and increasing the export of phenolic compounds. We tested this hypothesis by adding 13C-labelled cellobiose, vanillin, and catechol to control and N fertilized soils (30 and 80 kg ha-1) collected from three forests; two dominated by Acer Saccharum and one dominated by Quercus Alba and Quercus Velutina. While N deposition increased total microbial respiration, it decreased soil oxidative enzyme activities, resulting in slower degradation rates of all compounds, and larger DOC pools. This effect was larger in the oak forest, where fungi dominate C-cycling processes. DNA and 13C-phospolipid analyses showed that N addition altered the fungal community and reduced the activity of fungal and bacterial populations in soil, potentially explaining reduced soil enzyme activities and incomplete decomposition.

Activity of trypsin-like enzymes and gelatinases in rats with doxorubicin cardiomyopathy.

PubMed

Gordiienko, Iu A; Babets, Ya V; Kulinich, A O; Shevtsova, A I; Ushakova, G O

2014-01-01

Activity of trypsin-like enzymes (ATLE) and gelatinases A and B were studied in the blood plasma and extracts from cardiac muscle, cerebral cortex and cerebellum of rats with cardiomyopathy caused by anthracycline antibiotic doxorubicin against the background of preventive application of corvitin and α-ketoglutarate. ATLE significantly increased in blood plasma and extracts from cerebral cortex but decreased in extracts from cardiac muscle and cerebellum in doxorubicin cardiomyopathy (DCMP). In addition, a significant increase of activity of both gelatinases in plasma and tissue extracts was observed. Preventive administration of corvitin and α-ketoglutarate resulted in differently directed changes of activity of the above mentioned enzymes in heart and brain tissues. Obtained data confirm the hypothesis about activation of proteolysis under the influence of anthracycline antibiotics and testify to selective effect of corvitin and α-ketoglutarate on ATLE and gelatinases.

Creation of catalytically active particles from enzymes crosslinked with a natural bifunctional agent--homocysteine thiolactone.

PubMed

Stroylova, Yulia Y; Semenyuk, Pavel I; Asriyantz, Regina A; Gaillard, Cedric; Haertlé, Thomas; Muronetz, Vladimir I

2014-09-01

The current study describes an approach to creation of catalytically active particles with increased stability from enzymes by N-homocysteinylation, a naturally presented protein modification. Enzymatic activities and properties of two globular tetrameric enzymes glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and lactate dehydrogenase (LDH) were studied before and after N-homocysteinylation. Modification of these proteins concerns the accessible lysine residues and introduces an average of 2-2,5 homocysteine residues per protein monomer. Formation of a range of aggregates was observed for both enzymes, which assemble via formation of intermolecular noncovalent bonds and by disulfide bonds. It was demonstrated that both studied enzymes retain their catalytic activities on modification and the subsequent formation of oligomeric forms. At low concentrations of homocysteine thiolactone, modification of GAPDH leads not only to prevention of spontaneous inactivation but also increases thermal stability of this enzyme on heating to 80°C. A moderate reduction of the activity of GAPDH observed in case of its crosslinking with 50-fold excess of homocysteine thiolactone per lysine is probably caused by hindered substrate diffusion. Spherical particles of 100 nm and larger diameters were observed by transmission electron microscopy and atomic force microscope techniques after modification of GAPDH with different homocysteine thiolactone concentrations. In case of LDH, branched fibril-like aggregates were observed under the same conditions. Interestingly, crosslinked samples of both proteins were found to have reversible thermal denaturation profiles, indicating that modification with homocysteine thiolactone stabilizes the spatial structure of these enzymes. © 2014 Wiley Periodicals, Inc.

Immobilization of alkaline phosphatase on solid surface through self-assembled monolayer and by active-site protection.

PubMed

Gao, En-Feng; Kang, Kyung Lhi; Kim, Jeong Hee

2014-06-01

Retaining biological activity of a protein after immobilization is an important issue and many studies reported to enhance the activity of proteins after immobilization. We recently developed a new immobilization method of enzyme using active-site protection and minimization of the cross-links between enzyme and surface with a DNA polymerase as a model system. In this study, we extended the new method to an enzyme with a small mono-substrate using alkaline phosphatase (AP) as another model system. A condition to apply the new method is that masking agents, in this case its own substrate needs to stay at the active-site of the enzyme to be immobilized in order to protect the active-site during the harsh immobilization process. This could be achieved by removal of essential divalent ion, Zn2+ that is required for full enzyme activity of AP from the masking solution while active-site of AP was protected with p-nitrophenyl phosphate (pNPP). Approximately 40% of the solution-phase activity was acquired with active-site protected immobilized AP. In addition to protection active-site of AP, the number of immobilization links was kinetically controlled. When the mole fraction of the activated carboxyl group of the linker molecule in self-assembled monolayer (SAM) of 12-mercaptododecanoic acid and 6-mercapto-1-ethanol was varied, 10% of 12-mercaptododecanoic acid gave the maximum enzyme activity. Approximately 51% increase in enzyme activity of the active-site protected AP was observed compared to that of the unprotected group. It was shown that the concept of active-site protection and kinetic control of the number of covalent immobilization bonds can be extended to enzymes with small mono-substrates. It opens the possibility of further extension of the new methods of active-site protection and kinetic control of immobilization bond to important enzymes used in research and industrial fields.

Physiological role of glucose-6-phosphate dehydrogenase in cold acclimation of strawberry (Fragaria × ananassa)

NASA Astrophysics Data System (ADS)

Zhang, Yong; Yu, Dingqun; Luo, Ya; Wang, Xiaorong; Chen, Qing; Sun, Bo; Wang, Yan; Liu, Zejing; Tang, Haoru

2018-04-01

In recent years, there has been an increasing interest in study of new resistance mechanism in fruit trees. All these regard the climate change and subsequent fruit production. Glucose-6-phosphate dehydrogenase (G6PDH) catalyzes the first and rate-limiting step of the oxidative pentose phosphate pathway (OPPP), and the expression of this enzyme is related to different biotic and abiotic stresses. Under accumulation of low temperature stress, the significant increase in G6PDH activity was found to be closely correlated to the levels of antioxidant enzymes, malondialdehyde (MDA) contents, sugar contents as well as changes of superoxide (O2•-). It is suggested that the enhancement of cold resistance of strawberry, which induced by cold acclimation, related to the significant increase in G6PDH activity. On one hand, G6PDH activates NADPH oxidase to generate reactive oxygen species (ROS); on the other hand, it may be involved in the activation of antioxidant enzymes, and accelerates many other important NADPH-dependent enzymatic reactions. Then further result in the elevation of membrane stability and cold resistance of strawberry. Interestingly, even though the plants were placed again under a temperature of 25°C for 1 d, the higher cold resistance, enzyme activities and soluble sugar content acquired.

[Role of vitamin E in regulation of cholecalciferol hydroxylation in hypovitaminosis D and hypervitaminosis D].

PubMed

Apukhovs'ka, L I; Velykyĭ, M M; Lotots'ka, O Iu; Khomenko, A V

2009-01-01

It is established, that dose-dependent influence of vitamin E on vitamin D3 metabolism, is conditioned by degree of cholecalciferol sufficiency. Under a condition of D-hypovitaminosis, contents of 25OHD3 in blood serum is 2-fold reduced and vitamin D3 25-hydroxylase enzymes activity increased in rat hepatocytes. Vitamin E (0.726-7.26 IU) significantly stimulated the effect of vitamin D3 (40 IU) in animals with D-hypovitaminosis and led to further increase of 25OHD3 content in the blood serum and activity of vitamin D3 25-hydroxylase enzymes in hepatocytes. In D-hypervitaminosis the contents of 25OHD3 in blood serum was more than 3-fold increased and vitamin D3 25-hydroxylase enzymes activity was inhibited. Vitamin E (0,726-7,26 IU) lowered the vitamin D toxicity, decreased contents of 25OHD3 in blood serum and activity of vitamin D3 25-hydroxylase enzymes in hepatocytes. High doses of vitamin E (36.3 IU) under these conditions demonstrated negative effect on vitamin D3 metabolism. The mechanism of vitaminE participation in the vitamin D3 metabolism under D-hypovitaminosis and D-hypervitaminosis may be its influence on the activity of different vitamin D3 25-hydroxylase systems of hepatocytes.

Effect of neohesperidin dihydrochalcone on the activity and stability of alpha-amylase: a comparative study on bacterial, fungal, and mammalian enzymes.

PubMed

Kashani-Amin, Elaheh; Ebrahim-Habibi, Azadeh; Larijani, Bagher; Moosavi-Movahedi, Ali Akbar

2015-10-01

Neohesperidin dihydrochalcone (NHDC) was recently introduced as an activator of mammalian alpha-amylase. In the current study, the effect of NHDC has been investigated on bacterial and fungal alpha-amylases. Enzyme assays and kinetic analysis demonstrated the capability of NHDC to significantly activate both tested alpha-amylases. The ligand activation pattern was found to be more similar between the fungal and mammalian enzyme in comparison with the bacterial one. Further, thermostability experiments indicated a stability increase in the presence of NHDC for the bacterial enzyme. In silico (docking) test locates a putative binding site for NHDC on alpha-amylase surface in domain B. This domain shows differences in various alpha-amylase types, and the different behavior of the ligand toward the studied enzymes may be attributed to this fact. Copyright © 2015 John Wiley & Sons, Ltd.

Consecutive emamectin benzoate and deltamethrin treatments affect the expressions and activities of detoxification enzymes in the rainbow trout (Oncorhynchus mykiss).

PubMed

Cárcamo, Juan Guillermo; Aguilar, Marcelo N; Carreño, Constanza F; Vera, Tamara; Arias-Darraz, Luis; Figueroa, Jaime E; Romero, Alex P; Alvarez, Marco; Yañez, Alejandro J

2017-01-01

Rainbow trout (Oncorhynchus mykiss) subjected to three consecutive, alternating treatments with emamectin benzoate (EMB) and deltamethrin (DM) during outbreaks of Caligus rogercresseyi in a farm located in southern Chile (Hornopiren, Chiloé), were studied to determine the effects of these treatments on the protein and enzymatic activity levels of cytochrome P450 1A (CYP1A), flavin-containing monooxygenase (FMO) and glutathione S-transferase (GST) in different tissues. Consecutive and alternating EMB/DM treatments resulted in a 10-fold increase and 3-fold decrease of CYP1A protein levels in the intestine and gills, respectively. Notably, CYP1A activity levels decreased in most of the analyzed tissues. FMO protein and activity levels markedly increased in the kidney and the intestine. GST was up-regulated in all tissues, either as protein or enzyme activity. When comparing consecutive EMB/DM treatments against previous studies of EMB treatment alone, CYP1A activity levels were similarly diminished, except in muscle. Likewise, FMO activity levels were increased in most of the analyzed tissues, particularly in the muscle, kidney, and intestine. The increases observed for GST were essentially unchanged between consecutive EMB/DM and EMB only treatments. These results indicate that consecutive EMB/DM treatments in rainbow trout induce the expression and activity of FMO and GST enzymes and decrease CYP1A activity. These altered activities of detoxification enzymes could generate imbalances in metabolic processes, synthesis, degradation of hormones and complications associated with drug interactions. It is especially important when analyzing possible effects of consecutive antiparasitic treatments on withholding periods and salmon farming yields. Copyright © 2016 Elsevier Inc. All rights reserved.

Lignin Peroxidase from Streptomyces viridosporus T7A: Enzyme Concentration Using Ultrafiltration

NASA Astrophysics Data System (ADS)

Gottschalk, Leda M. F.; Bon, Elba P. S.; Nobrega, Ronaldo

It is well known that lignin degradation is a key step in the natural process of biomass decay whereby oxidative enzymes such as laccases and high redox potential ligninolytic peroxidases and oxidases play a central role. More recently, the importance of these enzymes has increased because of their prospective industrial use for the degradation of the biomass lignin to increase the accessibility of the cellulose and hemicellulose moieties to be used as renewable material for the production of fuels and chemicals. These biocatalysts also present potential application on environmental biocatalysis for the degradation of xenobiotics and recalcitrant pollutants. However, the cost for these enzymes production, separation, and concentration must be low to permit its industrial use. This work studied the concentration of lignin peroxidase (LiP), produced by Streptomyces viridosporus T7A, by ultrafiltration, in a laboratory-stirred cell, loaded with polysulfone (PS) or cellulose acetate (CA) membranes with molecular weight cutoffs (MWCO) of 10, 20, and 50 KDa. Experiments were carried out at 25 °C and pH 7.0 in accordance to the enzyme stability profile. The best process conditions and enzyme yield were obtained using a PS membrane with 10 KDa MWCO, whereby it was observed a tenfold LiP activity increase, reaching 1,000 U/L and 90% enzyme activity upholding.

Enzyme-assisted supercritical carbon dioxide extraction of black pepper oleoresin for enhanced yield of piperine-rich extract.

PubMed

Dutta, Sayantani; Bhattacharjee, Paramita

2015-07-01

Black pepper (Piper nigrum L.), the King of Spices is the most popular spice globally and its active ingredient, piperine, is reportedly known for its therapeutic potency. In this work, enzyme-assisted supercritical carbon dioxide (SC-CO2) extraction of black pepper oleoresin was investigated using α-amylase (from Bacillus licheniformis) for enhanced yield of piperine-rich extract possessing good combination of phytochemical properties. Optimization of the extraction parameters (without enzyme), mainly temperature and pressure, was conducted in both batch and continuous modes and the optimized conditions that provided the maximum yield of piperine was in the batch mode, with a sample size of 20 g of black pepper powder (particle diameter 0.42 ± 0.02 mm) at 60 °C and 300 bar at 2 L/min of CO2 flow. Studies on activity of α-amylase were conducted under these optimized conditions in both batch and continuous modes, with varying amounts of lyophilized enzyme (2 mg, 5 mg and 10 mg) and time of exposure of the enzyme to SC-CO2 (2.25 h and 4.25 h). The specific activity of the enzyme increased by 2.13 times when treated in the continuous mode than in the batch mode (1.25 times increase). The structural changes of the treated enzymes were studied by (1)H NMR analyses. In case of α-amylase assisted extractions of black pepper, both batch and continuous modes significantly increased the yields and phytochemical properties of piperine-rich extracts; with higher increase in batch mode than in continuous. Copyright © 2014 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

[Oxygen consumption rate and effects of hypoxia stress on enzyme activities of Sepiella maindron].

PubMed

Wang, Chun-lin; Wu, Dan-hua; Dong, Tian-ye; Jiang, Xia-min

2008-11-01

The oxygen consumption rate and suffocation point of Sepiella maindroni were determined through the measurement of dissolved oxygen in control and experimental respiration chambers by Winkler's method, and the changes of S. maindroni enzyme activities under different levels of hypoxia stress were studied. The results indicated that the oxygen consumption rate of S. maindroni exhibited an obvious diurnal fluctuation of 'up-down-up-down', and positively correlated with water temperature (16 degrees C-28 degrees C) and illumination (3-500 micromol x m(-2) x s(-1)) while negatively correlated with water pH (6.25-9.25). With increasing water salinity from 18.1 to 29.8, the oxygen consumption rate had a variation of 'up-down-up', being the lowest at salinity 24. 8. Female S. maindroni had a higher oxygen consumption rate than male S. maindroni. The suffocation point of S. maindroni decreased with its increasing body mass, and that of (38.70 +/- 0.52) g in mass was (0.9427 +/- 0.0318) mg x L(-1). With the increase of hypoxia stress, the activities of superoxide dismutase (SOD), catalase (CAT) and peroxidase (POD) decreased after an initial increase, lipase activity decreased, protease activity had a variation of 'decrease-increase-decrease', and lactate dehydrogenase (LDH) activity had a trend of increasing first and decreasing then. The enzyme activities were higher under hypoxia stress than under normal conditions.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tatsumi, Y.; Chachin, K.; Ogata, K.

The purpose of the experiment was to determine the changes of o- diphenol, ascorbic acid, and activities of polyphenol oxidase and peroxidase, accompanied with the browning by the low dose of gamma ray, in three parts of tuber tissue (cortex, vascular bundle, and pith), and to observe the relation between the browning and the changes of ihose compounds and enzyme activities. The odiphenol content increased in irradiated tabers and the increasing rate was greater in cortex and vascular bundle than in pith. The ascorbic acid content decreased with higher doses, and the decreasing rate was greater in cortex and vascularmore » bundle than in pith. The activities of polyphenol oxidase and peroxidase also increased in irradiated tubers. The activity of polyphenol oxidase increased more in cortex than in vascular bundle and pith and the activity of peroxidase increased more in vascular bundle than in cortex and pith. ln the potato tubers in which irradiation was conducted immediately after harvest, the browning was induced within several days after irradiation. However, in the potato tubers in which irradiation was conducted about 3 months, the browning did not occur after irradiation. The former showed the increase of o-diphenol content and the activities of these enzymes, and the decrease of ascorbic acid content, the latter did not show the changes of o-diphenol and ascorbic acid and activities of the enzymes. (auth)« less

Altering lipase activity and enantioselectivity in organic media using organo-soluble bases: Implication for rate-limiting proton transfer in acylation step.

PubMed

Chen, Chun-Chi; Chen, Teh-Liang; Tsai, Shau-Wei

2006-06-05

With the hydrolytic resolution of (R,S)-naproxen 2,2,2-trifluoroethyl esters via a partially purified papaya lipase (PCPL) in water-saturated isooctane as the model system, the enzyme activity, and enantioselectivty is altered by adding a variety of organo-soluble bases that act as either enzyme activators (i.e., TEA, MP, TOA, DPA, PY, and DMA) or enzyme inhibitors (i.e., PDP, DMAP, and PP). Triethylamine (TEA) is selected as the best enzyme activator as 2.24-fold increase of the initial rate for the (S)-ester is obtained when adding 120 mM of the base. By using an expanded Michaelis-Menten mechanism for the acylation step, the kinetic analysis indicates that the proton transfer for the breakdown of tetrahedral intermediates to acyl-enzyme intermediates is the rate-limiting step, or more sensitive than that for the formation of tetrahedral intermediates when the enzyme activators of different pKa are added. However, no correlation for the proton transfers in the acylation step is found when adding the bases acting as enzyme deactivators. Copyright 2006 Wiley Periodicals, Inc.

In Situ Observation of Chymotrypsin Catalytic Activity Change Actuated by Nonheating Low-Frequency Magnetic Field.

PubMed

Efremova, Maria V; Veselov, Maxim M; Barulin, Alexander V; Gribanovsky, Sergey L; Le-Deygen, Irina M; Uporov, Igor V; Kudryashova, Elena V; Sokolsky-Papkov, Marina; Majouga, Alexander G; Golovin, Yuri I; Kabanov, Alexander V; Klyachko, Natalia L

2018-04-24

Magnetomechanical modulation of biochemical processes is a promising instrument for bioengineering and nanomedicine. This work demonstrates two approaches to control activity of an enzyme, α-chymotrypsin immobilized on the surface of gold-coated magnetite magnetic nanoparticles (GM-MNPs) using a nonheating low-frequency magnetic field (LF MF). The measurement of the enzyme reaction rate was carried out in situ during exposure to the magnetic field. The first approach involves α-chymotrypsin-GM-MNPs conjugates, in which the enzyme undergoes mechanical deformations with the reorientation of the MNPs under LF MF (16-410 Hz frequency, 88 mT flux density). Such mechanical deformations result in conformational changes in α-chymotrypsin structure, as confirmed by infrared spectroscopy and molecular modeling, and lead to a 63% decrease of enzyme initial activity. The second approach involves an α-chymotrypsin-GM-MNPs/trypsin inhibitor-GM-MNPs complex, in which the activity of the enzyme is partially inhibited. In this case the reorientation of MNPs in the field leads to disruption of the enzyme-inhibitor complex and an almost 2-fold increase of enzyme activity. The results further demonstrate the utility of magnetomechanical actuation at the nanoscale for the remote modulation of biochemical reactions.

Directed Evolution of a Thermostable Quorum-quenching Lactonase from the Amidohydrolase Superfamily*

PubMed Central

Chow, Jeng Yeong; Xue, Bo; Lee, Kang Hao; Tung, Alvin; Wu, Long; Robinson, Robert C.; Yew, Wen Shan

2010-01-01

A thermostable quorum-quenching lactonase from Geobacillus kaustophilus HTA426 (GI: 56420041) was used as an initial template for in vitro directed evolution experiments. This enzyme belongs to the phosphotriesterase-like lactonase (PLL) group of enzymes within the amidohydrolase superfamily that hydrolyze N-acylhomoserine lactones (AHLs) that are involved in virulence pathways of quorum-sensing pathogenic bacteria. Here we have determined the N-butyryl-l-homoserine lactone-liganded structure of the catalytically inactive D266N mutant of this enzyme to a resolution of 1.6 Å. Using a tunable, bioluminescence-based quorum-quenching molecular circuit, the catalytic efficiency was enhanced, and the AHL substrate range increased through two point mutations on the loops at the C-terminal ends of the third and seventh β-strands. This E101N/R230I mutant had an increased value of kcat/Km of 72-fold toward 3-oxo-N-dodecanoyl-l-homoserine lactone. The evolved mutant also exhibited lactonase activity toward N-butyryl-l-homoserine lactone, an AHL that was previously not hydrolyzed by the wild-type enzyme. Both the purified wild-type and mutant enzymes contain a mixture of zinc and iron and are colored purple and brown, respectively, at high concentrations. The origin of this coloration is suggested to be because of a charge transfer complex involving the β-cation and Tyr-99 within the enzyme active site. Modulation of the charge transfer complex alters the lactonase activity of the mutant enzymes and is reflected in enzyme coloration changes. We attribute the observed enhancement in catalytic reactivity of the evolved enzyme to favorable modulations of the active site architecture toward productive geometries required for chemical catalysis. PMID:20980257

Directed evolution of a thermostable quorum-quenching lactonase from the amidohydrolase superfamily.

PubMed

Chow, Jeng Yeong; Xue, Bo; Lee, Kang Hao; Tung, Alvin; Wu, Long; Robinson, Robert C; Yew, Wen Shan

2010-12-24

A thermostable quorum-quenching lactonase from Geobacillus kaustophilus HTA426 (GI: 56420041) was used as an initial template for in vitro directed evolution experiments. This enzyme belongs to the phosphotriesterase-like lactonase (PLL) group of enzymes within the amidohydrolase superfamily that hydrolyze N-acylhomoserine lactones (AHLs) that are involved in virulence pathways of quorum-sensing pathogenic bacteria. Here we have determined the N-butyryl-L-homoserine lactone-liganded structure of the catalytically inactive D266N mutant of this enzyme to a resolution of 1.6 Å. Using a tunable, bioluminescence-based quorum-quenching molecular circuit, the catalytic efficiency was enhanced, and the AHL substrate range increased through two point mutations on the loops at the C-terminal ends of the third and seventh β-strands. This E101N/R230I mutant had an increased value of k(cat)/K(m) of 72-fold toward 3-oxo-N-dodecanoyl-L-homoserine lactone. The evolved mutant also exhibited lactonase activity toward N-butyryl-L-homoserine lactone, an AHL that was previously not hydrolyzed by the wild-type enzyme. Both the purified wild-type and mutant enzymes contain a mixture of zinc and iron and are colored purple and brown, respectively, at high concentrations. The origin of this coloration is suggested to be because of a charge transfer complex involving the β-cation and Tyr-99 within the enzyme active site. Modulation of the charge transfer complex alters the lactonase activity of the mutant enzymes and is reflected in enzyme coloration changes. We attribute the observed enhancement in catalytic reactivity of the evolved enzyme to favorable modulations of the active site architecture toward productive geometries required for chemical catalysis.

The effect of exogenous calcium on mitochondria, respiratory metabolism enzymes and ion transport in cucumber roots under hypoxia

PubMed Central

He, Lizhong; Li, Bin; Lu, Xiaomin; Yuan, Lingyun; Yang, Yanjuan; Yuan, Yinghui; Du, Jing; Guo, Shirong

2015-01-01

Hypoxia induces plant stress, particularly in cucumber plants under hydroponic culture. In plants, calcium is involved in stress signal transmission and growth. The ultimate goal of this study was to shed light on the mechanisms underlying the effects of exogenous calcium on the mitochondrial antioxidant system, the activity of respiratory metabolism enzymes, and ion transport in cucumber (Cucumis sativus L. cv. Jinchun No. 2) roots under hypoxic conditions. Our experiments revealed that exogenous calcium reduces the level of reactive oxygen species (ROS) and increases the activity of antioxidant enzymes in mitochondria under hypoxia. Exogenous calcium also enhances the accumulation of enzymes involved in glycolysis and the tricarboxylic acid (TCA) cycle. We utilized fluorescence and ultrastructural cytochemistry methods to observe that exogenous calcium increases the concentrations of Ca2+ and K+ in root cells by increasing the activity of plasma membrane (PM) H+-ATPase and tonoplast H+-ATPase and H+-PPase. Overall, our results suggest that hypoxic stress has an immediate and substantial effect on roots. Exogenous calcium improves metabolism and ion transport in cucumber roots, thereby increasing hypoxia tolerance in cucumber. PMID:26304855

Energetic and Informative Interaction of Microwaves with Neurons and Enzymes

NASA Astrophysics Data System (ADS)

Maharramov, A. A.; Babazade, S. N.; Yusifov, E. Yu.; Gajiyev, A. M.

2007-04-01

Besides Purkinje Cells (PC) in cat cerebellum, experiments on Microwave-Living System interaction have been performed on some antioxidant enzymes - Super oxide dismutase (SOD), Catalase (C) and Glutathione peroxidase (GP) in the eye structures (pigment epithelium and neuronal structure - retina) in frogs, and on Glucose-6-Phosphatedehydrogenase (GPD) and Pyruvate-kinase (PK) in different organs - cerebellum, hypothalamus, liver and erythrocytes - of wistar albino rats. Exposure parameters of Microwaves of decimetre range (DRM) - total exposure, λ=65 cm, duration of exposition 10-20 minutes. According to the data obtained it may be concluded that, PC increasing their impulse activity irregularity, may react to the energetic (thermal) component of DRM action, whereas the result of informative (subtle) interaction between DRM and PC leads to the increase of regularity in electrophysiological activity of the latter. In the case of enzymes, in identification of the character of interactions, the type of the enzymes, the structure where an enzyme activity is studied and the physiological conditions related to such a factor as hunger, for example, take places. In this paper the effects of DRM on PC, G6PD and PK have been presented.

Nitrate Reductase Activity and Polyribosomal Content of Corn (Zea mays L.) Having Low Leaf Water Potentials 1

PubMed Central

Morilla, Camila A.; Boyer, J. S.; Hageman, R. H.

1973-01-01

Desiccation of 8- to 13-day-old seedlings, achieved by withholding nutrient solution from the vermiculite root medium, caused a reduction in nitrate reductase activity of the leaf tissue. Activity declined when leaf water potentials decreased below −2 bars and was 25% of the control at a leaf water potential of −13 bars. Experiments were conducted to determine whether the decrease in nitrate reductase activity was due to reduced levels of nitrate in the tissue, direct inactivation of the enzyme by low leaf water potentials, or to changes in rates of synthesis or decay of the enzyme. Although tissue nitrate content decreased with the onset of desiccation, it did not continue to decline with tissue desiccation and loss of enzyme activity. Nitrate reductase activity recovered when the plants were rewatered with nitrate-free medium, suggesting that the nitrate in the plant was adequate for high nitrate reductase activity. The rate of decay of nitrate reductase activity from desiccated tissue was essentially identical to that of the control, in vivo or in vitro, regardless of the rapidity of desiccation of the tissue. Direct inactivation of the enzyme by the low water potentials was not detected. Polyribosomal content of the tissue declined with the decrease in water potential, prior to the decline in nitrate reductase activity. Changes in ribosomal profiles occurred during desiccation, regardless of whether the tissue had been excised or not and whether desiccation was rapid or slow. Reduction in polyribosomal content did not appear to be associated with changes in ribonuclease activity. Nitrate reductase activity and the polyribosomal content of the tissue recovered upon rewatering, following the recovery in water potential. The increase in polyribosomal content preceded the increase in nitrate reductase activity. Recovery of enzyme activity was prevented by cycloheximide. Based on these results, it appears that nitrate reductase activity was affected primarily by a decrease in the rate of enzyme synthesis at low leaf water potentials. PMID:16658419

[The influence of nitrates on platelet oxygen metabolism: in vitro studies].

PubMed

Buczyński, A; Dziedziczak-Buczyńska, M; Gnitecki, W; Kocur, J

1999-01-01

Our investigations were carried out on human blood platelets obtained from persons aged 20-23, free from any systemic diseases. Drugs were incubated with blood platelets. Changes of antioxidant enzymes were detected. Glyceryl trinitrate increased the activity of Zn Cu-SOD (4.62%) and GPx (275.91%), concentration of ATP (13.01%) and the blood platelets aggregations (17.88%). Izosorbide dinitrate increased the activity of ZnCu-SOD (19.46%), GPx (150.36%) and Cat (15.62%), increased concentration of ATP (23.73%) and blood platelets aggregation (3.64%). Both preparats decreased concentration of MDA (Sustonit--30.79%, Iso-Mack--35.04%). Gliceryl trinitrate decreased the activity of catalase otherwise izosorbide dinitrate increased the activity of this enzyme.

Influence of Tribulus terrestris on testicular enzyme in fresh water ornamental fish Poecilia latipinna.

PubMed

Kavitha, P; Subramanian, P

2011-12-01

The influence of Tribulus terrestris on the activities of testicular enzyme in Poecilia latipinna was assessed in lieu of reproductive manipulation. Different concentrations of (100, 150, 200, 250, and 300 mg) Tribulus terrestris extract and of a control were tested for testicular activity of enzymes in Poecilia latipinna for 2 months. The testis and liver were homogenized separately in 0.1 mol/l potassium phosphate buffer (0.1 mol/l, pH 7.2). The crude homogenate was centrifuged, and supernatant obtained was used as an enzyme extract for determination of activities. The activities of testicular functional enzyme ALP, ACP, SDH, LDH, and G6PDH levels were changed to different extent in treated groups compared with that of the control. The total body weight and testis weight were increased with the Tribulus terrestris-treated fish (Poecilia latipinna). These results suggest that Tribulus terrestris induced the testicular enzyme activity that may aid in the male reproductive functions. It is discernible from the present study that Tribulus terrestris has the inducing effect on reproductive system of Poecilia latipinna.

Purification and properties of rennin-like enzyme from Aspergillus ochraceus.

PubMed

Ismail, A A; Foda, M S; Khorshid, M A

1978-01-01

An active milk-clotting enzyme was purified some 40-fold from culture supernatant of Aspergillus ochraceus. The purification steps included ammonium sulfate precipitation, G-100 Sephadex gel filtration, and ion exchange chromatography, using DEAE Cellulose column. The enzyme exhibited milk-clotting activity and proteolytic behaviour, an optimum at pH 6.0 and in the range of 7--8.5, respectively. The purified enzyme was actively proteolytic against casein, haemoglobin, and bovine serum albumin at pH 8. The milk-clotting activity was greatly enhanced by manganous ions and by increasing concentrations of calcium chloride. Copper, zinc, and ammonium ions were potent inhibitors of the milk-curdling activity of the purified enzyme. Significant inhibition was also noted with sodium chloride at concentrations of 3% or more. Under the specified reaction condition, maximum rate of proteolysis against casein was obtained at 0.4% substrate concentration, whereas the milk-clotting time was linear proportional to dry skim milk concentration in the range of 8 to 24%. The results are discussed in comparison with other microbial milk-clotting enzymes, and limitations of applicability are also presented.

Biocatalysis with thermostable enzymes: structure and properties of a thermophilic 'ene'-reductase related to old yellow enzyme.

PubMed

Adalbjörnsson, Björn V; Toogood, Helen S; Fryszkowska, Anna; Pudney, Christopher R; Jowitt, Thomas A; Leys, David; Scrutton, Nigel S

2010-01-25

We report the crystal structure of a thermophilic "ene" reductase (TOYE) isolated from Thermoanaerobacter pseudethanolicus E39. The crystal structure reveals a tetrameric enzyme and an active site that is relatively large compared to most other structurally determined and related Old Yellow Enzymes. The enzyme adopts higher order oligomeric states (octamers and dodecamers) in solution, as revealed by sedimentation velocity and multiangle laser light scattering. Bead modelling indicates that the solution structure is consistent with the basic tetrameric structure observed in crystallographic studies and electron microscopy. TOYE is stable at high temperatures (T(m)>70 degrees C) and shows increased resistance to denaturation in water-miscible organic solvents compared to the mesophilic Old Yellow Enzyme family member, pentaerythritol tetranitrate reductase. TOYE has typical ene-reductase properties of the Old Yellow Enzyme family. There is currently major interest in using Old Yellow Enzyme family members in the preparative biocatalysis of a number of activated alkenes. The increased stability of TOYE in organic solvents is advantageous for biotransformations in which water-miscible organic solvents and biphasic reaction conditions are required to both deliver novel substrates and minimize product racemisation.

S-allylcysteine ameliorates isoproterenol-induced cardiac toxicity in rats by stabilizing cardiac mitochondrial and lysosomal enzymes.

PubMed

Padmanabhan, M; Mainzen Prince, P Stanely

2007-02-13

This study was aimed to evaluate the preventive role of S-allylcysteine (SAC) on mitochondrial and lysosomal enzymes in isoproterenol (ISO)-induced rats. Male albino Wistar rats were pretreated with SAC (50, 100 and 150 mg/kg) daily for a period of 45 days. After the treatment period, ISO (150 mg/kg) was subcutaneously injected to rats at an interval of 24 h for two days. The activities of heart mitochondrial enzymes (isocitrate dehydrogenase, succinate dehydrogenase, malate dehydrogenase and alpha-ketoglutarate dehydrogenase) and respiratory chain enzymes (NADH dehydrogenase and cytochrome C oxidase) were decreased significantly (p<0.05) in ISO-induced rats. The activities of lysosomal enzymes (beta-glucuronidase, beta-N-acetyl glucosaminidase, beta-galactosidase, cathepsin-D and acid phosphatase) were increased significantly (p<0.05) in serum and heart of ISO-induced rats. Pretreatment with SAC (100 mg/kg and 150 mg/kg) for a period of 45 days increased significantly (p<0.05) the activities of mitochondrial and respiratory chain enzymes and decreased the activities of lysosomal enzymes significantly (p<0.05) in ISO-induced rats. Oral administration of SAC (50, 100 and 150 mg/kg) for a period of 45 days to normal rats did not show any significant (p<0.05) effect in all the parameters studied. The altered electrocardiogram (ECG) of ISO-treated rats was also restored to near normal by treatment with SAC (100 and 150 mg/kg). These results confirm the efficacy of SAC in alleviating ISO-induced cardiac damage.

The effectiveness of dietary sunflower meal and exogenous enzyme on growth, digestive enzymes, carcass traits, and blood chemistry of broilers.

PubMed

Alagawany, Mahmoud; Attia, Adel I; Ibrahim, Zenat A; Mahmoud, Reda A; El-Sayed, Sabry A

2017-05-01

High costs of conventional protein feed sources including soybean meal (SBM) generated the need for finding other alternatives. Thus, the present study was designed to evaluate the impact of graded replacements of SBM by sunflower seed meal (SFM) with or without enzyme supplementation on growth performance, digestive enzymes, carcass traits, and blood profile of broiler chickens. A total of 240 unsexed 1-week-old broiler chicks (Hubbard) were randomly divided into eight treatment groups of 30 chicks each in five replicates each of six chicks in a factorial design (4 × 2) arrangement, including four levels of SFM (0, 25, 50, and 75% replacing SBM) and two levels of enzyme (0- or 0.1-g/kg diet) supplementation. Performance traits including feed conversion ratio, body weight, and weight gain were significantly (P < 0.01) improved with increasing SFM up to 50% substitution for SBM or with enzyme supplementation in broiler diet during the experiment. However, feed intake of broiler chicks was decreased with enzyme supplementation (P < 0.05). The activities of digestive enzymes (protease and amylase) were significantly (P < 0.05) influenced and enhanced by SFM and enzyme inclusion in diets, respectively. The activities of protease and amylase were improved with SFM diet supplemented with 0.1 g/kg enzyme in comparison with those with the un-supplemented diet. The evaluated carcass traits were not statistically (P > 0.05) influenced by feeding SFM meal or enzyme addition. Biochemical blood parameters were significantly (P < 0.01) affected by SFM, enzyme, or their interaction in broiler diets, except for globulin that was not affected by dietary enzyme. It is concluded that increasing SFM level in the diet up to 50% replacing SBM with the supplementation of enzyme improved the growth performance and enhanced positively carcass traits as well as the activity of digestive enzymes in broiler chickens.

Partial purification of saccharifying and cell wall-hydrolyzing enzymes from malt in waste from beer fermentation broth.

PubMed

Khattak, Waleed Ahmad; Kang, Minkyung; Ul-Islam, Mazhar; Park, Joong Kon

2013-06-01

A number of hydrolyzing enzymes that are secreted from malt during brewing, including cell wall-hydrolyzing, saccharide-hydrolyzing, protein-degrading, lipid-hydrolyzing, and polyphenol and thiol-hydrolyzing enzymes, are expected to exist in an active form in waste from beer fermentation broth (WBFB). In this study, the existence of these enzymes was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis, after which enzyme extract was partially purified through a series of purification steps. The hydrolyzing enzyme activity was then measured under various conditions at each purification step using carboxymethyl cellulose as a substrate. The best hydrolyzing activities of partially purified enzymes were found at pH 4.5 and 50 °C in a citrate buffer system. The enzymes showed highest thermal stability at 30 °C when exposed for prolonged time. As the temperature increased gradually from 25 to 70 °C, yeast cells in the chemically defined medium with enzyme extract lost their cell wall and viability earlier than those without enzyme extract. Cell wall degradation and the release of cell matrix into the culture media at elevated temperature (45-70 °C) in the presence of enzyme extract were monitored through microscopic pictures. Saccharification enzymes from malt were relatively more active in the original WBFB than supernatant and diluted sediments. The presence of hydrolyzing enzymes from malt in WBFB is expected to play a role in bioethanol production using simultaneous saccharification and fermentation without the need for additional enzymes, nutrients, or microbial cells via a cell-free enzyme system.

Effects of interferon-gamma and tumor necrosis factor-alpha on macrophage enzyme levels

NASA Technical Reports Server (NTRS)

Pierangeli, Silvia S.; Sonnenfeld, Gerald

1989-01-01

Murine peritoneal macrophages were treated with interferon-gamma (IFN-gamma) or tumor necrosis factor-alpha (TNF). Measurements of changes in acid phosphatase and beta-glucuronidase levels were made as an indication of activation by cytokine treatment. IFN-gamma or TNF-gamma treatment resulted in a significant increase in the activities of both enzymes measured in the cell lysates. This increase was observable after 6 h of incubation, but reached its maximum level after 24 h of incubation. The effect of the treatment of the cell with both cytokines together was additive. No synergistic effect of addition of both cytokines on the enzyme levels was observed.

Nanotechnology Enabled Enhancement of Enzyme Activity and Thermostability: Study on Impaired Pectate Lyase from Attenuated Macrophomina phaseolina in Presence of Hydroxyapatite Nanoparticle

PubMed Central

Dutta, Nalok; Mukhopadhyay, Arka; Dasgupta, Anjan Kr.; Chakrabarti, Krishanu

2013-01-01

In this paper we show that hydroxyapatite nanoparticles (NP) can not only act as a chaperon (by imparting thermostability) but can serve as a synthetic enhancer of activity of an isolated extracellular pectate lyase (APL) with low native state activity. The purified enzyme (an attenuated strain of Macrophomina phaseolina) showed feeble activity at 50°C and pH 5.6. However, on addition of 10.5 µg/ml of hydroxyapatite nanoparticles (NP), APL activity increased 27.7 fold with a 51 fold increase in half-life at a temperature of 90°C as compared to untreated APL. The chaperon like activity of NP was evident from entropy–enthalpy compensation profile of APL. The upper critical temperature for such compensation was elevated from 50°C to 90°C in presence of NP. This dual role of NP in enhancing activity and conferring thermostability to a functionally impaired enzyme is reported for the first time. PMID:23691068

Scaffolding Function of PI3Kgamma Emerges from Enzyme's Shadow.

PubMed

Mohan, Maradumane L; Naga Prasad, Sathyamangla V

2017-03-24

Traditionally, an enzyme is a protein that mediates biochemical action by binding to the substrate and by catalyzing the reaction that translates external cues into biological responses. Sequential dissemination of information from one enzyme to another facilitates signal transduction in biological systems providing for feed-forward and feed-back mechanisms. Given this viewpoint, an enzyme without its catalytic activity is generally considered to be an inert organizational protein without catalytic function and has classically been termed as pseudo-enzymes. However, pseudo-enzymes still have biological function albeit non-enzymatic like serving as a chaperone protein or an interactive platform between proteins. In this regard, majority of the studies have focused solely on the catalytic role of enzymes in biological function, overlooking the potentially critical non-enzymatic roles. Increasing evidence from recent studies implicate that the scaffolding function of enzymes could be as important in signal transduction as its catalytic activity, which is an antithesis to the definition of enzymes. Recognition of non-enzymatic functions could be critical, as these unappreciated roles may hold clues to the ineffectiveness of kinase inhibitors in pathology, which is characteristically associated with increased enzyme expression. Using an established enzyme phosphoinositide 3-kinase γ, we discuss the insights obtained from the scaffolding function and how this non-canonical role could contribute to/alter the outcomes in pathology like cancer and heart failure. Also, we hope that with this review, we provide a forum and a starting point to discuss the idea that catalytic function alone may not account for all the actions observed with increased expression of the enzyme. Copyright © 2017 Elsevier Ltd. All rights reserved.

Purification and substrate specificity of Staphylococcus hyicus lipase.

PubMed

van Oort, M G; Deveer, A M; Dijkman, R; Tjeenk, M L; Verheij, H M; de Haas, G H; Wenzig, E; Götz, F

1989-11-28

The Staphylococcus hyicus lipase gene has been cloned and expressed in Staphylococcus carnosus. From the latter organism the enzyme was secreted into the medium as a protein with an apparent molecular mass of 86 kDa. This protein was purified, and the amino-terminal sequence showed that the primary gene product was indeed cleaved at the proposed signal peptide cleavage site. The protein was purified from large-scale preparations after tryptic digestion. This limited proteolysis reduced the molecular mass to 46 kDa and increased the specific activity about 3-fold. Although the enzyme had a low specific activity in the absence of divalent cations, the activity increased about 40-fold in the presence of Sr2+ or Ca2+ ions. The purified lipase has a broad substrate specificity. The acyl chains were removed from the primary and secondary positions of natural neutral glycerides and from a variety of synthetic glyceride analogues. Thus triglycerides were fully hydrolyzed to free fatty acid and glycerol. The enzyme hydrolyzed naturally occurring phosphatidylcholines, their synthetic short-chain analogues, and lysophospholipids to free fatty acids and water-soluble products. The enzyme had a 2-fold higher activity on micelles of short-chain D-lecithins than on micelles composed of the L-isomers. Thus the enzyme from S. hyicus has lipase activity and also high phospholipase A and lysophospholipase activity.

The Activity of Cholinesterases in Diapausing and Flying Red Mason Bees Osmia bicornis (Megachilidae).

PubMed

Dmochowska-Slezak, Kamila; Zaobidna, Ewa; Domeracka, Joanna; Swiatkowska, Marta; Rusznica, Małgorzata; Zółtowska, Krystyna

2015-01-01

The red mason bee (Osmia bicornis) is a highly effective pollinator that is exposed to various xenobiotics. The organism's potential resistance to the toxic effects of xenobiotics can be determined based on cholinesterase activity. The activity of cholinesterases (ChEs) towards acetylcholine (ACh) and butyrylcholine (BCh) was determined in extracts of diapausing (between October and late March) and flying bees (May). In both males and females, enzyme activity was higher towards ACh than towards BCh. The ratio of ACh/BCh activity was determined in the range of 1.43 to 4.15 in diapausing females and 3.00 to 7.18 in diapausing males. No significant changes in ChE activity towards ACh were observed in females before December and in males before February. Enzyme activity towards ACh increased dynamically in the second half of March. Enzyme activity towards BCh remained stable in both sexes until mid-March, after which it increased significantly. Excluding mid-March, enzyme BCh activity was significantly higher in females than in males. The activity of carboxylesterase towards 4-p-nitrophenyl butyrate was determined in females to assess the involvement of non-specific esterases in the hydrolysis of choline esters. Carboxylesterase activity was low in comparison with cholinesterase activity, and it remained practically unchanged throughout diapause, suggesting that choline esters in female O. bicornis extracts were hydrolyzed mainly by acetylcholinesterases.

Studies of UMP synthase in orotic aciduria fibroblasts

DOE Office of Scientific and Technical Information (OSTI.GOV)

Perry, M.E.; Jones, M.E.

UMP synthase catalyzes the final two reactions of de novo pyrimidine biosynthesis in mammals. UMP synthase activities are low in fibroblasts from a patient with hereditary orotic aciduria, but increase 80-100 fold to normal levels when the cells are incubated in the presence of 6-azauridine (6-azaU). Normal fibroblasts exhibit at most a two-fold increase in UMP synthase activities in response to 6-azaU. The increase in mutant cell enzyme activity is accompanied by increased UMP synthase protein in immunoprecipitates from (/sup 3//sub 5/S)-methionine-labeled cell extracts. This 6-azaU-dependent protein is precipitated by several monoclonal antibodies and polyclonal antibody raised against pure humanmore » UMP synthase. UMP synthase from normal and mutant fibroblasts comigrate on SDS gels and are stable for at least 2 1/2 hrs at 37/sup 0/C in the presence of a substrate, OMP. However, in the absence of substrate, at 57/sup 0/C, they have different inactivation patterns. Stability of the enzyme derived from normal cells > that of the enzyme from mutant cells cultured with 6-azaU > that of the enzyme from mutant cells. Southern blots of DNA from normal and mutant cells show identical restriction patterns with five enzymes. These results are consistent with the theory that the low level of UMP synthase in mutant cells reflects an increased susceptibility to proteolytic degradation which can be blocked by administration of 6-azaU to the cells in culture.« less

Effect of growth substrate, method of fermentation, and nitrogen source on lignocellulose-degrading enzymes production by white-rot basidiomycetes.

PubMed

Elisashvili, Vladimir; Kachlishvili, Eva; Penninckx, Michel

2008-11-01

The exploration of seven physiologically different white rot fungi potential to produce cellulase, xylanase, laccase, and manganese peroxidase (MnP) showed that the enzyme yield and their ratio in enzyme preparations significantly depends on the fungus species, lignocellulosic growth substrate, and cultivation method. The fruit residues were appropriate growth substrates for the production of hydrolytic enzymes and laccase. The highest endoglucanase (111 U ml(-1)) and xylanase (135 U ml(-1)) activities were revealed in submerged fermentation (SF) of banana peels by Pycnoporus coccineus. In the same cultivation conditions Cerrena maxima accumulated the highest level of laccase activity (7,620 U l(-1)). The lignified materials (wheat straw and tree leaves) appeared to be appropriate for the MnP secretion by majority basidiomycetes. With few exceptions, SF favored to hydrolases and laccase production by fungi tested whereas SSF was appropriate for the MnP accumulation. Thus, the Coriolopsis polyzona hydrolases activity increased more than threefold, while laccase yield increased 15-fold when tree leaves were undergone to SF instead SSF. The supplementation of nitrogen to the control medium seemed to have a negative effect on all enzyme production in SSF of wheat straw and tree leaves by Pleurotus ostreatus. In SF peptone and ammonium containing salts significantly increased C. polyzona and Trametes versicolor hydrolases and laccase yields. However, in most cases the supplementation of media with additional nitrogen lowered the fungi specific enzyme activities. Especially strong repression of T. versicolor MnP production was revealed.

Effect of chlorpyrifos and enrofloxacin on selected enzymes in rats.

PubMed

Barski, D; Spodniewska, A

2018-03-01

This study examined the effect of chlorpyrifos and/or enrofloxacin on the activity of acetylcholinesterase (AChE) in the blood and brain, and the activity of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in serum. The experiment was conducted on Wistar strain rats. Chlorpyrifos was administered with a stomach tube at a dose of 0.04 LD50 for 28 days and enrofloxacin at a dose of 5 mg/kg bw for 5 consecutive days. The experiment found that enrofloxacin changed the activity of the enzymes under study only to a small extent. At the dose applied in the experiment, chlorpyrifos decreased the activity of AChE significantly, both in blood and in the brain, and increased the activity of ALT and AST in rat serum. The administration of chlorpyrifos in combination with enrofloxacin changed the activity of the enzymes under study only slightly. A weaker, but longer, inhibition of AChE activity in both blood and the brain was observed in this group compared to the animals exposed only to chlorpyrifos. However, although enrofloxacin, like chlorpyrifos, increases the activity of ALT and AST in serum, their combined administration did not increase the hepatotoxic effect. Copyright© by the Polish Academy of Sciences.

Enhanced hepatic and kidney cytochrome p-450 activities in nandrolone decanoate treated albino mice.

PubMed

Acharjee, B K; Mahanta, R

2009-04-01

Anabolic androgenic steroids are the xenobiotic substrates that are metabolized in the body by the protective enzyme systems. Mixed function oxygenase enzymes include a group of enzymes which play an essential role in the metabolism of a broad range of xenobiotics including endogenous and exogenous substrates. Cytochrome P-450, a member of mixed function oxygenase enzymes, plays an important role in oxidative metabolism of drugs and xenobiotics entering human body. Various anabolic steroids are found either to increase or decrease the activity of cytochrome P-450. However, effect of nandrolone decanoate, most commonly abused anabolic steroid, on cytochrome P-450 activity is still fragmentary. In the present study, albino mice were administered intramuscular 2.5 mg of nandrolone decanoate injection at 15 days interval. Cytochrome P-450 activity is determined by following the method of Omura and Sato (1964) in liver and kidney tissues of both normal and experimental groups upto 90 days. Investigation shows a significant (p <0.01) increase of cytochrome P-450 (nmol/mg) activity in liver tissue as compared to that of kidney tissues. A tissue specific and dose specific increase of cytochrome P-450 activity is observed. Mean cytochrome P-450 is found highest in liver tissue on 45(th) day whereas the activity in kidney tissue is noticed on 90(th) day of treatment. From the above observation, nandrolone decanoate can be suggested as a potent inducer of cytochrome P-450 activity like other anabolic steroids.

Immobilization of polygalacturonase from Aspergillus niger onto activated polyethylene and its application in apple juice clarification.

PubMed

Saxena, Shivalika; Shukla, Surendra; Thakur, Akhilesh; Gupta, Reena

2008-03-01

The present work is focused on efficient immobilization of polygalacturonase on polyethylene matrix, followed by its application in apple juice clarification. Immobilization of polygalacturonase on activated polyethylene and its use in apple juice clarification was not reported so far. Aspergillus niger Van Tieghem (MTCC 3323) produced polygalacturonase when grown in modified Riviere's medium containing pectin as single carbon source by fed-batch culture. The enzyme was precipitated with ethanol and purified by gel filtration chromatography (Sephacryl S-100) and immobilized onto glutaraldehyde-activated polyethylene. The method is very simple and time saving for enzyme immobilization. Various characteristics of immobilized enzyme such as optimum reaction temperature and pH, temperature and pH stability, binding kinetics, efficiency of binding, reusability and metal ion effect on immobilized enzymes were evaluated in comparison to the free enzyme. Both the free and immobilized enzyme showed maximum activity at a temperature of 45 degrees C and pH 4.8. Maximum binding efficiency was 38%. The immobilized enzyme was reusable for 3 cycles with 50% loss of activity after the third cycle. Twenty-four U of immobilized enzyme at 45 degrees C and 1 h incubation time increased the transmittance of the apple juice by about 55% at 650 nm. The immobilized enzyme can be of industrial advantage in terms of sturdiness, availability, inertness, low price, reusability and temperature stability.

Elevation of Glucose 6-Phosphate Dehydrogenase Activity Induced by Amplified Insulin Response in Low Glutathione Levels in Rat Liver

PubMed Central

Taniguchi, Misako; Mori, Nobuko; Iramina, Chizuru

2016-01-01

Weanling male Wistar rats were fed on a 10% soybean protein isolate (SPI) diet for 3 weeks with or without supplementing 0.3% sulfur-containing amino acids (SAA; methionine or cystine) to examine relationship between glutathione (GSH) levels and activities of NADPH-producing enzymes, glucose 6-phosphate dehydrogenase (G6PD) and malic enzyme (ME), in the liver. Of rats on the 10% SPI diet, GSH levels were lower and the enzyme activities were higher than of those fed on an SAA-supplemented diet. Despite the lower GSH level, γ-glutamylcysteine synthetase (γ-GCS) activity was higher in the 10% SPI group than other groups. Examination of mRNAs of G6PD and ME suggested that the GSH-suppressing effect on enzyme induction occurred prior to and/or at transcriptional levels. Gel electrophoresis of G6PD indicated that low GSH status caused a decrease in reduced form and an increase in oxidized form of the enzyme, suggesting an accelerated turnover rate of the enzyme. In primary cultured hepatocytes, insulin response to induce G6PD activity was augmented in low GSH levels manipulated in the presence of buthionine sulfoximine. These findings indicated that elevation of the G6PD activity in low GSH levels was caused by amplified insulin response for expression of the enzyme and accelerated turnover rate of the enzyme molecule. PMID:27597985

Hydroxycinnamic acid decarboxylase activity of Brettanomyces bruxellensis involved in volatile phenol production: relationship with cell viability.

PubMed

Laforgue, R; Lonvaud-Funel, A

2012-12-01

Brettanomyces bruxellensis populations have been correlated with an increase in phenolic off-flavors in wine. The volatile phenols causing the olfactory defect result from the successive decarboxylation and reduction of hydroxycinnamic acids that are normal components of red wines. The growth of B. bruxellensis is preventable by adding sulfur dioxide (SO(2)), with variable effectiveness. Moreover, it was hypothesized that SO(2) was responsible for the entry of B. bruxellensis into a viable but non-culturable (VBNC) state. The aim of this project was to investigate the effects of SO(2) on the remaining enzyme activities of B. bruxellensis populations according to their viability and cultivability, focusing on the hydroxycinnamate decarboxylase enzyme, the first enzyme needed, rather than the metabolites produced. Enzyme activity was determined both in cell-free extracts and resting cells after various SO(2) treatments in synthetic media. After slight sulfiting (around 50 mg/L total SO(2)), the yeasts had lost part of their enzyme activity but not their cultivability. At higher doses (at least 75 mg/L total SO(2)) the majority of yeasts had lost their cultivability but still retained part of their enzyme activity. These results suggested that non culturable cells retained some enzyme activity. Copyright © 2012 Elsevier Ltd. All rights reserved.

Mounting evidence validates Ursolic Acid directly activates SIRT1: A powerful STAC which mimic endogenous activator of SIRT1.

PubMed

Bakhtiari, Nuredin; Mirzaie, Sako; Hemmati, Roohullah; Moslemee-Jalalvand, Elham; Noori, Ali Reza; Kazemi, Jahanfard

2018-07-15

Ursolic Acid (UA), a pentacyclic triterpenoid compound, plays a vital role in aging process. However, the role of UA in the regulation of aging and longevity is still controversial as we have previously demonstrated that UA increases SIRT1 protein level in aged-mice. Here, we reveal that UA directly activates SIRT1 in silico, in vitro and in vivo. We have identified that UA binds to outer surface of SIRT1 and leads to tight binding of substrates to enzyme in comparison with Resveratrol (RSV) and control. Furthermore, our results indicate that UA drives the structure of SIRT1 toward a closed state (an active form of enzyme). Interestingly, our experimental findings are in agreement with the molecular dynamic results. Based on our data, UA increases the affinity of enzyme for both substrates with decreasing Km value, while enhances the Vmax of enzyme. Additionally, we have determined that UA heightened SIRT1 catalytic efficiency by 2 folds compared with RSV. Thereby, to identify the endogenous activator of SIRT1, UA was administrated to aged-mice and then the tissues were isolated. According to our results, it can be concluded that UA increases SIRT1 activity and mimics Lamin A and AROS behavior in the living cells. Copyright © 2018 Elsevier Inc. All rights reserved.

Modelling the Effects of Ageing Time of Starch on the Enzymatic Activity of Three Amylolytic Enzymes

PubMed Central

Guerra, Nelson P.; Pastrana Castro, Lorenzo

2012-01-01

The effect of increasing ageing time (t) of starch on the activity of three amylolytic enzymes (Termamyl, San Super, and BAN) was investigated. Although all the enzymatic reactions follow michaelian kinetics, v max decreased significantly (P < 0.05) and K M increased (although not always significantly) with the increase in t. The conformational changes produced in the starch chains as a consequence of the ageing seemed to affect negatively the diffusivity of the starch to the active site of the enzymes and the release of the reaction products to the medium. A similar effect was observed when the enzymatic reactions were carried out with unaged starches supplemented with different concentrations of gelatine [G]. The inhibition in the amylolytic activities was best mathematically described by using three modified forms of the Michaelis-Menten model, which included a term to consider, respectively, the linear, exponential, and hyperbolic inhibitory effects of t and [G]. PMID:22666116

INHIBITION OF THE DEVELOPMENT OF HEPATIC MICROSOMAL DETOXIFICATION ENZYMES BY X-IRRADIATION.

DTIC Science & Technology

of young, male rats, on the activity of these enzymes in the livers of adult animals, and on induced enzyme synthesis by phenobarbital . Exposure of 23...caused by phenobarbital administration. The results of these studies indicate that radiation specifically inhibits the synthesis of increased microsomal

K+ Stimulation of ATPase Activity Associated with the Chloroplast Inner Envelope 1

PubMed Central

Wu, Weihua; Berkowitz, Gerald A.

1992-01-01

Studies were conducted to characterize ATPase activity associated with purified chloroplast inner envelope preparations from spinach (Spinacea oleracea L.) plants. Comparison of free Mg2+ and Mg·ATP complex effects on ATPase activity revealed that any Mg2+ stimulation of activity was likely a function of the use of the Mg·ATP complex as a substrate by the enzyme; free Mg2+ may be inhibitory. In contrast, a marked (one- to twofold) stimulation of ATPase activity was noted in the presence of K+. This stimulation had a pH optimum of approximately pH 8.0, the same pH optimum found for enzyme activity in the absence of K+. K+ stimulation of enzyme activity did not follow simple Michaelis-Menton kinetics. Rather, K+ effects were consistent with a negative cooperativity-type binding of the cation to the enzyme, with the Km increasing at increasing substrate. Of the total ATPase activity associated with the chloroplast inner envelope, the K+-stimulated component was most sensitive to the inhibitors oligomycin and vanadate. It was concluded that K+ effects on this chloroplast envelope ATPase were similar to this cation's effects on other transport ATPases (such as the plasmalemma H+-ATPase). Such ATPases are thought to be indirectly involved in active K+ uptake, which can be facilitated by ATPase-dependent generation of an electrical driving force. Thus, K+ effects on the chloroplast enzyme in vitro were found to be consistent with the hypothesized role of this envelope ATPase in facilitating active cation transport in vivo. ImagesFigure 3 PMID:16668922

Activating Intrinsic Carbohydrate-Active Enzymes of the Smut Fungus Ustilago maydis for the Degradation of Plant Cell Wall Components

PubMed Central

Geiser, Elena; Reindl, Michèle; Blank, Lars M.; Feldbrügge, Michael

2016-01-01

ABSTRACT The microbial conversion of plant biomass to valuable products in a consolidated bioprocess could greatly increase the ecologic and economic impact of a biorefinery. Current strategies for hydrolyzing plant material mostly rely on the external application of carbohydrate-active enzymes (CAZymes). Alternatively, production organisms can be engineered to secrete CAZymes to reduce the reliance on externally added enzymes. Plant-pathogenic fungi have a vast repertoire of hydrolytic enzymes to sustain their lifestyle, but expression of the corresponding genes is usually highly regulated and restricted to the pathogenic phase. Here, we present a new strategy in using the biotrophic smut fungus Ustilago maydis for the degradation of plant cell wall components by activating its intrinsic enzyme potential during axenic growth. This fungal model organism is fully equipped with hydrolytic enzymes, and moreover, it naturally produces value-added substances, such as organic acids and biosurfactants. To achieve the deregulated expression of hydrolytic enzymes during the industrially relevant yeast-like growth in axenic culture, the native promoters of the respective genes were replaced by constitutively active synthetic promoters. This led to an enhanced conversion of xylan, cellobiose, and carboxymethyl cellulose to fermentable sugars. Moreover, a combination of strains with activated endoglucanase and β-glucanase increased the release of glucose from carboxymethyl cellulose and regenerated amorphous cellulose, suggesting that mixed cultivations could be a means for degrading more complex substrates in the future. In summary, this proof of principle demonstrates the potential applicability of activating the expression of native CAZymes from phytopathogens in a biocatalytic process. IMPORTANCE This study describes basic experiments that aim at the degradation of plant cell wall components by the smut fungus Ustilago maydis. As a plant pathogen, this fungus contains a set of lignocellulose-degrading enzymes that may be suited for biomass degradation. However, its hydrolytic enzymes are specifically expressed only during plant infection. Here, we provide the proof of principle that these intrinsic enzymes can be synthetically activated during the industrially relevant yeast-like growth. The fungus is known to naturally synthesize valuable compounds, such as itaconate or glycolipids. Therefore, it could be suited for use in a consolidated bioprocess in which more complex and natural substrates are simultaneously converted to fermentable sugars and to value-added compounds in the future. PMID:27316952

Small-Angle Neutron Scattering Reveals pH-Dependent Conformational Changes in Trichoderma reesei Cellobiohydrolase I: Implications for Enzymatic Activity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pingali, Sai Venkatesh; O'Neill, Hugh Michael; McGaughey, Joseph

2011-01-01

Cellobiohydrolase I (Cel7A) of the fungus Trichoderma reesei (now classified as an anamorph of Hypocrea jecorina) hydrolyzes crystalline cellulose to soluble sugars, making it of key interest for producing fermentable sugars from biomass for biofuel production. The activity of the enzyme is pH-dependent, with its highest activity occurring at pH 4 5. To probe the response of the solution structure of Cel7A to changes in pH, we measured small angle neutron scattering of it in a series of solutions having pH values of 7.0, 6.0, 5.3, and 4.2. As the pH decreases from 7.0 to 5.3, the enzyme structure remainsmore » well defined, possessing a spatial differentiation between the cellulose binding domain and the catalytic core that only changes subtly. At pH 4.2, the solution conformation of the enzyme changes to a structure that is intermediate between a properly folded enzyme and a denatured, unfolded state, yet the secondary structure of the enzyme is essentially unaltered. The results indicate that at the pH of optimal activity, the catalytic core of the enzyme adopts a structure in which the compact packing typical of a fully folded polypeptide chain is disrupted and suggest that the increased range of structures afforded by this disordered state plays an important role in the increased activity of Cel7A through conformational selection.« less

Effects of contraceptive agents on drug metabolism in various animal species.

PubMed Central

Briatico, G; Guiso, G; Jori, A; Ravazzani, C

1976-01-01

The effect on liver microsomal enzyme activity of three steroid contraceptive drug (SCD) combinations was compared in rats, mice and guinea-pigs. Lynestrenol plus mestranol, norethisterone plus mestranol and norethynodrel plus mestranol were given orally for 4 consecutive days (acute treatment) or 30 days (chronic treatment) at various doses eliciting an experimentally controlled antifertility activity which varied in its extent. In rats and mice all the combined treatments (with the exception of norethynodrel plus mestranol in mice) were active as inducers of liver microsomal enzymes. This induction seems to be mediated mainly by the progestogenic compounds. Oestrogens showed a very poor effect bordering on significance only in a few cases. No effect on liver microsomal protein or cytochrome P 450 concentration was obtained after treatment with doses capable of increasing the microsomal enzyme activity. The activity of the liver microsomal enzymes did not appear to be reduced immediately (2 h) after the last administration of the SCD given during 4 or 30 days. Contraceptive treatments at doses capable of eliciting complete antifertility activity were inactive on liver microsomal enzyme activity in guinea-pigs. PMID:987822

Profiling the orphan enzymes

PubMed Central

2014-01-01

The emergence of Next Generation Sequencing generates an incredible amount of sequence and great potential for new enzyme discovery. Despite this huge amount of data and the profusion of bioinformatic methods for function prediction, a large part of known enzyme activities is still lacking an associated protein sequence. These particular activities are called “orphan enzymes”. The present review proposes an update of previous surveys on orphan enzymes by mining the current content of public databases. While the percentage of orphan enzyme activities has decreased from 38% to 22% in ten years, there are still more than 1,000 orphans among the 5,000 entries of the Enzyme Commission (EC) classification. Taking into account all the reactions present in metabolic databases, this proportion dramatically increases to reach nearly 50% of orphans and many of them are not associated to a known pathway. We extended our survey to “local orphan enzymes” that are activities which have no representative sequence in a given clade, but have at least one in organisms belonging to other clades. We observe an important bias in Archaea and find that in general more than 30% of the EC activities have incomplete sequence information in at least one superkingdom. To estimate if candidate proteins for local orphans could be retrieved by homology search, we applied a simple strategy based on the PRIAM software and noticed that candidates may be proposed for an important fraction of local orphan enzymes. Finally, by studying relation between protein domains and catalyzed activities, it appears that newly discovered enzymes are mostly associated with already known enzyme domains. Thus, the exploration of the promiscuity and the multifunctional aspect of known enzyme families may solve part of the orphan enzyme issue. We conclude this review with a presentation of recent initiatives in finding proteins for orphan enzymes and in extending the enzyme world by the discovery of new activities. Reviewers This article was reviewed by Michael Galperin, Daniel Haft and Daniel Kahn. PMID:24906382

Contribution of the mu loop to the structure and function of rat glutathione transferase M1-1

PubMed Central

Hearne, Jennifer L.; Colman, Roberta F.

2006-01-01

The “mu loop,” an 11-residue loop spanning amino acid residues 33–43, is a characteristic structural feature of the mu class of glutathione transferases. To assess the contribution of the mu loop to the structure and function of rat GST M1-1, amino acid residues 35–44 (35GDAPDYDRSQ44) were excised by deletion mutagenesis, resulting in the “Deletion Enzyme.” Kinetic studies reveal that the Km values of the Deletion Enzyme are markedly increased compared with those of the wild-type enzyme: 32-fold for 1-chloro-2,4-dinitrobenzene, 99-fold for glutathione, and 880-fold for monobromobimane, while the Vmax value for each substrate is increased only modestly. Results from experiments probing the structure of the Deletion Enzyme, in comparison with that of the wild-type enzyme, suggest that the secondary and quaternary structures have not been appreciably perturbed. Thermostability studies indicate that the Deletion Enzyme is as stable as the wild-type enzyme at 4°C and 10°C, but it rapidly loses activity at 25°C, unlike the wild-type enzyme. In the temperature range of 4°C through 25°C, the loss of activity of the Deletion Enzyme is not the result of a change in its structure, as determined by circular dichroism spectroscopy and sedimentation equilibrium centrifugation. Collectively, these results indicate that the mu loop is not essential for GST M1-1 to maintain its structure nor is it required for the enzyme to retain some catalytic activity. However, it is an important determinant of the enzyme's affinity for its substrates. PMID:16672236

Copper-induced overexpression of genes encoding antioxidant system enzymes and metallothioneins involve the activation of CaMs, CDPKs and MEK1/2 in the marine alga Ulva compressa.

PubMed

Laporte, Daniel; Valdés, Natalia; González, Alberto; Sáez, Claudio A; Zúñiga, Antonio; Navarrete, Axel; Meneses, Claudio; Moenne, Alejandra

2016-08-01

Transcriptomic analyses were performed in the green macroalga Ulva compressa cultivated with 10μM copper for 24h. Nucleotide sequences encoding antioxidant enzymes, ascorbate peroxidase (ap), dehydroascorbate reductase (dhar) and glutathione reductase (gr), enzymes involved in ascorbate (ASC) synthesis l-galactose dehydrogenase (l-gdh) and l-galactono lactone dehydrogenase (l-gldh), in glutathione (GSH) synthesis, γ-glutamate-cysteine ligase (γ-gcl) and glutathione synthase (gs), and metal-chelating proteins metallothioneins (mt) were identified. Amino acid sequences encoded by transcripts identified in U. compressa corresponding to antioxidant system enzymes showed homology mainly to plant and green alga enzymes but those corresponding to MTs displayed homology to animal and plant MTs. Level of transcripts encoding the latter proteins were quantified in the alga cultivated with 10μM copper for 0-12 days. Transcripts encoding enzymes of the antioxidant system increased with maximal levels at day 7, 9 or 12, and for MTs at day 3, 7 or 12. In addition, the involvement of calmodulins (CaMs), calcium-dependent protein kinases (CDPKs), and the mitogen-activated protein kinase kinase (MEK1/2) in the increase of the level of the latter transcripts was analyzed using inhibitors. Transcript levels decreased with inhibitors of CaMs, CDPKs and MEK1/2. Thus, copper induces overexpression of genes encoding antioxidant enzymes, enzymes involved in ASC and GSH syntheses and MTs. The increase in transcript levels may involve the activation of CaMs, CDPKs and MEK1/2 in U. compressa. Copyright © 2016 Elsevier B.V. All rights reserved.

Is the alkaline tide a signal to activate metabolic or ionoregulatory enzymes in the dogfish shark (Squalus acanthias)?

PubMed

Wood, Chris M; Kajimura, Makiko; Mommsen, Thomas P; Walsh, Patrick J

2008-01-01

Experimental metabolic alkalosis is known to stimulate whole-animal urea production and active ion secretion by the rectal gland in the dogfish shark. Furthermore, recent evidence indicates that a marked alkaline tide (systemic metabolic alkalosis) follows feeding in this species and that the activities of the enzymes of the ornithine-urea cycle (OUC) for urea synthesis in skeletal muscle and liver and of energy metabolism and ion transport in the rectal gland are increased at this time. We therefore evaluated whether alkalosis and/or NaCl/volume loading (which also occurs with feeding) could serve as a signal for activation of these enzymes independent of nutrient loading. Fasted dogfish were infused for 20 h with either 500 mmol L(-1) NaHCO3 (alkalosis + volume expansion) or 500 mmol L(-1) NaCl (volume expansion alone), both isosmotic to dogfish plasma, at a rate of 3 mL kg(-1) h(-1). NaHCO3 infusion progressively raised arterial pH to 8.28 (control = 7.85) and plasma [HCO3-] to 20.8 mmol L(-1) (control = 4.5 mmol L(-1)) at 20 h, with unchanged arterial P(CO2), whereas NaCl/volume loading had no effect on blood acid-base status. Rectal gland Na+,K+-ATPase activity was increased 50% by NaCl loading and more than 100% by NaHCO3 loading, indicating stimulatory effects of both volume expansion and alkalosis. Rectal gland lactate dehydrogenase activity was elevated 25% by both treatments, indicating volume expansion effects only, whereas neither treatment increased the activities of the aerobic enzymes citrate synthase, NADP-isocitrate dehydrogenase, or the ketone body-utilizing enzyme beta-hydroxybutyrate dehydrogenase in the rectal gland or liver. The activity of ornithine-citrulline transcarbamoylase in skeletal muscle was doubled by NaHCO3 infusion, but neither treatment altered the activities of other OUC-related enzymes (glutamine synthetase, carbamoylphosphate synthetase III). We conclude that both the alkaline tide and salt loading/volume expansion act as signals to activate some but not all of the elevated metabolic pathways and ionoregulatory mechanisms needed during processing of a meal.

Directed evolution of new and improved enzyme functions using an evolutionary intermediate and multidirectional search.

PubMed

Porter, Joanne L; Boon, Priscilla L S; Murray, Tracy P; Huber, Thomas; Collyer, Charles A; Ollis, David L

2015-02-20

The ease with which enzymes can be adapted from their native roles and engineered to function specifically for industrial or commercial applications is crucial to enabling enzyme technology to advance beyond its current state. Directed evolution is a powerful tool for engineering enzymes with improved physical and catalytic properties and can be used to evolve enzymes where lack of structural information may thwart the use of rational design. In this study, we take the versatile and diverse α/β hydrolase fold framework, in the form of dienelactone hydrolase, and evolve it over three unique sequential evolutions with a total of 14 rounds of screening to generate a series of enzyme variants. The native enzyme has a low level of promiscuous activity toward p-nitrophenyl acetate but almost undetectable activity toward larger p-nitrophenyl esters. Using p-nitrophenyl acetate as an evolutionary intermediate, we have generated variants with altered specificity and catalytic activity up to 3 orders of magnitude higher than the native enzyme toward the larger nonphysiological p-nitrophenyl ester substrates. Several variants also possess increased stability resulting from the multidimensional approach to screening. Crystal structure analysis and substrate docking show how the enzyme active site changes over the course of the evolutions as either a direct or an indirect result of mutations.

Transformation of cyclodextrin glucanotransferase (CGTase) from aqueous suspension to fine solid particles via electrospraying.

PubMed

Saallah, Suryani; Naim, M Nazli; Mokhtar, Mohd Noriznan; Abu Bakar, Noor Fitrah; Gen, Masao; Lenggoro, I Wuled

2014-10-01

In this study, the potential of electrohydrodynamic atomization or electrospraying to produce nanometer-order CGTase particles from aqueous suspension was demonstrated. CGTase enzyme was prepared in acetate buffer solution (1% v/v), followed by electrospraying in stable Taylor cone-jet mode. The deposits were collected on aluminium foil (collector) at variable distances from the tip of spraying needle, ranging from 10 to 25 cm. The Coulomb fission that occurs during electrospraying process successfully transformed the enzyme to the solid state without any functional group deterioration. The functional group verification was conducted by FTIR analysis. Comparison between the deposit and the as-received enzyme in dry state indicates almost identical spectra. By increasing the distance of the collector from the needle tip, the average particle size of the solidified enzyme was reduced from 200±117 nm to 75±34 nm. The average particle sizes produced from the droplet fission were in agreement with the scaling law models. Enzyme activity analysis showed that the enzyme retained its initial activity after the electrospraying process. The enzyme particles collected at the longest distance (25 cm) demonstrated the highest enzyme activity, which indicates that the activity was controlled by the enzyme particle size. Copyright © 2014 Elsevier Inc. All rights reserved.

[Effects of infrasound exposure on several enzymes activities of spleen and liver in rats].

PubMed

Chen, Yao-ming; Ye, Lin; Gao, Shuang-bin; Zhu, Dong-hai; Luo, Weng-jing; Liu, Xiu-hong; Chen, Jing-yuan; Chen, Jing-zao

2004-05-01

To investigate the changes of several enzymes activities in the spleen and liver of rats after exposure to 8 Hz 130 dB infrasound for different time. Thirty-five male SD rats were randomly divided into five groups. Rats of group 1 served as control, rats from group 2 to 5 were exposed to 8 Hz 130 dB infrasound, 2 hours per day, for 1 wk, 2 wk, 3 wk, and 4 wk, respectively. The changes of enzymes activities in spleen and liver of rats were observed. Monoamine oxidase activities in spleen were significantly increased at 1 wk and 2 wk, it was decreased at 3 wk, and increased again at 4 wk (P < 0.05). There were no changes in the liver compared with the control group. Glutathione peroxides activities in spleen were significantly increased at 4 wk (P < 0.05) and it also increased in liver at 1 wk (P < 0.05). Superoxide dismutase activities in spleen were increased significantly from 1 wk to 4 wk, but there were no markedly changes in liver. The level of malondialdehyde in spleen were increased at 3 wk and 4 wk. In the liver, it were increased at 1 wk and 2 wk, and decreased at 3 wk, but it increased again at 4 wk (P < 0.05). The results indicated that lipid peroxidation and oxygen free radicals in spleen and liver were increased after infrasound exposure and it might induce the damage in tissue or cells.

The Effect of Exogenous Spermidine Concentration on Polyamine Metabolism and Salt Tolerance in Zoysiagrass (Zoysia japonica Steud) Subjected to Short-Term Salinity Stress.

PubMed

Li, Shucheng; Jin, Han; Zhang, Qiang

2016-01-01

Salt stress, particularly short-term salt stress, is among the most serious abiotic factors limiting plant survival and growth in China. It has been established that exogenous spermidine (Spd) stimulates plant tolerance to salt stress. The present study utilized two zoysiagrass cultivars commonly grown in China that exhibit either sensitive (cv. Z081) or tolerant (cv. Z057) adaptation capacity to salt stress. The two cultivars were subjected to 200 mM salt stress and treated with different exogenous Spd concentrations for 8 days. Polyamine [diamine putrescine (Put), tetraamine spermine (Spm), and Spd], H2O2 and malondialdehyde (MDA) contents and polyamine metabolic (ADC, ODC, SAMDC, PAO, and DAO) and antioxidant (superoxide dismutase, catalase, and peroxidase) enzyme activities were measured. The results showed that salt stress induced increases in Spd and Spm contents and ornithine decarboxylase (ODC), S-adenosylmethionine decarboxylase (SAMDC), and diamine oxidase (DAO) activities in both cultivars. Exogenous Spd application did not alter polyamine contents via regulation of polyamine-degrading enzymes, and an increase in polyamine biosynthetic enzyme levels was observed during the experiment. Increasing the concentration of exogenous Spd resulted in a tendency of the Spd and Spm contents and ODC, SAMDC, DAO, and antioxidant enzyme activities to first increase and then decrease in both cultivars. H2O2 and MDA levels significantly decreased in both cultivars treated with Spd. Additionally, in both cultivars, positive correlations between polyamine biosynthetic enzymes (ADC, SAMDC), DAO, and antioxidant enzymes (SOD, POD, CAT), but negative correlations with H2O2 and MDA levels, and the Spd + Spm content were observed with an increase in the concentration of exogenous Spd.

The Effect of Exogenous Spermidine Concentration on Polyamine Metabolism and Salt Tolerance in Zoysiagrass (Zoysia japonica Steud) Subjected to Short-Term Salinity Stress

PubMed Central

Li, Shucheng; Jin, Han; Zhang, Qiang

2016-01-01

Salt stress, particularly short-term salt stress, is among the most serious abiotic factors limiting plant survival and growth in China. It has been established that exogenous spermidine (Spd) stimulates plant tolerance to salt stress. The present study utilized two zoysiagrass cultivars commonly grown in China that exhibit either sensitive (cv. Z081) or tolerant (cv. Z057) adaptation capacity to salt stress. The two cultivars were subjected to 200 mM salt stress and treated with different exogenous Spd concentrations for 8 days. Polyamine [diamine putrescine (Put), tetraamine spermine (Spm), and Spd], H2O2 and malondialdehyde (MDA) contents and polyamine metabolic (ADC, ODC, SAMDC, PAO, and DAO) and antioxidant (superoxide dismutase, catalase, and peroxidase) enzyme activities were measured. The results showed that salt stress induced increases in Spd and Spm contents and ornithine decarboxylase (ODC), S-adenosylmethionine decarboxylase (SAMDC), and diamine oxidase (DAO) activities in both cultivars. Exogenous Spd application did not alter polyamine contents via regulation of polyamine-degrading enzymes, and an increase in polyamine biosynthetic enzyme levels was observed during the experiment. Increasing the concentration of exogenous Spd resulted in a tendency of the Spd and Spm contents and ODC, SAMDC, DAO, and antioxidant enzyme activities to first increase and then decrease in both cultivars. H2O2 and MDA levels significantly decreased in both cultivars treated with Spd. Additionally, in both cultivars, positive correlations between polyamine biosynthetic enzymes (ADC, SAMDC), DAO, and antioxidant enzymes (SOD, POD, CAT), but negative correlations with H2O2 and MDA levels, and the Spd + Spm content were observed with an increase in the concentration of exogenous Spd. PMID:27582752

Increased production of biomass-degrading enzymes by double deletion of creA and creB genes involved in carbon catabolite repression in Aspergillus oryzae.

PubMed

Ichinose, Sakurako; Tanaka, Mizuki; Shintani, Takahiro; Gomi, Katsuya

2018-02-01

In a previous study, we reported that a double gene deletion mutant for CreA and CreB, which constitute the regulatory machinery involved in carbon catabolite repression, exhibited improved production of α-amylase compared with the wild-type strain and single creA or creB deletion mutants in Aspergillus oryzae. Because A. oryzae can also produce biomass-degrading enzymes, such as xylolytic and cellulolytic enzymes, we examined the production levels of those enzymes in deletion mutants in this study. Xylanase and β-glucosidase activities in the wild-type were hardly detected in submerged culture containing xylose as the carbon source, whereas those enzyme activities were significantly increased in the single creA deletion (ΔcreA) and double creA and creB deletion (ΔcreAΔcreB) mutants. In particular, the ΔcreAΔcreB mutant exhibited >100-fold higher xylanase and β-glucosidase activities than the wild-type. Moreover, in solid-state culture, the β-glucosidase activity of the double deletion mutant was >7-fold higher than in the wild-type. These results suggested that deletion of both creA and creB genes could also efficiently improve the production levels of biomass-degrading enzymes in A. oryzae. Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

New glycyl radical enzymes catalysing key metabolic steps in anaerobic bacteria.

PubMed

Selmer, Thorsten; Pierik, Antonio J; Heider, Johann

2005-10-01

During the last decade, an increasing number of new enzymes containing glycyl radicals in their active sites have been identified and biochemically characterised. These include benzylsuccinate synthase (Bss), 4-hydroxyphenylacetate decarboxylase (Hpd) and the coenzyme B12-independent glycerol dehydratase (Gdh). These are involved in metabolic pathways as different as anaerobic toluene metabolism, fermentative production of p-cresol and glycerol fermentation. Some features of these newly discovered enzymes are described and compared with those of the previously known glycyl radical enzymes pyruvate formate-lyase (Pfl) and anaerobic ribonucleotide reductase (Nrd). Among the new enzymes, Bss and Hpd share the presence of small subunits, the function of which in the catalytic mechanisms is still enigmatic, and both enzymes contain metal centres in addition to the glycyl radical prosthetic group. The activating enzymes of the novel systems also deviate from the standard type, containing at least one additional Fe-S cluster. Finally, the available whole-genome sequences of an increasing number of strictly or facultative anaerobic bacteria revealed the presence of many more hitherto unknown glycyl radical enzyme (GRE) systems. Recent studies suggest that the particular types of these enzymes represent the ends of different evolutionary lines, which emerged early in evolution and diversified to yield remarkably versatile biocatalysts for chemical reactions that are otherwise difficult to perform in anoxic environments.

Catalytic properties of thermophilic lactate dehydrogenase and halophilic malate dehydrogenase at high temperature and low water activity.

PubMed

Hecht, K; Wrba, A; Jaenicke, R

1989-07-15

Thermophilic lactate dehydrogenases from Thermotoga maritima and Bacillus stearothermophilus are stable up to temperature limits close to the optimum growth temperature of their parent organisms. Their catalytic properties are anomalous in that Km shows a drastic increase with increasing temperature. At low temperatures, the effect levels off. Extreme halophilic malate dehydrogenase from Halobacterium marismortui exhibits a similar anomaly. Increasing salt concentration (NaCl) leads to an optimum curve for Km, oxaloacctate while Km, NADH remains constant. Previous claims that the activity of halophilic malate dehydrogenase shows a maximum at 1.25 M NaCl are caused by limiting substrate concentration; at substrate saturation, specific activity of halophilic malate dehydrogenase reaches a constant value at ionic strengths I greater than or equal to 1 M. Non-halophilic (mitochondrial) malate dehydrogenase shows Km characteristics similar to those observed for the halophilic enzyme. The drastic decrease in specific activity of the mitochondrial enzyme at elevated salt concentrations is caused by the salt-induced increase in rigidity of the enzyme, rather than gross structural changes.

Ultrastructural and biochemical studies of the effect of polychlorinated biphenyl on mouse parotid gland cells.

PubMed

Kawasaki, G; Mataki, S; Mizuno, A

1995-01-01

These effects of polychlorinated biphenyl (PCB) were examined by light and electron microscopy and biochemical analysis of lysosomal enzyme activities. Several experimental protocols with dosage schedules of either 0.2, 2.0, or 20 mg/kg of PCB were used. Typical histological changes were observed in mice given 2 mg/kg of PCB in a single injection. There were no remarkable changes until 4 days after PCB administration; marked cytoplasmic vacuolation was observed in parotid acinar cells at 7 days. The activities of lysosomal enzymes increased after the PCB injection and their maximum values appeared consistently at 4 days after the treatment; the increases were threefold for acid phosphatase, twofold for beta-glucuronidase, threefold for cathepsin D, fivefold for cathepsin H and twofold for cathepsin L. As vacuolation was preceded by a large increase in lysosomal enzyme activities and the vacuoles co-localized with lysosomes, it is suggested that an increase in these activities induced by PCB may be closely related to the development of vacuolation in the parotid acinar cells as a subacute effect of PCB.

Improved production of a recombinant Rhizomucor miehei lipase expressed in Pichia pastoris and its application for conversion of microalgae oil to biodiesel.

PubMed

Huang, Jinjin; Xia, Ji; Yang, Zhen; Guan, Feifei; Cui, Di; Guan, Guohua; Jiang, Wei; Li, Ying

2014-01-01

We previously cloned a 1,3-specific lipase gene from the fungus Rhizomucor miehei and expressed it in methylotrophic yeast Pichia pastoris strain GS115. The enzyme produced (termed RML) was able to catalyze methanolysis of soybean oil and showed strong position specificity. However, the enzyme activity and amount of enzyme produced were not adequate for industrial application. Our goal in the present study was to improve the enzyme properties of RML in order to apply it for the conversion of microalgae oil to biofuel. Several new expression plasmids were constructed by adding the propeptide of the target gene, optimizing the signal peptide, and varying the number of target gene copies. Each plasmid was transformed separately into P. pastoris strain X-33. Screening by flask culture showed maximal (21.4-fold increased) enzyme activity for the recombinant strain with two copies of the target gene; the enzyme was termed Lipase GH2. The expressed protein with the propeptide (pRML) was a stable glycosylated protein, because of glycosylation sites in the propeptide. Quantitative real-time RT-PCR analysis revealed two major reasons for the increase in enzyme activity: (1) the modified recombinant expression system gave an increased transcription level of the target gene (rml), and (2) the enzyme was suitable for expression in host cells without causing endoplasmic reticulum (ER) stress. The modified enzyme had improved thermostability and methanol or ethanol tolerance, and was applicable directly as free lipase (fermentation supernatant) in the catalytic esterification and transesterification reaction. After reaction for 24 hours at 30°C, the conversion rate of microalgae oil to biofuel was above 90%. Our experimental results show that signal peptide optimization in the expression plasmid, addition of the gene propeptide, and proper gene dosage significantly increased RML expression level and enhanced the enzymatic properties. The target enzyme was the major component of fermentation supernatant and was stable for over six months at 4°C. The modified free lipase is potentially applicable for industrial-scale conversion of microalgae oil to biodiesel.

Identification and modification of dynamical regions in proteins for alteration of enzyme catalytic effect

DOEpatents

Agarwal, Pratul K.

2015-11-24

A method for analysis, control, and manipulation for improvement of the chemical reaction rate of a protein-mediated reaction is provided. Enzymes, which typically comprise protein molecules, are very efficient catalysts that enhance chemical reaction rates by many orders of magnitude. Enzymes are widely used for a number of functions in chemical, biochemical, pharmaceutical, and other purposes. The method identifies key protein vibration modes that control the chemical reaction rate of the protein-mediated reaction, providing identification of the factors that enable the enzymes to achieve the high rate of reaction enhancement. By controlling these factors, the function of enzymes may be modulated, i.e., the activity can either be increased for faster enzyme reaction or it can be decreased when a slower enzyme is desired. This method provides an inexpensive and efficient solution by utilizing computer simulations, in combination with available experimental data, to build suitable models and investigate the enzyme activity.

Identification and modification of dynamical regions in proteins for alteration of enzyme catalytic effect

DOEpatents

Agarwal, Pratul K.

2013-04-09

A method for analysis, control, and manipulation for improvement of the chemical reaction rate of a protein-mediated reaction is provided. Enzymes, which typically comprise protein molecules, are very efficient catalysts that enhance chemical reaction rates by many orders of magnitude. Enzymes are widely used for a number of functions in chemical, biochemical, pharmaceutical, and other purposes. The method identifies key protein vibration modes that control the chemical reaction rate of the protein-mediated reaction, providing identification of the factors that enable the enzymes to achieve the high rate of reaction enhancement. By controlling these factors, the function of enzymes may be modulated, i.e., the activity can either be increased for faster enzyme reaction or it can be decreased when a slower enzyme is desired. This method provides an inexpensive and efficient solution by utilizing computer simulations, in combination with available experimental data, to build suitable models and investigate the enzyme activity.

Functional and structural characterization of the pentapeptide insertion of Theileria annulata lactate dehydrogenase by site-directed mutagenesis, comparative modeling and molecular dynamics simulations.

PubMed

Erdemir, Aysegul; Mutlu, Ozal

2017-06-01

Lactate dehydrogenase (LDH) is an important metabolic enzyme in glycolysis and it has been considered as the main energy source in many organisms including apicomplexan parasites. Differences at the active site loop of the host and parasite LDH's makes this enzyme an attractive target for drug inhibitors. In this study, five amino acid insertions in the active site pocket of Theileria annulata LDH (TaLDH) were deleted by PCR-based site-directed mutagenesis, expression and activity analysis of mutant and wild type TaLDH enzymes were performed. Removal of the insertion at the active site loop caused production of an inactive enzyme. Furthermore, structures of wild and mutant enzymes were predicted by comparative modeling and the importance of the insertions at the active site loop were also assigned by molecular docking and dynamics simulations in order to evaluate essential role of this loop for the enzymatic activity. Pentapeptide insertion removal resulted in loss of LDH activity due to deletion of Trp96 and conformational change of Arg98 because of loop instability. Analysis of wild type and mutant enzymes with comparative molecular dynamics simulations showed that the fluctuations of the loop residues increase in mutant enzyme. Together with in silico studies, in vitro results revealed that active site loop has a vital role in the enzyme activity and our findings promise hope for the further drug design studies against theileriosis and other apicomplexan parasite diseases. Copyright © 2017 Elsevier Inc. All rights reserved.

Gene expression and activity of antioxidant enzymes in rice plants, cv. BRS AG, under saline stress.

PubMed

Rossatto, Tatiana; do Amaral, Marcelo Nogueira; Benitez, Letícia Carvalho; Vighi, Isabel Lopes; Braga, Eugenia Jacira Bolacel; de Magalhães Júnior, Ariano Martins; Maia, Mara Andrade Colares; da Silva Pinto, Luciano

2017-10-01

The rice cultivar ( Oryza sativa L.) BRS AG, developed by Embrapa Clima Temperado, is the first cultivar designed for purposes other than human consumption. It may be used in ethanol production and animal feed. Different abiotic stresses negatively affect plant growth. Soil salinity is responsible for a serious reduction in productivity. Therefore, the objective of this study was to evaluate the gene expression and the activity of antioxidant enzymes (SOD, CAT, APX and GR) and identify their functions in controlling ROS levels in rice plants, cultivar BRS AG, after a saline stress period. The plants were grown in vitro with two NaCl concentrations (0 and 136 mM), collected at 10, 15 and 20 days of cultivation. The results indicated that the activity of the enzymes evaluated promotes protection against oxidative stress. Although, there was an increase of reactive oxygen species, there was no increase in MDA levels. Regarding genes encoding isoforms of antioxidant enzymes, it was observed that OsSOD3 - CU/Zn , OsSOD2 - Cu/Zn , OsSOD - Cu/Zn , OsSOD4 - Cu/Zn , OsSODCc1 - Cu/Zn , OsSOD - Fe , OsAPX1 , OsCATB and OsGR2 were the most responsive. The increase in the transcription of all genes among evaluated isoforms, except for OsAPX6 , which remained stable, contributed to the increase or the maintenance of enzyme activity. Thus, it is possible to infer that the cv. BRS AG has defense mechanisms against salt stress.

Enhanced enzyme kinetic stability by increasing rigidity within the active site.

PubMed

Xie, Yuan; An, Jiao; Yang, Guangyu; Wu, Geng; Zhang, Yong; Cui, Li; Feng, Yan

2014-03-14

Enzyme stability is an important issue for protein engineers. Understanding how rigidity in the active site affects protein kinetic stability will provide new insight into enzyme stabilization. In this study, we demonstrated enhanced kinetic stability of Candida antarctica lipase B (CalB) by mutating the structurally flexible residues within the active site. Six residues within 10 Å of the catalytic Ser(105) residue with a high B factor were selected for iterative saturation mutagenesis. After screening 2200 colonies, we obtained the D223G/L278M mutant, which exhibited a 13-fold increase in half-life at 48 °C and a 12 °C higher T50(15), the temperature at which enzyme activity is reduced to 50% after a 15-min heat treatment. Further characterization showed that global unfolding resistance against both thermal and chemical denaturation also improved. Analysis of the crystal structures of wild-type CalB and the D223G/L278M mutant revealed that the latter formed an extra main chain hydrogen bond network with seven structurally coupled residues within the flexible α10 helix that are primarily involved in forming the active site. Further investigation of the relative B factor profile and molecular dynamics simulation confirmed that the enhanced rigidity decreased fluctuation of the active site residues at high temperature. These results indicate that enhancing the rigidity of the flexible segment within the active site may provide an efficient method for improving enzyme kinetic stability.

Dried blood spots for the enzymatic diagnosis of lysosomal storage diseases in dogs and cats.

PubMed

Sewell, Adrian C; Haskins, Mark E; Giger, Urs

2012-12-01

In people, lysosomal storage diseases (LSD) can be diagnosed by assaying enzyme activities in dried blood spots (DBS). The aim of this study was to evaluate the feasibility of using DBS samples from dogs and cats to measure lysosomal enzymatic activities and diagnose LSD. Drops of fresh whole blood collected in EDTA from dogs and cats with known or suspected LSD and from clinically healthy dogs and cats were placed on neonatal screening cards, dried, and mailed to the Metabolic Laboratory, University Children's Hospital, Frankfurt, Germany. Activities of selected lysosomal enzymes were measured using fluorescent substrates in a 2-mm diameter disk (~2.6 μL blood) punched from the DBS. Results were expressed as nmol substrate hydrolyzed per mL of blood per minute or hour. Reference values were established for several lysosomal enzyme activities in DBS from dogs and cats; for most enzymes, activities were higher than those published for human samples. Activities of β-glucuronidase, N-acetylglucosamine-4-sulfatase (arylsulfatase B), α-mannosidase, α-galactosidase, α-fucosidase, and hexosaminidase A were measureable in DBS from healthy cats and dogs; α-iduronidase activity was measureable only in cats. In samples from animals with LSD, markedly reduced activity of a specific enzyme was found. In contrast, in samples from cats affected with mucolipidosis II, activities of lysosomal enzymes were markedly increased. Measurement of lysosomal enzyme activities in DBS provides an inexpensive, simple, and convenient method to screen animals for suspected LSD and requires only a small sample volume. For diseases in which the relevant enzyme activity can be measured in DBS, a specific diagnosis can be made. © 2012 American Society for Veterinary Clinical Pathology.

Gene expression and activity of digestive enzymes of Daphnia pulex in response to food quality differences.

PubMed

Schwarzenberger, Anke; Fink, Patrick

2018-04-01

Food quality is an important factor influencing organisms' well-being. In freshwater ecosystems, food quality has been studied extensively for the keystone herbivore genus Daphnia, as they form the critical trophic link between primary producers and higher order consumers such as fish. For Daphnia, the edible fraction of phytoplankton in lakes (consisting mostly of unicellular algae and cyanobacteria) is extraordinarily diverse. To be able to digest different food particles, Daphnia possess a set of digestive enzymes that metabolize carbohydrates, lipids and proteins. Recent studies have found a connection between gene expression and activity of single digestive enzyme types of Daphnia, i.e. lipases and proteases, and transcriptome studies have shown that a variety of genes coding for gut enzymes are differentially expressed in response to different food algae. However, never before has a set of digestive enzymes been studied simultaneously both on the gene expression and the enzyme activity level in Daphnia. Here, we investigated several digestive enzymes of Daphnia pulex in a comparison between a high-quality (green algal) and a low-quality (cyanobacterial) diet. Diet significantly affected the expression of all investigated digestive enzyme genes and enzyme activity was altered between treatments. Furthermore, we found that gene expression and enzyme activity were significantly correlated in cellulase, triacylglycerol lipase and β-glucosidase when switched from high to low-quality food. We conclude that one of the factors causing the often observed low biomass and energy transfer efficiency from cyanobacteria to Daphnia is probably the switch to a cost-effective overall increase of gene expression and activity of digestive enzymes of this herbivore. Copyright © 2018 Elsevier Inc. All rights reserved.

Induction of antioxidant enzyme activity and lipid peroxidation level in ion-beam-bombarded rice seeds

NASA Astrophysics Data System (ADS)

Semsang, Nuananong; Yu, LiangDeng

2013-07-01

Low-energy ion beam bombardment has been used to mutate a wide variety of plant species. To explore the indirect effects of low-energy ion beam on biological damage due to the free radical production in plant cells, the increase in antioxidant enzyme activities and lipid peroxidation level was investigated in ion-bombarded rice seeds. Local rice seeds were bombarded with nitrogen or argon ion beams at energies of 29-60 keV and ion fluences of 1 × 1016 ions cm-2. The activities of the antioxidant enzymes; superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), dehydroascorbate reductase (DHAR), glutathione reductase (GR), glutathione S-transferase (GST) and lipid peroxidation level were assayed in the germinated rice seeds after ion bombardment. The results showed most of the enzyme activities and lipid peroxidation levels in both the argon and nitrogen bombarded samples were higher than those in the natural control. N-ion bombardment could induce higher levels of antioxidant enzyme activities in the rice samples than the Ar-ion bombardment. Additional effects due to the vacuum condition were found to affect activities of some antioxidant enzymes and lipid peroxidation level. This study demonstrates that ion beam bombardment and vacuum condition could induce the antioxidant enzyme activity and lipid peroxidation level which might be due to free radical production in the bombarded rice seeds.

Cajanus cajan Linn. (Leguminosae) prevents alcohol-induced rat liver damage and augments cytoprotective function.

PubMed

Kundu, Rakesh; Dasgupta, Suman; Biswas, Anindita; Bhattacharya, Anirban; Pal, Bikas C; Bandyopadhyay, Debashis; Bhattacharya, Shelley; Bhattacharya, Samir

2008-08-13

Cajanus cajan Linn. (Leguminosae) is a nontoxic edible herb, widely used in Indian folk medicine for the prevention of various liver disorders. In the present study we have demonstrated that methanol-aqueous fraction (MAF2) of Cajanus cajan leaf extract could prevent the chronically treated alcohol induced rat liver damage. Chronic doses of alcohol (3.7 g/ kg) orally administered to rats for 28 days and liver function marker enzymes such as GPT, GOT, ALP and anti-oxidant enzyme activities were determined. Effect of MAF2 at a dose of 50mg/kg body weight on alcohol treated rats was noted. Alcohol effected significant increase in liver marker enzyme activities and reduced the activities of anti-oxidant enzymes. Co-administration of MAF2 reversed the liver damage due to alcohol; it decreased the activities of liver marker enzymes and augmented antioxidant enzyme activities. We also demonstrate significant decrease of the phase II detoxifying enzyme, UDP-glucuronosyl transferase (UGT) activity along with a three- and two-fold decrease of UGT2B gene and protein expression respectively. MAF2 co-administration normalized UGT activity and revived the expression of UGT2B with a concomitant expression and nuclear translocation of Nrf2, a transcription factor that regulates the expression of many cytoprotective genes. Cajanus cajan extract therefore shows a promise in therapeutic use in alcohol induced liver dysfunction.

Mineralogical impact on long-term patterns of soil nitrogen and phosphorus enzyme activities

NASA Astrophysics Data System (ADS)

Mikutta, Robert; Turner, Stephanie; Meyer-Stüve, Sandra; Guggenberger, Georg; Dohrmann, Reiner; Schippers, Axel

2014-05-01

Soil chronosequences provide a unique opportunity to study microbial activity over time in mineralogical diverse soils of different ages. The main objective of this study was to test the effect of mineralogical properties, nutrient and organic matter availability over whole soil pro-files on the abundance and activity of the microbial communities. We focused on microbio-logical processes involved in nitrogen and phosphorus cycling at the 120,000-year Franz Josef soil chronosequence. Microbial abundances (microbial biomass and total cell counts) and enzyme activities (protease, urease, aminopeptidase, and phosphatase) were determined and related to nutrient contents and mineralogical soil properties. Both, microbial abundances and enzyme activities decreased with soil depth at all sites. In the organic layers, microbial biomass and the activities of N-hydrolyzing enzymes showed their maximum at the intermediate-aged sites, corresponding to a high aboveground biomass. In contrast, the phosphatase activity increased with site age. The activities of N-hydrolyzing enzymes were positively correlated with total carbon and nitrogen contents, whereas the phosphatase activity was negatively correlated with the phosphorus content. In the mineral soil, the enzyme activities were generally low, thus reflecting the presence of strongly sorbing minerals. Sub-strate-normalized enzyme activities correlated negatively to clay content as well as poorly crystalline Al and Fe oxyhydroxides, supporting the view that the evolution of reactive sec-ondary mineral phases alters the activity of the microbial communities by constraining sub-strate availability. Our data suggest a strong mineralogical influence on nutrient cycling par-ticularly in subsoil environments.

Chickpea seeds germination rational parameters optimization

NASA Astrophysics Data System (ADS)

Safonova, Yu A.; Ivliev, M. N.; Lemeshkin, A. V.

2018-05-01

The paper presents the influence of chickpea seeds bioactivation parameters on their enzymatic activity experimental results. Optimal bioactivation process modes were obtained by regression-factor analysis: process temperature - 13.6 °C, process duration - 71.5 h. It was found that in the germination process, the proteolytic, amylolytic and lipolytic enzymes activity increased, and the urease enzyme activity is reduced. The dependences of enzyme activity on chickpea seeds germination conditions were obtained by mathematical processing of experimental data. The calculated data are in good agreement with the experimental ones. This confirms the optimization efficiency based on experiments mathematical planning in order to determine the enzymatic activity of chickpea seeds germination optimal parameters of bioactivated seeds.

Caloric restriction counteracts age-related changes in the activities of sorbitol metabolizing enzymes from mouse liver

PubMed Central

Hagopian, Kevork; Ramsey, Jon J.; Weindruch, Richard

2009-01-01

The influence of caloric restriction (CR) on hepatic sorbitol-metabolizing enzyme activities was investigated in young and old mice. Aldose reductase and sorbitol dehydrogenase activities were significantly lower in old CR mice than in old controls. Young CR mice showed decreased aldose reductase activity and a trend towards decreased sorbitol dehydrogenase when compared to controls. Metabolites of the pathway, namely sorbitol, glucose and fructose were decreased by CR in young and old mice. Pyruvate levels were decreased by CR in both young and old mice, while lactate decreased only in old CR. Malate levels increased in old CR but remained unchanged in young CR, when compared with controls. Accordingly, the lactae/pyruvate and malate/pyruvate ratios in young and old CR mice were increased, indicating increased NADH/NAD and NADPH/NADP redox couples, respectively. The results indicate that decreased glucose levels under CR conditions lead to decreased sorbitol pathway enzyme activities and metabolite levels, and could contribute to the beneficial effects of long-term CR through decreased sorbitol levels and NADPH sparing. PMID:18953666

Visualization of enzyme activities inside earthworm biopores by in situ soil zymography

NASA Astrophysics Data System (ADS)

Thu Duyen Hoang, Thi; Razavi, Bahar. S.; Blagodatskaya, Evgenia; Kuzyakov, Yakov

2015-04-01

Earthworms can strongly activate microorganisms, increase microbial and enzyme activities and consequently the turnover of native soil organic matter. In extremely dynamic microhabitats and hotspots as biopores made by earthworms, the in situ enzyme activities are a footprint of complex biotic interactions. The effect of earthworms on the alteration of enzyme activities inside biopores and the difference between bio-pores and earthworm-free soil was visualized by in situ soil zymography (Spohn and Kuzyakov, 2014). For the first time, we prepared quantitative imaging of enzyme activities in biopores. Furthermore, we developed the zymography technique by direct application of a substrate saturated membrane to the soil to obtain better spatial resolution. Lumbricus terrestris L. was placed into transparent box (15×20×15cm). Simultaneously, maize seed was sown in the soil. Control soil box with maize and without earthworm was prepared in the same way. After two weeks when bio-pore systems were formed by earthworm, we visualized in situ enzyme activities of five hydrolytic enzymes (β-glucosidase, cellobiohydrolase, chitinase, xylanase, leucine aminopeptidase) and phosphatase. Followed by non-destructive zymography, biopore samples and control soil were destructively collected to assay enzyme kinetics by fluorogenically labeled substrates method. Zymography showed higher activity of β-glucosidase, chitinase, xylanase and phosphatase in biopores comparing to bulk soil. These differences were further confirmed by fluorimetric microplate enzyme assay detected significant difference of Vmax in four above mentioned enzymes. Vmax of β-glucosidase, chitinase, xylanase and phosphatase in biopores is 68%, 108%, 50% and 49% higher than that of control soil. However, no difference in cellobiohydrolase and leucine aminopeptidase kinetics between biopores and control soil were detected. This indicated little effect of earthworms on protein and cellulose transformation in soil. In conclusion, earthworms contribute to the decomposition of carbohydrates through promoting enzyme activities involved in the C-cycle except for leucine aminopeptidase and cellobiohydrolase. References Spohn M, Kuzyakov Y. (2014) Spatial and temporal dynamics of hotspots of enzyme activity in soil as affected by living and dead roots - a soil zymography analysis, Plant Soil 379: 67-77

Antioxidant enzyme activity and malondialdehyde levels can be modulated by Piper betle, tocotrienol rich fraction and Chlorella vulgaris in aging C57BL/6 mice.

PubMed

Aliahmat, Nor Syahida; Noor, Mohd Razman Mohd; Yusof, Wan Junizam Wan; Makpol, Suzana; Ngah, Wan Zurinah Wan; Yusof, Yasmin Anum Mohd

2012-12-01

The aim of this study was to determine the erythrocyte antioxidant enzyme activity and the superoxide dismutase, catalase, glutathione peroxidase, and plasma malondialdehyde levels in aging mice and to evaluate how these measures are modulated by potential antioxidants, including the tocotrienol-rich fraction, Piper betle, and Chlorella vulgaris. One hundred and twenty male C57BL/6 inbred mice were divided into three age groups: young (6 months old), middle-aged (12 months old), and old (18 months old). Each age group consisted of two control groups (distilled water and olive oil) and three treatment groups: Piper betle (50 mg/kg body weight), tocotrienol-rich fraction (30 mg/kg), and Chlorella vulgaris (50 mg/kg). The duration of treatment for all three age groups was two months. Blood was withdrawn from the orbital sinus to determine the antioxidant enzyme activity and the malondialdehyde level. Piper betle increased the activities of catalase, glutathione peroxidase, and superoxide dismutase in the young, middle, and old age groups, respectively, when compared to control. The tocotrienol-rich fraction decreased the superoxide dismutase activity in the middle and the old age groups but had no effect on catalase or glutathione peroxidase activity for all age groups. Chlorella vulgaris had no effect on superoxide dismutase activity for all age groups but increased glutathione peroxidase and decreased catalase activity in the middle and the young age groups, respectively. Chlorella vulgaris reduced lipid peroxidation (malondialdehyde levels) in all age groups, but no significant changes were observed with the tocotrienol-rich fraction and the Piper betle treatments. We found equivocal age-related changes in erythrocyte antioxidant enzyme activity when mice were treated with Piper betle, the tocotrienol-rich fraction, and Chlorella vulgaris. However, Piper betle treatment showed increased antioxidant enzymes activity during aging.

Antioxidant enzyme activity and malondialdehyde levels can be modulated by Piper betle, tocotrienol rich fraction and Chlorella vulgaris in aging C57BL/6 mice

PubMed Central

Aliahmat, Nor Syahida; Noor, Mohd Razman Mohd; Yusof, Wan Junizam Wan; Makpol, Suzana; Ngah, Wan Zurinah Wan; Yusof, Yasmin Anum Mohd

2012-01-01

OBJECTIVE: The aim of this study was to determine the erythrocyte antioxidant enzyme activity and the superoxide dismutase, catalase, glutathione peroxidase, and plasma malondialdehyde levels in aging mice and to evaluate how these measures are modulated by potential antioxidants, including the tocotrienol-rich fraction, Piper betle, and Chlorella vulgaris. METHOD: One hundred and twenty male C57BL/6 inbred mice were divided into three age groups: young (6 months old), middle-aged (12 months old), and old (18 months old). Each age group consisted of two control groups (distilled water and olive oil) and three treatment groups: Piper betle (50 mg/kg body weight), tocotrienol-rich fraction (30 mg/kg), and Chlorella vulgaris (50 mg/kg). The duration of treatment for all three age groups was two months. Blood was withdrawn from the orbital sinus to determine the antioxidant enzyme activity and the malondialdehyde level. RESULTS: Piper betle increased the activities of catalase, glutathione peroxidase, and superoxide dismutase in the young, middle, and old age groups, respectively, when compared to control. The tocotrienol-rich fraction decreased the superoxide dismutase activity in the middle and the old age groups but had no effect on catalase or glutathione peroxidase activity for all age groups. Chlorella vulgaris had no effect on superoxide dismutase activity for all age groups but increased glutathione peroxidase and decreased catalase activity in the middle and the young age groups, respectively. Chlorella vulgaris reduced lipid peroxidation (malondialdehyde levels) in all age groups, but no significant changes were observed with the tocotrienol-rich fraction and the Piper betle treatments. CONCLUSION: We found equivocal age-related changes in erythrocyte antioxidant enzyme activity when mice were treated with Piper betle, the tocotrienol-rich fraction, and Chlorella vulgaris. However, Piper betle treatment showed increased antioxidant enzymes activity during aging. PMID:23295600

[Features of cholecalciferol hydroxylation in the liver of rats in conditions of D-hypervitaminosis and activity of alpha-tocopherol].

PubMed

Velykyĭ, M M; Apukhovs'ka, L I; Vasylevs'ka, V M; Lotots'ka, O Iu; Besusiak, A I; Khomenko, A V

2010-01-01

It is shown, that hepatocytes contain two (microsomal and mitochondrial) vitamin D3 25-hydroxylase enzymes, which differ as to their activity and function with maximal activity at different concentrations to substrate, namely at 15 microM and 100 microM of vitamin D3, accordingly. Activity of vitamin D3 25-hydroxylase enzymes of hepatocytes is regulated by cholecalciferol and alpha-tocopherol. The general and microsomal vitamin D3 25-hydroxylase enzymes activity of hepatocytes is lowered, but mitochondrial isoform is increased under D-hypervitaminosis conditions. Vitamin E increases microsomal vitamin D3 25-hydroxylase activity and decreases mitochondrial isoform activity of rats hepatocytes under D-hypervitaminosis conditions. It is established that D-hypervitaminosis is accompanied by expressed hypercalcemia and hyperphosphatemia, by decreased contents of mineral components in the bone tissue and high activity of alkaline phosphatase in the blood serum. The physiological doses of vitamin E under these conditions normalized the mineral metabolism, contents of calcium, phosphates and activity of alkaline phosphatase isoform in the blood serum.

FT-Raman spectroscopic analysis of enhanced activity of supercritical carbon dioxide treated bacterial alpha-amylase.

PubMed

Paul, Kaninika; Dutta, Sayantani; Bhattacharjee, Paramita

2017-09-01

Our previous investigation on high pressure supercritical carbon dioxide treatment of a bacterial α-amylase had revealed enhanced activity of the same. 1 H NMR analysis of the activity enhanced enzyme led the authors to hypothesize that the enhancement was possibly owing to alterations in the active site of the enzyme. In the present study, the changes in the active site of the treated enzyme was analysed by Fourier-transform Raman (FT-Raman) spectroscopy. The spectra obtained revealed shifting of bands in the active site of α-amylase indicating a nudging effect of the bonds in this region consequent to high pressure treatment. Also, shifts in bands in the OH stretching vibration of water were observed in the enzyme spectra. These variations in the spectra confirmed changes in the active site as well as in the water associated with the same that perhaps had a concerted effect on the increased activity of α-amylase. Copyright © 2017 Elsevier Inc. All rights reserved.

Crosslinked Enzyme Aggregates in Hierarchically-Ordered Mesoporous Silica: A Simple and Effective Method for Enzyme Stabilization

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kim, Moon Il; Kim, Jungbae; Lee, Jinwoo

2007-02-01

alpha-chymotrypsin (CT) and lipase (LP) were immobilized in hierarchically-ordered mesocellular mesoporous silica (HMMS) in a simple but effective way for the enzyme stabilization, which was achieved by the enzyme adsorption followed by glutaraldehyde (GA) crosslinking. This resulted in the formation of nanometer scale crosslinked enzyme aggregates (CLEAs) entrapped in the mesocellular pores of HMMS (37 nm), which did not leach out of HMMS through narrow mesoporous channels (13 nm). CLEA of alpha-chymotrypsin (CLEA-CT) in HMMS showed a high enzyme loading capacity and significantly increased enzyme stability. No activity decrease of CLEA-CT was observed for two weeks under even rigorously shakingmore » condition, while adsorbed CT in HMMS and free CT showed a rapid inactivation due to the enzyme leaching and presumably autolysis, respectively. With the CLEA-CT in HMMS, however, there was no tryptic digestion observed suggesting that the CLEA-CT is not susceptible to autolysis. Moreover, CLEA of lipase (CLEA-LP) in HMMS retained 30% specific activity of free lipase with greatly enhanced stability. This work demonstrates that HMMS can be efficiently employed as host materials for enzyme immobilization leading to highly enhanced stability of the immobilized enzymes with high enzyme loading and activity.« less

Dietary Immunogen® modulated digestive enzyme activity and immune gene expression in Litopenaeus vannamei post larvae.

PubMed

Miandare, Hamed Kolangi; Mirghaed, Ali Taheri; Hosseini, Marjan; Mazloumi, Nastaran; Zargar, Ashkan; Nazari, Sajad

2017-11-01

Pacific white shrimp Litopenaeus vannamei (Boone, 1931) is an important economical shrimp species worldwide, especially in the Middle East region, and farming activities of this species have been largely affected by diseases, mostly viral and bacterial diseases. Scientists have started to use prebiotics for bolstering the immune status of the animal. This study aimed to investigate the influence of Immunogen ® on growth, digestive enzyme activity and immune related gene expression of Litopenaeus vannamei post-larvae. All post-larvae were acclimated to the laboratory condition for 14 days. Upon acclimation, shrimps were fed on different levels of Immunogen ® (0, 0.5, 1 and 1.5 g kg -1 ) for 60 days. No significant differences were detected in weight gain, specific growth rate (SGR) and food conversion ratio (FCR) in shrimp post-larvae in which fed with different levels of Immunogen ® and control diet. The results showed that digestive enzymes activity including protease and lipase increased with different amounts of Immunogen ® in the shrimp diet. Protease activity increased with 1.5 g kg -1 Immunogen ® after 60 days and lipase activity increased with 1 and 1.5 g kg -1 Immunogen ® after 30 and 60 days of the trial respectively (P < 0.05), while amylase activity did not change in response to different levels of Immunogen ® (P > 0.05). The expression of immune related genes including, prophenoloxidase, crustin and g-type lysozyme increased with diet 1.5 g kg -1 Immunogen ® (P < 0.05) while expression of penaeidin gene increased only with experimental diet 1 g kg -1 of Immunogen ® . These results indicated that increase in digestive enzymes activity and expression of immune related genes could modulate the Immunogen ® in the innate immune system in L. vannamei in this study. Copyright © 2017. Published by Elsevier Ltd.

Experiment K-6-21. Effect of microgravity on 1) metabolic enzymes of type 1 and type 2 muscle fibers and on 2) metabolic enzymes, neutransmitter amino acids, and neurotransmitter associated enzymes in motor and somatosensory cerebral cortex. Part 1: Metabolic enzymes of individual muscle fibers; part 2: metabolic enzymes of hippocampus and spinal cord

NASA Technical Reports Server (NTRS)

Lowry, O.; Mcdougal, D., Jr.; Nemeth, Patti M.; Maggie, M.-Y. Chi; Pusateri, M.; Carter, J.; Manchester, J.; Norris, Beverly; Krasnov, I.

1990-01-01

The individual fibers of any individual muscle vary greatly in enzyme composition, a fact which is obscured when enzyme levels of a whole muscle are measured. The purpose of this study was therefore to assess the changes due to weightless on the enzyme patterns composed by the individual fibers within the flight muscles. In spite of the limitation in numbers of muscles examined, it is apparent that: (1) that the size of individual fibers (i.e., their dry weight) was reduced about a third, (2) that this loss in dry mass was accompanied by changes in the eight enzymes studied, and (3) that these changes were different for the two muscles, and different for the two enzyme groups. In the soleus muscle the absolute amounts of the three enzymes of oxidative metabolism decreased about in proportion to the dry weight loss, so that their concentration in the atrophic fibers was almost unchanged. In contrast, there was little loss among the four enzymes of glycogenolysis - glycolysis so that their concentrations were substantially increased in the atrophic fibers. In the TA muscle, these seven enzymes were affected in just the opposite direction. There appeared to be no absolute loss among the oxidative enzymes, whereas the glycogenolytic enzymes were reduced by nearly half, so that the concentrations of the first metabolic group were increased within the atrophic fibers and the concentrations of the second group were only marginally decreased. The behavior of hexokinase was exceptional in that it did not decrease in absolute terms in either type of muscle and probably increased as much as 50 percent in soleus. Thus, their was a large increase in concentration of this enzyme in the atrophied fibers of both muscles. Another clear-cut finding was the large increase in the range of activities of the glycolytic enzymes among individual fibers of TA muscles. This was due to the emergence of TA fibers with activities for enzymes of this group extending down to levels as low as those found in control soleus muscles. It would be interesting to know if this represents a transition stage, and whether with prolonged weightlessness most of the fibers would be transformed into a low glycogenolytic type.

Regulation of accumulation of ammonium-inducible glutamate dehydrogenase catalytic activity and antigen during the cell cycle of fully induced, synchronous Chlorella sorokiniana cells.

PubMed

Yeung, A T; Bascomb, N F; Turner, K J; Schmidt, R R

1981-05-01

By use of a rocket immunoelectrophoresis-activity stain procedure, it was shown that catalytic activity of an ammonium-inducible nicotinamide adenine dinucleotide phosphate-specific glutamate dehydrogenase (NADP-GDH) was accompanied by a coincident increase in enzyme antigen during the cell cycle of preinduced synchronous Chlorella sorokiniana cells growing in the continuous presence of ammonia. Between the fourth and fifth hours of the G-1 phase of the cell cycle, a three- to fourfold increase in linear accumulation of enzyme antigen was observed. Pulse-chase studies with [35S]sulfate, coupled with a specific indirect immunoadsorption procedure for enzyme antigen, showed that NADP-GDH antigen undergoes continuous degradation (i.e., a half-life of 88 to 110 min) during its linear pattern of accumulation during the cell cycle. The apparent half-life of the enzyme increased by approximately 23% of the 4.5-h positive rate change in antigen accumulation during the cell cycle. This increase in half-life is insufficient in itself to account for the large change in rate of NADP-GDH antigen accumulation. The data from immunoelectrophoresis, pulse-chase, and initial 35S incorporation rate experiments taken together support the inference that changes in the rate of NADP-GDH synthesis are primarily responsible for the accumulation patterns of NADP-GDH activity during the C. sorokiniana cell cycle.

Energetic aspects of the light activation of two chloroplast enzymes: fructose-1,6-bisphosphatase and NADP-malate dehydrogenase.

PubMed

Miginiac-Maslow, M; Jacquot, J P; Droux, M

1985-09-01

The light energy requirements for photoactivation of two chloroplast enzymes: fructose-1,6-bisphosphatase and NADP-malate dehydrogenase were studied in a reconstituted chloroplast system. This system comprised isolated pea thylakoids, ferredoxin (Fd), ferredoxin-thioredoxin reductase (FTR) thioredoxinm and f (Tdm, Tdf) and the photoactivatable enzyme. Light-saturation curves of the photoactivation process were established with once washed thylakoids which did not require the addition of Td for light activation. They exhibited a plateau at 10 W·m(-2) under nitrogen and 50 W·m(-2) under air, while NADP photoreduction was saturated at 240 W·m(-2). Cyclic and pseudocyclic phosphorylations saturated at identical levels as enzyme photoactivations. All these observations suggested that the shift of the light saturation plateau towards higher values under air was due to competing oxygen-dependent reactions. With twice washed thylakoids, which required Td for enzyme light-activation, photophosphorylation was stimulated under N2 by the addition of the components of the photoactivation system. Its rate increased with increasing Td concentrations, just as did the enzyme photoactivation rate, while varying the target enzyme concentration had only a weak effect. Considering that Td concentrations were in a large excess over target enzyme concentrations, it may be assumed that the observed ATP synthesis was essentially dependent on the rate of Td reduction.Under air, Fd-dependent pseudo-cyclic photophosphorylation was not stimulated by the addition of the other enzyme photoactivation components, suggesting that an important site of action of O2 was located at the level of Fd.

Characterization of Sugar Contents and Sucrose Metabolizing Enzymes in Developing Leaves of Hevea brasiliensis

PubMed Central

Zhu, Jinheng; Qi, Jiyan; Fang, Yongjun; Xiao, Xiaohu; Li, Jiuhui; Lan, Jixian; Tang, Chaorong

2018-01-01

Sucrose-metabolizing enzymes in plant leaves have hitherto been investigated mainly in temperate plants, and rarely conducted in tandem with gene expression and sugar analysis. Here, we investigated the sugar content, gene expression, and the activity of sucrose-metabolizing enzymes in the leaves of Hevea brasiliensis, a tropical tree widely cultivated for natural rubber. Sucrose, fructose and glucose were the major sugars detected in Hevea leaves at four developmental stages (I to IV), with starch and quebrachitol as minor saccharides. Fructose and glucose contents increased until stage III, but decreased strongly at stage IV (mature leaves). On the other hand, sucrose increased continuously throughout leaf development. Activities of all sucrose-cleaving enzymes decreased markedly at maturation, consistent with transcript decline for most of their encoding genes. Activity of sucrose phosphate synthase (SPS) was low in spite of its high transcript levels at maturation. Hence, the high sucrose content in mature leaves was not due to increased sucrose-synthesizing activity, but more to the decline in sucrose cleavage. Gene expression and activities of sucrose-metabolizing enzymes in Hevea leaves showed striking differences compared with other plants. Unlike in most other species where vacuolar invertase predominates in sucrose cleavage in developing leaves, cytoplasmic invertase and sucrose synthase (cleavage direction) also featured prominently in Hevea. Whereas SPS is normally responsible for sucrose synthesis in plant leaves, sucrose synthase (synthesis direction) was comparable or higher than that of SPS in Hevea leaves. Mature Hevea leaves had an unusually high sucrose:starch ratio of about 11, the highest reported to date in plants. PMID:29449852

Complex of vitamins and antioxidants protects low-density lipoproteins in blood plasma from free radical oxidation and activates antioxidants enzymes in erythrocytes from patients with coronary heart disease.

PubMed

Konovalova, G G; Lankin, V Z; Tikhaze, A K; Nezhdanova, I B; Lisina, M O; Kukharchuk, V V

2003-08-01

We studied the effect of a complex containing antioxidant vitamins C and E, provitamin A, and antioxidant element selenium on the contents of primary (lipid peroxides) and secondary products (malonic dialdehyde) of free radical lipid oxidation in low-density lipoproteins isolated from the plasma of patients with coronary heart disease and hypercholesterolemia by means of preparative ultracentrifugation. Activity of key antioxidant enzymes in the blood was measured during treatment with the antioxidant preparation. Combination treatment with antioxidant vitamins and antioxidant element selenium sharply decreased the contents of primary and secondary free radical oxidation products in circulating low-density lipoproteins and increased activity of antioxidant enzymes in erythrocytes. Activities of superoxide dismutase and selenium-containing glutathione peroxidase increased 1 and 2 months after the start of therapy, respectively.

Reconciling Apparent Variability in Effects of Biochar Amendment on Soil Enzyme Activities by Assay Optimization

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bailey, Vanessa L.; Fansler, Sarah J.; Smith, Jeffery L.

2011-02-01

Applying biochar to soils as an ameliorative substance and mechanism for C sequestration has received a great deal of interest in light of the sustained fertility observed in the Terra Preta soils of Brazil. The effects of synthetic biochars on biochemical processes needs to be better understood in order to determine if this is a reasonable practice in managed systems. The biochar studied was formed from the fast-pyrolysis of a switchgrass feedstock. Four soil enzymes were studied: β-glucosidase, β-N-acetylglucosaminidase, lipase, and leucine aminopeptidase. Both colorimetric and fluorescent assays were used for β-glucosidase and β-N-acetylglucosaminidase. Seven days after biochar was addedmore » to microcosms of a Palouse silt loam, the fluorescence-based assays indicated increased activities of the four enzymes, compared to non-amended soil. To clarify the mechanisms of the observed effects,in the absence of soil, purified enzymes or substrates were briefly exposed to biochar and then assayed. Except for β-N-acetylglucosaminidase, the exposure of substrate to biochar reduced the apparent activity of the remaining three enzymes in vitro, suggesting that sorption reactions between the substrate and biochar either removed the substrate from the assays or impeded the enzyme binding. The activity of purified β-N-acetylglucosaminidase increased significantly following biochar exposure, suggesting a chemical stimulation of enzyme functioning. We conclude that biochar added to soil acts as a substrate that can stimulate the soil microbial biomass and its activity. Our in vitro study suggests that biochar is not biochemically inert. Biochar amendments are likely to have effects that are currently difficult to predict, and that could impact overall soil function.« less

Influence of protoplast fusion between two Trichoderma spp. on extracellular enzymes production and antagonistic activity.

PubMed

Hassan, Mohamed M

2014-11-02

Biological control plays a crucial role in grapevine pathogens disease management. The cell-wall degrading enzymes chitinase, cellulase and β-glucanase have been suggested to be essential for the mycoparasitism activity of Trichoderma species against grapevine fungal pathogens. In order to develop a useful strain as a single source of these vital enzymes, it was intended to incorporate the characteristics of two parental fungicides tolerant mutants of Trichoderma belonging to the high chitinase producing species T. harzianum and the high cellulase producing species T. viride , by fusing their protoplasts. The phylogeny of the parental strains was carried out using a sequence of the 5.8S-ITS region. The BLAST of the obtained sequence identified these isolates as T. harzianum and T. viride . Protoplasts were isolated using lysing enzymes and were fused using polyethylene glycol. The fused protoplasts have been regenerated on protoplast regeneration minimal medium supplemented with two selective fungicides. Among the 40 fast growing fusants, 17 fusants were selected based on their enhanced growth on selective media for further studies. The fusant strains were growing 60%-70% faster than the parents up to third generation. All the 17 selected fusants exhibited morphological variations. Some fusant strains displayed threefold increased chitinase enzyme activity and twofold increase in β-glucanase enzyme activity compared to the parent strains. Most fusants showed powerful antagonistic activity against Macrophomin aphaseolina , Pythium ultimum and Sclerotium rolfsii pathogens. Fusant number 15 showed the highest inhibition percentage (92.8%) against M. phaseolina and P. ultimum, while fusant number 9 showed the highest inhibition percentage (98.2%) against the growth of S. rolfsii. A hyphal intertwining and degradation phenomenon was observed by scanning electron microscope. The Trichoderma antagonistic effect against pathogenic fungal mycelia was due to the mycoparasitism effect of the extracellular enzymes.

Statistical Optimization of Fibrinolytic Enzyme Production Using Agroresidues by Bacillus cereus IND1 and Its Thrombolytic Activity In Vitro

PubMed Central

Prakash Vincent, Samuel Gnana

2014-01-01

A potent fibrinolytic enzyme-producing Bacillus cereus IND1 was isolated from the Indian food, rice. Solid-state fermentation was carried out using agroresidues for the production of fibrinolytic enzyme. Among the substrates, wheat bran supported more enzyme production and has been used for the optimized enzyme production by statistical approach. Two-level full-factorial design demonstrated that moisture, supplementation of beef extract, and sodium dihydrogen phosphate have significantly influenced enzyme production (P < 0.05). A central composite design resulted in the production of 3699 U/mL of enzyme in the presence of 0.3% (w/w) beef extract and 0.05% (w/w) sodium dihydrogen phosphate, at 100% (v/w) moisture after 72 h of fermentation. The enzyme production increased fourfold compared to the original medium. This enzyme was purified to homogeneity by ammonium sulfate precipitation, diethylaminoethyl-cellulose ion-exchange chromatography, Sephadex G-75 gel filtration chromatography, and casein-agarose affinity chromatography and had an apparent molecular mass of 29.5 kDa. The optimum pH and temperature for the activity of fibrinolytic enzyme were found to be 8.0 and 60°C, respectively. This enzyme was highly stable at wide pH range (7.0–9.0) and showed 27% ± 6% enzyme activity after initial denaturation at 60°C for 1 h. In vitro assays revealed that the enzyme could activate plasminogen and significantly degraded the fibrin net of blood clot, which suggests its potential as an effective thrombolytic agent. PMID:25003130

The effect of alloxan diabetes on the activity of some mixed function oxidases in male rats.

PubMed

Nedjar, A; Stoytchev, T

1990-01-01

The effect of alloxan-induced diabetes on the duration of hexobarbital sleep (HB sleep) the activity of ethylmorphine-N-demethylase (EMND), aniline hydroxylase (AH), the content of microsomal cytochrome P-450 and b5, on the activity of ethoxycumarine-0-deethylase (ECOD) and ethoxyresorufine-0-deethylase (EROD) after induction with beta naphthoflavone (beta-NF), as well as the activity of benzphetamine-N-demethylase and pentoxyresorufine-O-dealkylase (PROD) after induction with phenobarbital (PB), was studied in experiments on male Wistar rats. In rats with alloxan diabetes there was a significant prolongation of HB sleep (by 106%) and inhibition of the liver EMND (by 54%), while the AH activity increased by 131%, with a parallel rise in the content of microsomal cytochromes P-450 (by 67%) and b5 (by 113%). In rats with alloxan diabetes the enzyme-inducing effect of beta-NF with respect to the activities of EROD and ECOD is reduced, although diabetes by itself causes a rise in the ECOD activity in untreated animals. When induced with PB, the PROD and benzphetamine-N-demethylase activity in diabetic rats is lower than in the healthy animals. However, if the enzyme activity after the application of inducers is referred to the respective starting enzyme activities of the two groups of animals, it is found that the enzyme-inducing effect of PB is preserved and even slightly potentiated in the diabetic rats compared with the healthy ones: the increases in the benzphetamine-N-demethylase activity is by 60% in the diabetic rats, compared with a rise of 28% in the healthy animals, of the PROD activity 19 times for the diabetic compared with 16 times increase for the healthy rats.

Litter type affects the activity of aerobic decomposers in a boreal peatland more than site nutrient and water table regimes

NASA Astrophysics Data System (ADS)

Straková, P.; Niemi, R. M.; Freeman, C.; Peltoniemi, K.; Toberman, H.; Heiskanen, I.; Fritze, H.; Laiho, R.

2011-09-01

Peatlands are carbon (C) storage ecosystems sustained by a high water table (WT). High WT creates anoxic conditions that suppress the activity of aerobic decomposers and provide conditions for peat accumulation. Peatland function can be dramatically affected by WT drawdown caused by climate and/or land-use change. Aerobic decomposers are directly affected by WT drawdown through environmental factors such as increased oxygenation and nutrient availability. Additionally, they are indirectly affected via changes in plant community composition and litter quality. We studied the relative importance of direct and indirect effects of WT drawdown on aerobic decomposer activity in plant litter at two stages of decomposition (incubated in the field for 1 or 2 years). We did this by profiling 11 extracellular enzymes involved in the mineralization of organic C, nitrogen (N), phosphorus (P) and sulphur. Our study sites represented a three-stage chronosequence from pristine to short-term (years) and long-term (decades) WT drawdown conditions under two nutrient regimes (bog and fen). The litter types included reflected the prevalent vegetation: Sphagnum mosses, graminoids, shrubs and trees. Litter type was the main factor shaping microbial activity patterns and explained about 30 % of the variation in enzyme activities and activity allocation. Overall, enzyme activities were higher in vascular plant litters compared to Sphagnum litters, and the allocation of enzyme activities towards C or nutrient acquisition was related to the initial litter quality (chemical composition). Direct effects of WT regime, site nutrient regime and litter decomposition stage (length of incubation period) summed to only about 40 % of the litter type effect. WT regime alone explained about 5 % of the variation in enzyme activities and activity allocation. Generally, enzyme activity increased following the long-term WT drawdown and the activity allocation turned from P and N acquisition towards C acquisition. This caused an increase in the rate of litter decomposition. The effects of the short-term WT drawdown were minor compared to those of the long-term WT drawdown: e.g., the increase in the activity of C-acquiring enzymes was up to 120 % (bog) or 320 % (fen) higher after the long-term WT drawdown compared to the short-term WT drawdown. In general, the patterns of microbial activity as well as their responses to WT drawdown depended on peatland type: e.g., the shift in activity allocation to C-acquisition was up to 100 % stronger at the fen compared to the bog. Our results imply that changes in plant community composition in response to persistent WT drawdown will strongly affect the C dynamics of peatlands. The predictions of decomposer activity under changing climate and/or land-use thus cannot be based on the direct effects of the changed environment only, but need to consider the indirect effects of environmental changes: the changes in plant community composition, their dependence on peatland type, and their time scale.

Effect of additives on the activity of tannase from Aspergillus awamori MTCC9299.

PubMed

Chhokar, Vinod; Sangwan, Meenakshi; Beniwal, Vikas; Nehra, Kiran; Nehra, Kaur S

2010-04-01

Tannase from Aspergillus awamori MTCC 9299 was purified using ammonium sulfate precipitation followed by ion-exchange chromatography. A purification fold of 19.5 with 13.5% yield was obtained. Temperature of 30 degrees C and pH of 5.5 were found optimum for tannase activity. The effects of metals and organic solvents on the activity of tannase were also studied. Metal ions Mg(+2), Mn(+2), Ca(+2), Na(+), and K(+) stimulated the tannase activity, while Cu(+2), Fe(+3), and Co(+2) acted as inhibitors of the enzyme. The addition of organic solvents like acetic acid, isoamylalcohol, chloroform, isopropyl alcohol, and ethanol completely inhibited the enzyme activity. However, butanol and benzene increased the enzyme activity.

Impact of peptidoglycan O-acetylation on autolytic activities of the Enterococcus faecalis N-acetylglucosaminidase AtlA and N-acetylmuramidase AtlB.

PubMed

Emirian, Aurélie; Fromentin, Sophie; Eckert, Catherine; Chau, Françoise; Dubost, Lionel; Delepierre, Muriel; Gutmann, Laurent; Arthur, Michel; Mesnage, Stéphane

2009-09-17

Autolysins are potentially lethal enzymes that partially hydrolyze peptidoglycan for incorporation of new precursors and septum cleavage after cell division. Here, we explored the impact of peptidoglycan O-acetylation on the enzymatic activities of Enterococcus faecalis major autolysins, the N-acetylglucosaminidase AtlA and the N-acetylmuramidase AtlB. We constructed isogenic strains with various O-acetylation levels and used them as substrates to assay E. faecalis autolysin activities. Peptidoglycan O-acetylation had a marginal inhibitory impact on the activities of these enzymes. In contrast, removal of cell wall glycopolymers increased the AtlB activity (37-fold), suggesting that these polymers negatively control the activity of this enzyme.

Selective activation of heme oxygenase-2 by menadione.

PubMed

Vukomanovic, Dragic; McLaughlin, Brian E; Rahman, Mona N; Szarek, Walter A; Brien, James F; Jia, Zongchao; Nakatsu, Kanji

2011-11-01

While substantial progress has been made in elucidating the roles of heme oxygenases-1 (HO-1) and -2 (HO-2) in mammals, our understanding of the functions of these enzymes in health and disease is still incomplete. A significant amount of our knowledge has been garnered through the use of nonselective inhibitors of HOs, and our laboratory has recently described more selective inhibitors for HO-1. In addition, our appreciation of HO-1 has benefitted from the availability of tools for increasing its activity through enzyme induction. By comparison, there is a paucity of information about HO-2 activation, with only a few reports appearing in the literature. This communication describes our observations of the up to 30-fold increase in the in-vitro activation of HO-2 by menadione. This activation was due to an increase in Vmax and was selective, in that menadione did not increase HO-1 activity.

Hepatoprotective effects of raspberry (Rubus coreanus Miq.) seed oil and its major constituents.

PubMed

Teng, Hui; Lin, Qiyang; Li, Kang; Yuan, Benyao; Song, Hongbo; Peng, Hongquan; Yi, Lunzhao; Wei, Ming-Chi; Yang, Yu-Chiao; Battino, Maurizio; Cespedes Acuña, Carlos L; Chen, Lei; Xiao, Jianbo

2017-12-01

Raspberry seed is a massive byproduct of raspberry juice and wine but usually discarded. The present study employed a microwave-assisted method for extraction of raspberry seed oil (RSO). The results revealed that omega-6 fatty acids (linoleic acid and γ-linolenic acid) were the major constituents in RSO. Cellular antioxidant enzyme activity such as superoxide dismutase (SOD), glutathione peroxidase (GPx), and catalase (CAT) were investigated in HepG2 cells treated with RSO. Induction of the synthesis of several antioxidants in H 2 O 2 -exposed HepG2 cells was found. RSO increased the enzyme activity of SOD, CAT, and GPx in H 2 O 2 -exposed HepG2. Furthermore, RSO inhibited the phosphorylation of upstream mitogen-activated protein kinases (MAPK) such as c-Jun N-terminal kinase (c-JNK) and extracellular signal-regulated kinase (ERK). Taken together, the possible mechanisms to increase antioxidant enzyme activities in HepG2 may through the suppression of ERK and JNK phosphorylation. Raspberry seed oil exhibited good effects on the activities of the intracellular antioxidant enzymes and seems to protect the liver from oxidative stress through the inhibition of MAPKs. Copyright © 2017. Published by Elsevier Ltd.

Glycolytic adjustments in tissues of frog Rana ridibunda and land snail Helix lucorum during seasonal hibernation.

PubMed

Michaelidis, Basile; Kyriakopoulou-Sklavounou, Pasqualina; Staikou, Alexandra; Papathanasiou, Ioanna; Konstantinou, Kiriaki

2008-12-01

The present work aimed to contribute to the understanding of the adaptation of the glycolytic pathway in tissues of frog Rana ridibunda and land snail species Helix lucorum during seasonal hibernation. Moreover responses of glycolytic enzymes from cold acclimated R. ridibunda and H. lucorum were studied as well. The drop in Po(2) in the blood of hibernated frogs and land snails indicated lower oxygen consumption and a decrease in their metabolic rate. The activities of glycolytic enzymes indicated that hibernation had a differential effect on the glycolyis in the two species studied and also in the tissues of the same species. The activity of l-LDH decreased significantly in the skeletal muscle and heart of hibernated R. ridibunda indicating a low glycolytic potential. Similar biochemical responses were observed in the same tissues during cold acclimation. The continuous increase in the activities of glycolytic enzymes studied, except for HK, might indicate a compensation for the impacts of low temperature on the enzymatic activities. In contrast to R. ridibunda, the activities of the enzymes increased and remained at higher levels than those of the prehibernation controls indicating maintenance of glycolytic potential in the tissues of hibernating land snails.

Effects of ribonuclease A on amino acid transport in Neurospora crassa.

PubMed

Stuart, W D; Woodward, D O

1975-04-01

Incubation of Neurospora crassa conidia with ribonuclease (RNase) A reduces transport of L-phenylalanine by those cells. Under similar conditions, oxidized RNase A, RNase T1, and RNase T2 do not have this effect. Incubation of conidia with active RNase covalently attached to polyacrylamide beads reduces L-phenylalanine transport. This indicates that the site of enzymatic action is at the cell surface. At the lower concentration of enzyme used in this study, incubation with RNase A reduces transport of L-phenylalanine by the general (G) amino acid permease. Increasing the enzyme concentration results in reduction of transport by the neutral aromatic (N)-specific permease. The increased transport activity that accompanies onset of conidial germination is also sensitive to incubation with RNase A. Application of the enzyme to actively transporting cells does not release amino acid transported prior to enzyme addition. Cells cultured on media supplemented with [2-14C] uridine release isotopic activity after RNase A incubation. Analogous treatments with Pronase, RNase T1, RNase T2, or deoxyribonuclease I do not release isotope activity. Pronase treatment does reduce L-phenylalanine transport. Incubation of conidia with RNase A also inhibits germination of those conidia.

Arrhenius plot for a reaction catalyzed by a single molecule of β-galactosidase.

PubMed

Craig, Douglas B; Chase, Linden N

2012-02-21

The activity of a single enzyme molecule of Escherichia coli β-galactosidase was measured using a capillary electrophoresis continuous flow assay. As the enzyme molecule traversed the capillary the incubation temperature was increased from 27 to 37 °C, providing a continuous record of the change in rate with temperature. This data was used to develop a single enzyme molecule Arrhenius plot, from which the activation energy of the reaction was determined to be 31 kJ mol(-1).

Targeting Tryptophan Catabolism: A Novel Method to Block Triple-Negative Breast Cancer Metastasis

DTIC Science & Technology

2016-04-01

in many immune cell types and its activation decreases T-cell activity leading to tumor immune escape. Since the rate limiting enzyme TDO2 increases...What were the major goals of the project? Our overall goals were to test the hypotheses that the ability to upregulate kynurenine via the enzyme TDO2...discipline(s) of the project? " o This research is strongly suggesting that TDO2 is likely the primary enzyme that catabolizes tryptophan that should

Molecular imprinting of enzymes with water-insoluble ligands for nonaqueous biocatalysis.

PubMed

Rich, Joseph O; Mozhaev, Vadim V; Dordick, Jonathan S; Clark, Douglas S; Khmelnitsky, Yuri L

2002-05-15

Attaining higher levels of catalytic activity of enzymes in organic solvents is one of the major challenges in nonaqueous enzymology. One of the most successful strategies for enhancing enzyme activity in organic solvents involves tuning the enzyme active site by molecular imprinting with substrates or their analogues. Unfortunately, numerous imprinters of potential importance are poorly soluble in water, which significantly limits the utility of this method. In the present study, we have developed strategies that overcome this limitation of the molecular-imprinting technique and that thus expand its applicability beyond water-soluble ligands. The solubility problem can be addressed either by converting the ligands into a water-soluble form or by adding relatively high concentrations of organic cosolvents, such as tert-butyl alcohol and 1,4-dioxane, to increase their solubility in the lyophilization medium. We have succeeded in applying both of these strategies to produce imprinted thermolysin, subtilisin, and lipase TL possessing up to 26-fold higher catalytic activity in the acylation of paclitaxel and 17beta-estradiol compared to nonimprinted enzymes. Furthermore, we have demonstrated for the first time that molecular imprinting and salt activation, applied in combination, produce a strong additive activation effect (up to 110-fold), suggesting different mechanisms of action involved in these enzyme activation techniques.

How exogenous nitric oxide regulates nitrogen assimilation in wheat seedlings under different nitrogen sources and levels

PubMed Central

Balotf, Sadegh; Islam, Shahidul; Kavoosi, Gholamreza; Kholdebarin, Bahman; Juhasz, Angela

2018-01-01

Nitrogen (N) is one of the most important nutrients for plants and nitric oxide (NO) as a signaling plant growth regulator involved in nitrogen assimilation. Understanding the influence of exogenous NO on nitrogen metabolism at the gene expression and enzyme activity levels under different sources of nitrogen is vitally important for increasing nitrogen use efficiency (NUE). This study investigated the expression of key genes and enzymes in relation to nitrogen assimilation in two Australian wheat cultivars, a popular high NUE cv. Spitfire and a normal NUE cv. Westonia, under different combinations of nitrogen and sodium nitroprusside (SNP) as the NO donor. Application of NO increased the gene expressions and activities of nitrogen assimilation pathway enzymes in both cultivars at low levels of nitrogen. At high nitrogen supplies, the expressions and activities of N assimilation genes increased in response to exogenous NO only in cv. Spitfire but not in cv. Westonia. Exogenous NO caused an increase in leaf NO content at low N supplies in both cultivars, while under high nitrogen treatments, cv. Spitfire showed an increase under ammonium nitrate (NH4NO3) treatment but cv. Westonia was not affected. N assimilation gene expression and enzyme activity showed a clear relationship between exogenous NO, N concentration and N forms in primary plant nitrogen assimilation. Results reveal the possible role of NO and different nitrogen sources on nitrogen assimilation in Triticum aestivum plants. PMID:29320529

How exogenous nitric oxide regulates nitrogen assimilation in wheat seedlings under different nitrogen sources and levels.

PubMed

Balotf, Sadegh; Islam, Shahidul; Kavoosi, Gholamreza; Kholdebarin, Bahman; Juhasz, Angela; Ma, Wujun

2018-01-01

Nitrogen (N) is one of the most important nutrients for plants and nitric oxide (NO) as a signaling plant growth regulator involved in nitrogen assimilation. Understanding the influence of exogenous NO on nitrogen metabolism at the gene expression and enzyme activity levels under different sources of nitrogen is vitally important for increasing nitrogen use efficiency (NUE). This study investigated the expression of key genes and enzymes in relation to nitrogen assimilation in two Australian wheat cultivars, a popular high NUE cv. Spitfire and a normal NUE cv. Westonia, under different combinations of nitrogen and sodium nitroprusside (SNP) as the NO donor. Application of NO increased the gene expressions and activities of nitrogen assimilation pathway enzymes in both cultivars at low levels of nitrogen. At high nitrogen supplies, the expressions and activities of N assimilation genes increased in response to exogenous NO only in cv. Spitfire but not in cv. Westonia. Exogenous NO caused an increase in leaf NO content at low N supplies in both cultivars, while under high nitrogen treatments, cv. Spitfire showed an increase under ammonium nitrate (NH4NO3) treatment but cv. Westonia was not affected. N assimilation gene expression and enzyme activity showed a clear relationship between exogenous NO, N concentration and N forms in primary plant nitrogen assimilation. Results reveal the possible role of NO and different nitrogen sources on nitrogen assimilation in Triticum aestivum plants.

Oxidative Stress and Antioxidants in Tomato (Solanum lycopersicum) Plants Subjected to Boron Toxicity

PubMed Central

Cervilla, Luis M.; Blasco, Begoña; Ríos, Juan J.; Romero, Luis; Ruiz, Juan M.

2007-01-01

Background and Aims Boron (B) toxicity triggers the formation of reactive oxygen species in plant tissues. However, there is still a lack of knowledge as to how B toxicity affects the plant antioxidant defence system. It has been suggested that ascorbate could be important against B stress, although existing information is limited in this respect. The objective of this study was to analyse how ascorbate and some other components of the antioxidant network respond to B toxicity. Methods Two tomato (Solanum lycopersicum) cultivars (‘Kosaco’ and ‘Josefina’) were subjected to 0·05 (control), 0·5 and 2 mm B. The following were studied in leaves: dry weight; relative leaf growth rate; total and free B; H2O2; malondialdehyde; ascorbate; glutathione; sugars; total non-enzymatic antioxidant activity, and the activity of superoxide dismutase, catalase, ascorbate peroxidase, monodehydroascorbate reductase, dehydroascorbate reductase, glutathione reductase, ascorbate oxidase and l-galactose dehydrogenase. Key Results The B-toxicity treatments diminished growth and boosted the amount of B, malondialdehyde and H2O2 in the leaves of the two cultivars, these trends being more pronounced in ‘Josefina’ than in ‘Kosaco’. B toxicity increased ascorbate concentration in both cultivars and increased glutathione only in ‘Kosaco’. Activities of antioxidant- and ascorbate-metabolizing enzymes were also induced. Conclusions High B concentration in the culture medium provokes oxidative damage in tomato leaves and induces a general increase in antioxidant enzyme activity. In particular, B toxicity increased ascorbate pool size. It also increased the activity of l-galactose dehydrogenase, an enzyme involved in ascorbate biosynthesis, and the activity of enzymes of the Halliwell–Asada cycle. This work therefore provides a starting point towards a better understanding of the role of ascorbate in the plant response against B stress. PMID:17660516

Comparison of the effects of agalsidase alfa and agalsidase beta on cultured human Fabry fibroblasts and Fabry mice.

PubMed

Sakuraba, Hitoshi; Murata-Ohsawa, Mai; Kawashima, Ikuo; Tajima, Youichi; Kotani, Masaharu; Ohshima, Toshio; Chiba, Yasunori; Takashiba, Minako; Jigami, Yoshifumi; Fukushige, Tomoko; Kanzaki, Tamotsu; Itoh, Kohji

2006-01-01

We compared two recombinant alpha-galactosidases developed for enzyme replacement therapy for Fabry disease, agalsidase alfa and agalsidase beta, as to specific alpha-galactosidase activity, stability in plasma, mannose 6-phosphate (M6P) residue content, and effects on cultured human Fabry fibroblasts and Fabry mice. The specific enzyme activities of agalsidase alfa and agalsidase beta were 1.70 and 3.24 mmol h(-1) mg protein(-1), respectively, and there was no difference in stability in plasma between them. The M6P content of agalsidase beta (3.6 mol/mol protein) was higher than that of agalsidase alfa (1.3 mol/mol protein). The administration of both enzymes resulted in marked increases in alpha-galactosidase activity in cultured human Fabry fibroblasts, and Fabry mouse kidneys, heart, spleen and liver. However, the increase in enzyme activity in cultured fibroblasts, kidneys, heart and spleen was higher when agalsidase beta was used. An immunocytochemical analysis revealed that the incorporated recombinant enzyme degraded the globotriaosyl ceramide accumulated in cultured Fabry fibroblasts in a dose-dependent manner, with the effect being maintained for at least 7 days. Repeated administration of agalsidase beta apparently decreased the number of accumulated lamellar inclusion bodies in renal tubular cells of Fabry mice.

Low operational stability of enzymes in dry organic solvents: changes in the active site might affect catalysis.

PubMed

Bansal, Vibha; Delgado, Yamixa; Legault, Marc; Barletta, Gabriel

2012-02-14

The potential of enzyme catalysis in organic solvents for synthetic applications has been overshadowed by the fact that their catalytic properties are affected by organic solvents. In addition, it has recently been shown that an enzyme's initial activity diminishes considerably after prolonged exposure to organic media. Studies geared towards understanding this last drawback have yielded unclear results. In the present work we decided to use electron paramagnetic resonance spectroscopy (EPR) to study the motion of an active site spin label (a nitroxide free radical) during 96 h of exposure of the serine protease subtilisin Carlsberg to four different organic solvents. Our EPR data shows a typical two component spectra that was quantified by the ratio of the anisotropic and isotropic signals. The isotropic component, associated with a mobile nitroxide free radical, increases during prolonged exposure to all solvents used in the study. The maximum increase (of 43%) was observed in 1,4-dioxane. Based on these and previous studies we suggest that prolonged exposure of the enzyme to these solvents provokes a cascade of events that could induce substrates to adopt different binding conformations. This is the first EPR study of the motion of an active-site spin label during prolonged exposure of an enzyme to organic solvents ever reported.

The effects of ingested petroleum on the maphthalene-metabolizing properties of the liver tissue in seawater-adapted mallard ducks (Anas platyrhynchos)

USGS Publications Warehouse

Gorsline, J.; Holmes, W.N.; Cronshaw, J.

1981-01-01

Hepatic mixed function oxidase activities were estimated in seawater-adapted mallard ducks (Anas platyrhynchos) that had been consuming food contaminated with one of five different types of crude oil. After 50 days of exposure to contaminated food, enzyme activities of liver microsomal preparations were assessed in terms of their naphthalenemetabolizing properties in vitro. Although dose-dependent increases in the total hepatic enzyme activities (nmole naphthalene metabolized per minute per unit mass body weight) were observed in birds consuming food contaminated with each type of crude oil, three patterns of response were apparent. Crude oils from South Louisiana and Kuwait stimulated large and significant increases in the specific activity of the enzyme system (nmole naphthalene metabolized per minute per unit mass microsomal protein), whereas little or no increase in either microsomal protein content or relative liver weight were observed. In contrast, two crude oils from Santa Barbara, Calif., induced only small increases in specific activity but significant increases occurred in hepatic microsomal protein concentration and relative liver weight. The crude oil from Prudhoe Bay, Ala., evoked intermediate patterns of response. The possible significance of these data is discussed in relation to the survival of seabirds consuming petroleum-contaminated food and drinking water.

Combined Effects of Lanthanum (III) and Acid Rain on Antioxidant Enzyme System in Soybean Roots.

PubMed

Zhang, Xuanbo; Du, Yuping; Wang, Lihong; Zhou, Qing; Huang, Xiaohua; Sun, Zhaoguo

2015-01-01

Rare earth element pollution (REEs) and acid rain (AR) pollution simultaneously occur in many regions, which resulted in a new environmental issue, the combined pollution of REEs and AR. The effects of the combined pollution on the antioxidant enzyme system of plant roots have not been reported. Here, the combined effects of lanthanum ion (La3+), one type of REE, and AR on the antioxidant enzyme system of soybean roots were investigated. In the combined treatment of La3+ (0.08 mM) and AR, the cell membrane permeability and the peroxidation of cell membrane lipid of soybean roots increased, and the superoxide dismutase, catalase, peroxidase and reduced ascorbic acid served as scavengers of reactive oxygen species. In other combined treatments of La3+ (0.40 mM, 1.20 mM) and AR, the membrane permeability, malonyldialdehyde content, superoxide dismutase activity, peroxidase activity and reduced ascorbic acid content increased, while the catalase activity decreased. The increased superoxide dismutase activity, peroxidase activity and reduced ascorbic acid content were inadequate to scavenge the excess hydrogen peroxide and superoxide, leading to the damage of the cell membrane, which was aggravated with the increase in the concentration of La3+ and the level of AR. The deleterious effects of the combined treatment of La3+ and AR were stronger than those of the single treatment of La3+ or AR. Moreover, the activity of antioxidant enzyme system in the combined treatment group was affected directly and indirectly by mineral element content in soybean plants.

Combined Effects of Lanthanum (III) and Acid Rain on Antioxidant Enzyme System in Soybean Roots

PubMed Central

Zhang, Xuanbo; Du, Yuping; Wang, Lihong; Zhou, Qing; Huang, Xiaohua; Sun, Zhaoguo

2015-01-01

Rare earth element pollution (REEs) and acid rain (AR) pollution simultaneously occur in many regions, which resulted in a new environmental issue, the combined pollution of REEs and AR. The effects of the combined pollution on the antioxidant enzyme system of plant roots have not been reported. Here, the combined effects of lanthanum ion (La3+), one type of REE, and AR on the antioxidant enzyme system of soybean roots were investigated. In the combined treatment of La3+ (0.08 mM) and AR, the cell membrane permeability and the peroxidation of cell membrane lipid of soybean roots increased, and the superoxide dismutase, catalase, peroxidase and reduced ascorbic acid served as scavengers of reactive oxygen species. In other combined treatments of La3+ (0.40 mM, 1.20 mM) and AR, the membrane permeability, malonyldialdehyde content, superoxide dismutase activity, peroxidase activity and reduced ascorbic acid content increased, while the catalase activity decreased. The increased superoxide dismutase activity, peroxidase activity and reduced ascorbic acid content were inadequate to scavenge the excess hydrogen peroxide and superoxide, leading to the damage of the cell membrane, which was aggravated with the increase in the concentration of La3+ and the level of AR. The deleterious effects of the combined treatment of La3+ and AR were stronger than those of the single treatment of La3+ or AR. Moreover, the activity of antioxidant enzyme system in the combined treatment group was affected directly and indirectly by mineral element content in soybean plants. PMID:26230263

Response of oxidative enzyme activities to nitrogen deposition affects soil concentrations of dissolved organic carbon

USGS Publications Warehouse

Waldrop, M.P.; Zak, D.R.

2006-01-01

Recent evidence suggests that atmospheric nitrate (NO3- ) deposition can alter soil carbon (C) storage by directly affecting the activity of lignin-degrading soil fungi. In a laboratory experiment, we studied the direct influence of increasing soil NO 3- concentration on microbial C cycling in three different ecosystems: black oak-white oak (BOWO), sugar maple-red oak (SMRO), and sugar maple-basswood (SMBW). These ecosystems span a broad range of litter biochemistry and recalcitrance; the BOWO ecosystem contains the highest litter lignin content, SMRO had intermediate lignin content, and SMBW leaf litter has the lowest lignin content. We hypothesized that increasing soil solution NO 3- would reduce lignolytic activity in the BOWO ecosystem, due to a high abundance of white-rot fungi and lignin-rich leaf litter. Due to the low lignin content of litter in the SMBW, we further reasoned that the NO3- repression of lignolytic activity would be less dramatic due to a lower relative abundance of white-rot basidiomycetes; the response in the SMRO ecosystem should be intermediate. We increased soil solution NO3- concentrations in a 73-day laboratory incubation and measured microbial respiration and soil solution dissolved organic carbon (DOC) and phenolics concentrations. At the end of the incubation, we measured the activity of ??-glucosidase, N-acetyl-glucosaminidase, phenol oxidase, and peroxidase, which are extracellular enzymes involved with cellulose and lignin degradation. We quantified the fungal biomass, and we also used fungal ribosomal intergenic spacer analysis (RISA) to gain insight into fungal community composition. In the BOWO ecosystem, increasing NO 3- significantly decreased oxidative enzyme activities (-30% to -54%) and increased DOC (+32% upper limit) and phenolic (+77% upper limit) concentrations. In the SMRO ecosystem, we observed a significant decrease in phenol oxidase activity (-73% lower limit) and an increase in soluble phenolic concentrations (+57% upper limit) in response to increasing NO 3- in soil solution, but there was no significant change in DOC concentration. In contrast to these patterns, increasing soil solution NO3- in the SMBW soil resulted in significantly greater phenol oxidase activity (+700% upper limit) and a trend toward lower DOC production (-52% lower limit). Nitrate concentration had no effect on microbial respiration or ??-glucosidase or N-acetyl-glucosaminidase activities. Fungal abundance and basidiomycete diversity tended to be highest in the BOWO soil and lowest in the SMBW, but neither displayed a consistent response to NO 3- additions. Taken together, our results demonstrate that oxidative enzyme production by microbial communities responds directly to NO3- deposition, controlling extracellular enzyme activity and DOC flux. The regulation of oxidative enzymes by different microbial communities in response to NO3- deposition highlights the fact that the composition and function of soil microbial communities directly control ecosystem-level responses to environmental change. ?? 2006 Springer Science+Business Media, Inc.

Regulation of aflatoxin biosynthesis: effect of glucose on activities of various glycolytic enzymes.

PubMed Central

Buchanan, R L; Lewis, D F

1984-01-01

Catabolism of carbohydrates has been implicated in the regulation of aflatoxin synthesis. To characterize this effect further, the activities of various enzymes associated with glucose catabolism were determined in Aspergillus parasiticus organisms that were initially cultured in peptone-mineral salts medium and then transferred to glucose-mineral salts and peptone-mineral salts media. After an initial increase in activity, the levels of glucose 6-phosphate dehydrogenase, mannitol dehydrogenase, and malate dehydrogenase were lowered in the presence of glucose. Phosphofructokinase activity was greater in the peptone-grown mycelium, but fructose diphosphatase was largely unaffected by carbon source. Likewise, carbon source had relatively little effect on the activities of pyruvate kinase, malic enzyme, isocitrate-NADP dehydrogenase, and isocitrate-NAD dehydrogenase. The results suggest that glucose may, in part, regulate aflatoxin synthesis via a carbon catabolite repression of NADPH-generating and tricarboxylic acid cycle enzymes. PMID:6091545

Epoxide metabolism in the liver of mice treated with clofibrate (ethyl-alpha-(p-chlorophenoxyisobutyrate)), a peroxisome proliferator.

PubMed

Moody, D E; Loury, D N; Hammock, B D

1985-05-01

An increase in cytosolic epoxide hydrolase (cEH) activity occurs in the livers of mice treated with peroxisome proliferating-hypolipidemic-nongenotoxic carcinogens. As increases in activity of epoxide metabolizing enzymes may reflect the carcinogenic mechanism, a detailed comparison of the response of cEH, microsomal epoxide hydrolase (mEH), and cytosolic glutathione S-transferase (cGST) activities using the geometrical isomers trans- and cis-stilbene oxide as substrates has been performed in livers from mice treated with clofibrate (ethyl-alpha-(p-chlorophenoxyisobutyrate]. The maximal increase of cEH activity occurred at lower dietary doses of clofibrate (0.5%) and within a shorter time (5 days) than mEH and cGST (2%, 14 days) activity. After 14 days at 0.5% clofibrate, cEH, mEH, and cGST activities were 250, 175, and 165% and 290, 220, and 75% of control values in male and female mice, respectively. Withdrawal of clofibrate from the diet resulted in a reversion of activities to control values within 7 days. Clofibrate treatment shifted the apparent subcellular compartmentation of all three enzymatic activities with an increase in the ratio of soluble to particulate activity. In particular, the relative specific activity of all three enzymes decreased in the light mitochondrial (peroxisomal) cell fraction, and an increase of a mEH-like activity (benzo[a]pyrene-4,5-oxide and cis-stilbene oxide hydrolysis) in the cytosol occurred. Both the increase of cEH activity and the appearance of mEH-like activity in the cytosol are novel responses of epoxide metabolizing enzymes, which may be related to the novel cellular responses that follow clofibrate treatment, peroxisome proliferation, hypolipidemia, and nongenotoxic carcinogenesis.

One-step combined focused epPCR and saturation mutagenesis for thermostability evolution of a new cold-active xylanase.

PubMed

Acevedo, Juan Pablo; Reetz, Manfred T; Asenjo, Juan A; Parra, Loreto P

2017-05-01

Enzymes active at low temperature are of great interest for industrial bioprocesses due to their high efficiency at a low energy cost. One of the particularities of naturally evolved cold-active enzymes is their increased enzymatic activity at low temperature, however the low thermostability presented in this type of enzymes is still a major drawback for their application in biocatalysis. Directed evolution of cold-adapted enzymes to a more thermostable version, appears as an attractive strategy to fulfill the stability and activity requirements for the industry. This paper describes the recombinant expression and characterization of a new and highly active cold-adapted xylanase from the GH-family 10 (Xyl-L), and the use of a novel one step combined directed evolution technique that comprises saturation mutagenesis and focused epPCR as a feasible semi-rational strategy to improve the thermostability. The Xyl-L enzyme was cloned from a marine-Antarctic bacterium, Psychrobacter sp. strain 2-17, recombinantly expressed in E. coli strain BL21(DE3) and characterized enzymatically. Molecular dynamic simulations using a homology model of the catalytic domain of Xyl-L were performed to detect flexible regions and residues, which are considered to be the possible structural elements that define the thermolability of this enzyme. Mutagenic libraries were designed in order to stabilize the protein introducing mutations in some of the flexible regions and residues identified. Twelve positive mutant clones were found to improve the T 50 15 value of the enzyme, in some cases without affecting the activity at 25°C. The best mutant showed a 4.3°C increase in its T 50 15 . The efficiency of the directed evolution approach can also be expected to work in the protein engineering of stereoselectivity. Copyright © 2017 Elsevier Inc. All rights reserved.

Rigidification of the autolysis loop enhances Na[superscript +] binding to thrombin

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pozzi, Nicola; Chen, Raymond; Chen, Zhiwei

2011-09-20

Binding of Na{sup +} to thrombin ensures high activity toward physiological substrates and optimizes the procoagulant and prothrombotic roles of the enzyme in vivo. Under physiological conditions of pH and temperature, the binding affinity of Na{sup +} is weak due to large heat capacity and enthalpy changes associated with binding, and the K{sub d} = 80 mM ensures only 64% saturation of the site at the concentration of Na{sup +} in the blood (140 mM). Residues controlling Na{sup +} binding and activation have been identified. Yet, attempts to improve the interaction of Na{sup +} with thrombin and possibly increase catalyticmore » activity under physiological conditions have so far been unsuccessful. Here we report how replacement of the flexible autolysis loop of human thrombin with the homologous rigid domain of the murine enzyme results in a drastic (up to 10-fold) increase in Na{sup +} affinity and a significant improvement in the catalytic activity of the enzyme. Rigidification of the autolysis loop abolishes the heat capacity change associated with Na{sup +} binding observed in the wild-type and also increases the stability of thrombin. These findings have general relevance to protein engineering studies of clotting proteases and trypsin-like enzymes.« less

Modified TMV Particles as Beneficial Scaffolds to Present Sensor Enzymes

PubMed Central

Koch, Claudia; Wabbel, Katrin; Eber, Fabian J.; Krolla-Sidenstein, Peter; Azucena, Carlos; Gliemann, Hartmut; Eiben, Sabine; Geiger, Fania; Wege, Christina

2015-01-01

Tobacco mosaic virus (TMV) is a robust nanotubular nucleoprotein scaffold increasingly employed for the high density presentation of functional molecules such as peptides, fluorescent dyes, and antibodies. We report on its use as advantageous carrier for sensor enzymes. A TMV mutant with a cysteine residue exposed on every coat protein (CP) subunit (TMVCys) enabled the coupling of bifunctional maleimide-polyethylene glycol (PEG)-biotin linkers (TMVCys/Bio). Its surface was equipped with two streptavidin [SA]-conjugated enzymes: glucose oxidase ([SA]-GOx) and horseradish peroxidase ([SA]-HRP). At least 50% of the CPs were decorated with a linker molecule, and all thereof with active enzymes. Upon use as adapter scaffolds in conventional “high-binding” microtiter plates, TMV sticks allowed the immobilization of up to 45-fold higher catalytic activities than control samples with the same input of enzymes. Moreover, they increased storage stability and reusability in relation to enzymes applied directly to microtiter plate wells. The functionalized TMV adsorbed to solid supports showed a homogeneous distribution of the conjugated enzymes and structural integrity of the nanorods upon transmission electron and atomic force microscopy. The high surface-increase and steric accessibility of the viral scaffolds in combination with the biochemical environment provided by the plant viral coat may explain the beneficial effects. TMV can, thus, serve as a favorable multivalent nanoscale platform for the ordered presentation of bioactive proteins. PMID:26734040

Enzyme Active Site Interactions by Raman/FTIR, NMR, and Ab Initio Calculations

PubMed Central

Deng, Hua

2017-01-01

Characterization of enzyme active site structure and interactions at high resolution is important for the understanding of the enzyme catalysis. Vibrational frequency and NMR chemical shift measurements of enzyme-bound ligands are often used for such purpose when X-ray structures are not available or when higher resolution active site structures are desired. This review is focused on how ab initio calculations may be integrated with vibrational and NMR chemical shift measurements to quantitatively determine high-resolution ligand structures (up to 0.001 Å for bond length and 0.01 Å for hydrogen bonding distance) and how interaction energies between bound ligand and its surroundings at the active site may be determined. Quantitative characterization of substrate ionic states, bond polarizations, tautomeric forms, conformational changes and its interactions with surroundings in enzyme complexes that mimic ground state or transition state can provide snapshots for visualizing the substrate structural evolution along enzyme-catalyzed reaction pathway. Our results have shown that the integration of spectroscopic studies with theoretical computation greatly enhances our ability to interpret experimental data and significantly increases the reliability of the theoretical analysis. PMID:24018325

The Activity of Carbohydrate-Degrading Enzymes in the Development of Brood and Newly Emerged workers and Drones of the Carniolan Honeybee, Apis mellifera carnica

PubMed Central

Żółtowska, Krystyna; Lipiński, Zbigniew; Łopieńska-Biernat, Elżbieta; Farjan, Marek; Dmitryjuk, Małgorzata

2012-01-01

The activity of glycogen Phosphorylase and carbohydrate hydrolyzing enzymes α-amylase, glucoamylase, trehalase, and sucrase was studied in the development of the Carniolan honey bee, Apis mellifera carnica Pollman (Hymenoptera: Apidae), from newly hatched larva to freshly emerged imago of worker and drone. Phosphorolytic degradation of glycogen was significantly stronger than hydrolytic degradation in all developmental stages. Developmental profiles of hydrolase activity were similar in both sexes of brood; high activity was found in unsealed larvae, the lowest in prepupae followed by an increase in enzymatic activity. Especially intensive increases in activity occurred in the last stage of pupae and newly emerged imago. Besides α-amylase, the activities of other enzymes were higher in drone than in worker broods. Among drones, activity of glucoamylase was particularly high, ranging from around three times higher in the youngest larvae to 13 times higher in the oldest pupae. This confirms earlier suggestions about higher rates of metabolism in drone broods than in worker broods. PMID:22943407

The activity of carbohydrate-degrading enzymes in the development of brood and newly emerged workers and drones of the Carniolan honeybee, Apis mellifera carnica.

PubMed

Żółtowska, Krystyna; Lipiński, Zbigniew; Łopieńska-Biernat, Elżbieta; Farjan, Marek; Dmitryjuk, Małgorzata

2012-01-01

The activity of glycogen Phosphorylase and carbohydrate hydrolyzing enzymes α-amylase, glucoamylase, trehalase, and sucrase was studied in the development of the Carniolan honey bee, Apis mellifera carnica Pollman (Hymenoptera: Apidae), from newly hatched larva to freshly emerged imago of worker and drone. Phosphorolytic degradation of glycogen was significantly stronger than hydrolytic degradation in all developmental stages. Developmental profiles of hydrolase activity were similar in both sexes of brood; high activity was found in unsealed larvae, the lowest in prepupae followed by an increase in enzymatic activity. Especially intensive increases in activity occurred in the last stage of pupae and newly emerged imago. Besides α-amylase, the activities of other enzymes were higher in drone than in worker broods. Among drones, activity of glucoamylase was particularly high, ranging from around three times higher in the youngest larvae to 13 times higher in the oldest pupae. This confirms earlier suggestions about higher rates of metabolism in drone broods than in worker broods.

Production, thermal stability and immobilisation of inulinase from Fusarium oxysporum.

PubMed

Gupta, A K; Rathore, P; Kaur, N; Singh, R

1990-01-01

Fusarium oxysporum produced maximum extracellular inulinase after 9 days of its growth at 25 degrees C on a medium (pH 5.5) containing 3% fructan and 0.2% sodium nitrate. The level of this enzyme decreased on the addition of either glucose, fructose, galactose or sucrose to F. oxysporum already growing on a fructan-containing medium. A significant increase in invertase production which resulted in an increase of the invertase/inulinase (S/I) ratio, was observed on addition of inulin to this fungus growing on other carbon sources. Glycerol (10%) gave better protection to inulinase against thermal denaturation at 50 degrees C compared to ethylene glycol and sorbitol. Inulinase immobilised in polyacrylamide gel retained 45% of its original activity. The immobilised enzyme showed a higher optimum temperature (45 degrees C) compared to free enzyme (37 degrees C). The immobilised enzyme after storage at 25 degrees C for 96 h showed 58% activity. Thermal stability of entrapped inulinase increased in the presence of inulin.

Biochemical responses of Gammarus pulex to malachite green solutions decolorized by Coriolus versicolor as a biosorbent under batch adsorption conditions optimized with response surface methodology.

PubMed

Yildirim, Nuran Cikcikoglu; Tanyol, Mehtap; Yildirim, Numan; Serdar, Osman; Tatar, Sule

2018-07-30

The current study was aimed to investigate the detoxifying and antioxidant enzyme response of Gammarus pulex exposed to malachite green (MG) after decolorization by Coriolus versicolor. Response surface methodology (RSM) was utilized to optimize the decolorization conditions of MG synthetic solutions by C. versicolor. Glutathione (GSH), malondialdehyde (MDA) levels and glutathione peroxidase (GP X ), catalase (CAT), superoxide dismutase (SOD), glutathione S-transferase (GST), cytochrome P450 1A1 (CYP1A1) activities in G. pulex exposed to undecolorized (A1) and decolorized (A2) MG synthetic solution during 24 and 96 h were tested by using ELISA method. SOD and GP X enzyme activity was increased after decolorization (p > 0.05). CAT enzyme activity was increased in A2 group during 24 h (p > 0.05) but decreased during 96 h (p < 0.05). GSH levels were increased in A2 group during 24 and 96 h (p < 0.05). GST, CYP1A1 enzyme activity and MDA levels were decreased after decolorization during 96 h (p < 0.05). In this study, GSH levels, CAT, GST and CYP1A1 activities in G. pulex approved the capability of C. versicolor in MG decolorization, optimized with RSM. Copyright © 2018 Elsevier Inc. All rights reserved.

Effects of Sublethal Exposure to a Glyphosate-Based Herbicide Formulation on Metabolic Activities of Different Xenobiotic-Metabolizing Enzymes in Rats.

PubMed

Larsen, Karen; Najle, Roberto; Lifschitz, Adrián; Maté, María L; Lanusse, Carlos; Virkel, Guillermo L

2014-07-01

The activities of different xenobiotic-metabolizing enzymes in liver subcellular fractions from Wistar rats exposed to a glyphosate (GLP)-based herbicide (Roundup full II) were evaluated in this work. Exposure to the herbicide triggered protective mechanisms against oxidative stress (increased glutathione peroxidase activity and total glutathione levels). Liver microsomes from both male and female rats exposed to the herbicide had lower (45%-54%, P < 0.01) hepatic cytochrome P450 (CYP) levels compared to their respective control animals. In female rats, the hepatic 7-ethoxycoumarin O-deethylase (a general CYP-dependent enzyme activity) was 57% higher (P < 0.05) in herbicide-exposed compared to control animals. Conversely, this enzyme activity was 58% lower (P < 0.05) in male rats receiving the herbicide. Lower (P < 0.05) 7-ethoxyresorufin O-deethlyase (EROD, CYP1A1/2 dependent) and oleandomycin triacetate (TAO) N-demethylase (CYP3A dependent) enzyme activities were observed in liver microsomes from exposed male rats. Conversely, in females receiving the herbicide, EROD increased (123%-168%, P < 0.05), whereas TAO N-demethylase did not change. A higher (158%-179%, P < 0.01) benzyloxyresorufin O-debenzylase (a CYP2B-dependent enzyme activity) activity was only observed in herbicide-exposed female rats. In herbicide-exposed rats, the hepatic S-oxidation of methimazole (flavin monooxygenase dependent) was 49% to 62% lower (P < 0.001), whereas the carbonyl reduction of menadione (a cytosolic carbonyl reductase-dependent activity) was higher (P < 0.05). Exposure to the herbicide had no effects on enzymatic activities dependent on carboxylesterases, glutathione transferases, and uridinediphospho-glucuronosyltransferases. This research demonstrated certain biochemical modifications after exposure to a GLP-based herbicide. Such modifications may affect the metabolic fate of different endobiotic and xenobiotic substances. The pharmacotoxicological significance of these findings remains to be clarified. © The Author(s) 2014.

Evaluation of a Hypocrea jecorina enzyme preparation for hydrolysis of Tifton 85 bermudagrass.

PubMed

Ximenes, E A; Brandon, S K; Doran-Peterson, J

2008-03-01

Tifton 85 bermudagrass, developed at the ARS-USDA in Tifton, GA, is grown on over ten million acres in the USA for hay and forage. Of the bermudagrass cultivars, Tifton 85 exhibits improved digestibility because the ratio of ether- to ester-linked phenolic acids has been lowered using traditional plant breeding techniques. A previously developed pressurized batch hot water (PBHW) method was used to treat Tifton 85 bermudagrass for enzymatic hydrolysis. Native grass (untreated) and PBHW-pretreated material were compared as substrates for fungal cultivation to produce enzymes. Cellulase activity, measured via the filter paper assay, was higher for fungi cultivated on PBHW-pretreated grass, whereas the other nine enzyme assays produced higher activities for the untreated grass. Ferulic acid and vanillin levels increased significantly for the enzyme preparations produced using PBHW-pretreated grass and the release of these phenolic compounds may have contributed to the observed reduction in enzyme activities. Culture supernatant from Tifton 85 bermudagrass-grown fungi were combined with two commercial enzyme preparations and the enzyme activity profiles are reported. The amount of reducing sugar liberated by the enzyme mixture from Hypocrea jecorina (after 192 h incubation with untreated bermudagrass) individually or in combination with feruloyl esterase was 72.1 and 84.8%, respectively, of the commercial cellulase preparation analyzed under the same conditions.

Evaluation of a Hypocrea jecorina Enzyme Preparation for Hydrolysis of Tifton 85 Bermudagrass

NASA Astrophysics Data System (ADS)

Ximenes, E. A.; Brandon, S. K.; Doran-Peterson, J.

Tifton 85 bermudagrass, developed at the ARS-USDA in Tifton, GA, is grown on over ten million acres in the USA for hay and forage. Of the bermudagrass cultivars, Tifton 85 exhibits improved digestibility because the ratio of ether- to ester-linked phenolic acids has been lowered using traditional plant breeding techniques. A previously developed pressurized batch hot water (PBHW) method was used to treat Tifton 85 bermudagrass for enzymatic hydrolysis. Native grass (untreated) and PBHW-pretreated material were compared as substrates for fungal cultivation to produce enzymes. Cellulase activity, measured via the filter paper assay, was higher for fungi cultivated on PBHW-pretreated grass, whereas the other nine enzyme assays produced higher activities for the untreated grass. Ferulic acid and vanillin levels increased significantly for the enzyme preparations produced using PBHW-pretreated grass and the release of these phenolic compounds may have contributed to the observed reduction in enzyme activities. Culture supernatant from Tifton 85 bermudagrass-grown fungi were combined with two commercial enzyme preparations and the enzyme activity profiles are reported. The amount of reducing sugar liberated by the enzyme mixture from Hypocrea jecorina (after 192 h incubation with untreated bermudagrass) individually or in combination with feruloyl esterase was 72.1 and 84.8%, respectively, of the commercial cellulase preparation analyzed under the same conditions.

Role of Disulfide Bridges in the Activity and Stability of a Cold-Active α-Amylase

PubMed Central

Siddiqui, Khawar Sohail; Poljak, Anne; Guilhaus, Michael; Feller, Georges; D'Amico, Salvino; Gerday, Charles; Cavicchioli, Ricardo

2005-01-01

The cold-adapted α-amylase from Pseudoalteromonas haloplanktis unfolds reversibly and cooperatively according to a two-state mechanism at 30°C and unfolds reversibly and sequentially with two transitions at temperatures below 12°C. To examine the role of the four disulfide bridges in activity and conformational stability of the enzyme, the eight cysteine residues were reduced with β-mercaptoethanol or chemically modified using iodoacetamide or iodoacetic acid. Matrix-assisted laser desorption-time of flight mass spectrometry analysis confirmed that all of the cysteines were modified. The iodoacetamide-modified enzyme reversibly folded/unfolded and retained approximately one-third of its activity. Removal of all disulfide bonds resulted in stabilization of the least stable region of the enzyme (including the active site), with a concomitant decrease in activity (increase in activation enthalpy). Disulfide bond removal had a greater impact on enzyme activity than on stability (particularly the active-site region). The functional role of the disulfide bridges appears to be to prevent the active site from developing ionic interactions. Overall, the study demonstrated that none of the four disulfide bonds are important in stabilizing the native structure of enzyme, and instead, they appear to promote a localized destabilization to preserve activity. PMID:16109962

Dual role of imidazole as activator/inhibitor of sweet almond (Prunus dulcis) β-glucosidase.

PubMed

Caramia, Sara; Gatius, Angela Gala Morena; Dal Piaz, Fabrizio; Gaja, Denis; Hochkoeppler, Alejandro

2017-07-01

The activity of Prunus dulcis (sweet almond) β-glucosidase at the expense of p -nitrophenyl-β-d-glucopyranoside at pH 6 was determined, both under steady-state and pre-steady-state conditions. Using crude enzyme preparations, competitive inhibition by 1-5 mM imidazole was observed under both kinetic conditions tested. However, when imidazole was added to reaction mixtures at 0.125-0.250 mM, we detected a significant enzyme activation. To further inspect this effect exerted by imidazole, β-glucosidase was purified to homogeneity. Two enzyme isoforms were isolated, i.e. a full-length monomer, and a dimer containing a full-length and a truncated subunit. Dimeric β-glucosidase was found to perform much better than the monomeric enzyme, independently of the kinetic conditions used to assay enzyme activity. In addition, the sensitivity towards imidazole was found to differ between the two isoforms. While monomeric enzyme was indeed found to be relatively insensitive to imidazole, dimeric β-glucosidase was observed to be significantly activated by 0.125-0.250 mM imidazole under pre-steady-state conditions. Further, steady-state assays revealed that the addition of 0.125 mM imidazole to reaction mixtures increases the K m of dimeric enzyme from 2.3 to 6.7 mM. The activation of β-glucosidase dimer by imidazole is proposed to be exerted via a conformational transition poising the enzyme towards proficient catalysis.

Entrapment of subtilisin in ceramic sol-gel coating for antifouling applications.

PubMed

Regina, Viduthalai Rasheedkhan; Søhoel, Helmer; Lokanathan, Arcot Raghupathi; Bischoff, Claus; Kingshott, Peter; Revsbech, Niels Peter; Meyer, Rikke Louise

2012-11-01

Enzymes with antifouling properties are of great interest in developing nontoxic antifouling coatings. A bottleneck in developing enzyme-based antifouling coatings is to immobilize the enzyme in a suitable coating matrix without compromising its activity and stability. Entrapment of enzymes in ceramics using the sol-gel method is known to have several advantages over other immobilization methods. The sol-gel method can be used to make robust coatings, and the aim of this study was to explore if sol-gel technology can be used to develop robust coatings harboring active enzymes for antifouling applications. We successfully entrapped a protease, subtilisin (Savinase, Novozymes), in a ceramic coating using a sol-gel method. The sol-gel formulation, when coated on a stainless steel surface, adhered strongly and cured at room temperature in less than 8 h. The resultant coating was smoother and less hydrophobic than stainless steel. Changes in the coating's surface structure, thickness and chemistry indicate that the coating undergoes gradual erosion in aqueous medium, which results in release of subtilisin. Subtilisin activity in the coating increased initially, and then gradually decreased. After 9 months, 13% of the initial enzyme activity remained. Compared to stainless steel, the sol-gel-coated surfaces with active subtilisin were able to reduce bacterial attachment of both Gram positive and Gram negative bacteria by 2 orders of magnitude. Together, our results demonstrate that the sol-gel method is a promising coating technology for entrapping active enzymes, presenting an interesting avenue for enzyme-based antifouling solutions.

Endopolygalacturonase in Apples (Malus domestica) and Its Expression during Fruit Ripening.

PubMed Central

Wu, Q.; Szakacs-Dobozi, M.; Hemmat, M.; Hrazdina, G.

1993-01-01

The activity of polygalacturonase (PG) has been detected in ripe McIntosh apples (Malus domestica Borkh. cv McIntosh) both by enzyme activity measurement and immunoblotting using an anti-tomato-PG antibody preparation. PG activity increased during fruit ripening and remained steady, or decreased slightly, after 5 months of controlled atmospheric storage. The enzyme had a relative molecular weight of 45,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 56,000 to 61,000 when determined by gel filtration. Viscosity and reducing end group measurements with a commercial pectin preparation showed that the enzyme is endo acting. In RNA and DNA blot hybridization experiments, a full-length tomato PG cDNA hybridized with the apple RNA and DNA, showing the identity of genes encoding the activity of the enzyme in tomato and apple. PMID:12231813

Proteolytic Enzymes Clustered in Specialized Plasma-Membrane Domains Drive Endothelial Cells’ Migration

PubMed Central

Salamone, Monica; Carfì Pavia, Francesco

2016-01-01

In vitro cultured endothelial cells forming a continuous monolayer establish stable cell-cell contacts and acquire a “resting” phenotype; on the other hand, when growing in sparse conditions these cells acquire a migratory phenotype and invade the empty area of the culture. Culturing cells in different conditions, we compared expression and clustering of proteolytic enzymes in cells having migratory versus stationary behavior. In order to observe resting and migrating cells in the same microscopic field, a continuous cell monolayer was wounded. Increased expression of proteolytic enzymes was evident in cell membranes of migrating cells especially at sprouting sites and in shed membrane vesicles. Gelatin zymography and western blotting analyses confirmed that in migrating cells, expression of membrane-bound and of vesicle-associated proteolytic enzymes are increased. The enzymes concerned include MMP-2, MMP-9, MT1-MMP, seprase, DPP4 (DiPeptidyl Peptidase 4) and uPA. Shed membrane vesicles were shown to exert degradative activity on ECM components and produce substrates facilitating cell migration. Vesicles shed by migrating cells degraded ECM components at an increased rate; as a result their effect on cell migration was amplified. Inhibiting either Matrix Metallo Proteases (MMPs) or Serine Integral Membrane Peptidases (SIMPs) caused a decrease in the stimulatory effect of vesicles, inhibiting the spontaneous migratory activity of cells; a similar result was also obtained when a monoclonal antibody acting on DPP4 was tested. We conclude that proteolytic enzymes have a synergistic stimulatory effect on cell migration and that their clustering probably facilitates the proteolytic activation cascades needed to produce maximal degradative activity on cell substrates during the angiogenic process. PMID:27152413

Substance P levels and neutral endopeptidase activity in acute burn wounds and hypertrophic scar.

PubMed

Scott, Jeffrey R; Muangman, Pornprom R; Tamura, Richard N; Zhu, Kathy Q; Liang, Zhi; Anthony, Joanne; Engrav, Loren H; Gibran, Nicole S

2005-04-01

Substance P, a cutaneous neuroinflammatory mediator released from peripheral nerves, plays a role in responses to injury. Neutral endopeptidase is a cell membrane-bound metallopeptidase enzyme that regulates substance P activity. The question of substance P involvement in hypertrophic scar development has been based on observations that hypertrophic scars have increased numbers of nerves. The authors hypothesized that hypertrophic scar has greater substance P levels and decreased neutral endopeptidase activity compared with uninjured skin and acute partial-thickness burns, which may contribute to an exuberant response to injury. The authors obtained small skin samples of deep partial-thickness burns (n = 7; postburn days 7 to 78) and uninjured skin (n = 14) from patients (eight male patients and six female patients; 2 to 71 years old) undergoing burn wound excision. Hypertrophic scar samples were obtained from six patients (three male patients and three female patients; 8 to 47 years old) undergoing surgical excision 13 to 64 months after burn injury. Protein concentrations were determined using a bicinchoninic acid assay. Substance P concentration was determined by means of indirect enzyme-linked immunosorbent assay. Neutral endopeptidase activity was measured using an enzymatic assay that quantifies a fluorescent degradation product, methoxy-2-naphthylamine (MNA). Substance P and neutral endopeptidase data were standardized to sample weight. Substance P levels were greater in hypertrophic scar (3506 pg/g) compared with uninjured skin (1698 pg/g; p < 0.03) and burned skin (958 pg/g; p < 0.01). Hypertrophic scar samples had decreased neutral endopeptidase enzyme activity (8.8 pM MNA/hour/microg) compared with normal skin (16.3 pM MNA/hour/microg; p < 0.05). Acute burn wounds (27.9 pM MNA/hour/microg) demonstrated increased neutral endopeptidase enzyme activity (p < 0.05). Increased substance P concentration in hypertrophic scar correlates with histologic findings of increased nerve numbers in hypertrophic scar samples. Decreased neutral endopeptidase enzyme activity in hypertrophic scar may contribute to increased available substance P that may result in an exuberant neuroinflammatory response.

Identification of parallel and divergent optimization solutions for homologous metabolic enzymes

DOE PAGES

Standaert, Robert F.; Giannone, Richard J.; Michener, Joshua K.

2018-04-18

Here, metabolic pathway assembly typically involves the expression of enzymes from multiple organisms in a single heterologous host. Ensuring that each enzyme functions effectively can be challenging, since many potential factors can disrupt proper pathway flux. Here, we compared the performance of two enzyme homologs in a pathway engineered to allow Escherichia coli to grow on 4-hydroxybenzoate (4-HB), a byproduct of lignocellulosic biomass deconstruction. Single chromosomal copies of the 4-HB 3-monooxygenase genes pobA and praI, from Pseudomonas putida KT2440 and Paenibacillus sp. JJ-1B, respectively, were introduced into a strain able to metabolize protocatechuate (PCA), the oxidation product of 4-HB. Neithermore » enzyme initially supported consistent growth on 4-HB. Experimental evolution was used to identify mutations that improved pathway activity. For both enzymes, silent mRNA mutations were identified that increased enzyme expression. With pobA, duplication of the genes for PCA metabolism allowed growth on 4-HB. However, with praI, growth required a mutation in the 4-HB/PCA transporter pcaK that increased intracellular concentrations of 4-HB, suggesting that flux through PraI was limiting. These findings demonstrate the value of directed evolution strategies to rapidly identify and overcome diverse factors limiting enzyme activity.« less

Identification of parallel and divergent optimization solutions for homologous metabolic enzymes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Standaert, Robert F.; Giannone, Richard J.; Michener, Joshua K.

Here, metabolic pathway assembly typically involves the expression of enzymes from multiple organisms in a single heterologous host. Ensuring that each enzyme functions effectively can be challenging, since many potential factors can disrupt proper pathway flux. Here, we compared the performance of two enzyme homologs in a pathway engineered to allow Escherichia coli to grow on 4-hydroxybenzoate (4-HB), a byproduct of lignocellulosic biomass deconstruction. Single chromosomal copies of the 4-HB 3-monooxygenase genes pobA and praI, from Pseudomonas putida KT2440 and Paenibacillus sp. JJ-1B, respectively, were introduced into a strain able to metabolize protocatechuate (PCA), the oxidation product of 4-HB. Neithermore » enzyme initially supported consistent growth on 4-HB. Experimental evolution was used to identify mutations that improved pathway activity. For both enzymes, silent mRNA mutations were identified that increased enzyme expression. With pobA, duplication of the genes for PCA metabolism allowed growth on 4-HB. However, with praI, growth required a mutation in the 4-HB/PCA transporter pcaK that increased intracellular concentrations of 4-HB, suggesting that flux through PraI was limiting. These findings demonstrate the value of directed evolution strategies to rapidly identify and overcome diverse factors limiting enzyme activity.« less

Identification of parallel and divergent optimization solutions for homologous metabolic enzymes.

PubMed

Standaert, Robert F; Giannone, Richard J; Michener, Joshua K

2018-06-01

Metabolic pathway assembly typically involves the expression of enzymes from multiple organisms in a single heterologous host. Ensuring that each enzyme functions effectively can be challenging, since many potential factors can disrupt proper pathway flux. Here, we compared the performance of two enzyme homologs in a pathway engineered to allow Escherichia coli to grow on 4-hydroxybenzoate (4-HB), a byproduct of lignocellulosic biomass deconstruction. Single chromosomal copies of the 4-HB 3-monooxygenase genes pobA and praI , from Pseudomonas putida KT2440 and Paenibacillus sp. JJ-1B, respectively, were introduced into a strain able to metabolize protocatechuate (PCA), the oxidation product of 4-HB. Neither enzyme initially supported consistent growth on 4-HB. Experimental evolution was used to identify mutations that improved pathway activity. For both enzymes, silent mRNA mutations were identified that increased enzyme expression. With pobA , duplication of the genes for PCA metabolism allowed growth on 4-HB. However, with praI , growth required a mutation in the 4-HB/PCA transporter pcaK that increased intracellular concentrations of 4-HB, suggesting that flux through PraI was limiting. These findings demonstrate the value of directed evolution strategies to rapidly identify and overcome diverse factors limiting enzyme activity.

Effects of ionizing radiation on the enzyme activities and ultrastructural changes of poultry

NASA Astrophysics Data System (ADS)

Hwang, H.-I.; Hau, L.-B.

1995-02-01

Enzyme-catalyzed changes are generally recognized as one of the major reasons for fresh meat deterioration after irradiation. In this study, the effects of ionizing radiation and storage on the enzyme activities of poultry as well as the ultrastructural change of muscle were evaluated. When chicken breasts were irradiated at 4°C and -20°C, both Ca 2+-dependent protease and cathepsin D showed some degree of resistance to irradiation. The activities of those two enzymes decreased with the increase of irradiation doses. During storage, Ca 2+-dependent proteases showed a marked decrease in activity. On the other hand, the cathepsin D activity was not significantly changed at either 4°C or -20°C after 20 days. Transmission electron microscope examination showed no structural changes of the myofibrils with a radiation dose of up to 10 kGy at either 4°C or -20°C. Freezing protected the irradiated chicken breasts from autolytic enzymes damage during storage. In contrast, considerable sarcomere degradation occurred in Z-line for irradiated samples when stored at 4°C for 20 days. The action of the proteolytic enzymes may have been responsible for the sarcomere degradation in irradiated chicken breasts.

Effects of isobutyrate supplementation on ruminal microflora, rumen enzyme activities and methane emissions in Simmental steers.

PubMed

Wang, C; Liu, Q; Zhang, Y L; Pei, C X; Zhang, S L; Wang, Y X; Yang, W Z; Bai, Y S; Shi, Z G; Liu, X N

2015-02-01

The objective of this study was to evaluate the effects of isobutyrate supplementation on rumen microflora, enzyme activities and methane emissions in Simmental steers consuming a corn stover-based diet. Eight ruminally cannulated Simmental steers were used in a replicated 4 × 4 Latin square experiment. The treatments were control (without isobutyrate), low isobutyrate (LIB), moderate isobutyrate (MIB) and high isobutyrate (HIB) with 8.4, 16.8 and 25.2 g isobutyrate per steer per day respectively. Isobutyrate was hand-mixed into the concentrate portion. Diet consisted of 60% corn stover and 40% concentrate [dry matter (DM) basis]. Dry matter intake (averaged 9 kg/day) was restricted to a maximum of 90% of ad libitum intake. Population of total bacteria, cellulolytic bacteria and anaerobic fungi were linearly increased, whereas that of protozoa and total methanogens was linearly reduced with increasing isobutyrate supplementation. Real-time PCR quantification of population of Ruminococcus albus, Ruminococcus flavefaciens, Butyrivibrio fibrisolvens and Fibrobacter succinogenes was linearly increased with increasing isobutyrate supplementation. Activities of carboxymethyl cellulase, xylanase and β-glucosidase were linearly increased, whereas that of protease was linearly reduced. Methane production was linearly decreased with increasing isobutyrate supplementation. Effective degradabilities of cellulose and hemicellulose of corn stover were linearly increased, whereas that of crude protein in diet was linearly decreased with increasing isobutyrate supplementation. The present results indicate that isobutyrate supplemented improved microflora, rumen enzyme activities and methane emissions in steers. It was suggested that the isobutyrate stimulated the digestive micro-organisms or enzymes in a dose-dependent manner. In the experimental conditions of this trial, the optimum isobutyrate dose was approximately 16.8 g isobutyrate per steer per day. Journal of Animal Physiology and Animal Nutrition © 2014 Blackwell Verlag GmbH.

Rapid bursts and slow declines: on the possible evolutionary trajectories of enzymes

PubMed Central

Newton, Matilda S.; Arcus, Vickery L.; Patrick, Wayne M.

2015-01-01

The evolution of enzymes is often viewed as following a smooth and steady trajectory, from barely functional primordial catalysts to the highly active and specific enzymes that we observe today. In this review, we summarize experimental data that suggest a different reality. Modern examples, such as the emergence of enzymes that hydrolyse human-made pesticides, demonstrate that evolution can be extraordinarily rapid. Experiments to infer and resurrect ancient sequences suggest that some of the first organisms present on the Earth are likely to have possessed highly active enzymes. Reconciling these observations, we argue that rapid bursts of strong selection for increased catalytic efficiency are interspersed with much longer periods in which the catalytic power of an enzyme erodes, through neutral drift and selection for other properties such as cellular energy efficiency or regulation. Thus, many enzymes may have already passed their catalytic peaks. PMID:25926697

Modification of erythrocyte membrane proteins, enzymes and transport mechanisms in chronic alcoholics: an in vivo and in vitro study.

PubMed

Maturu, Paramahamsa; Vaddi, Damodara Reddy; Pannuru, Padmavathi; Nallanchakravarthula, Varadacharyulu

2013-01-01

The aim of the study was to elucidate the molecular mechanisms underlying the alcohol perturbation leading to deleterious effects on erythrocyte membrane transport in chronic alcoholics. Membrane bound enzyme activities such as Na(+), K(+)-ATPase, Ca(2+),Mg(2+)-ATPase and acetylcholine esterase and membrane transport analysis by in vitro and erythrocyte membrane profile analysis in controls and chronic alcoholic red cells were analyzed. It was observed that decreased Na(+), K(+)-ATPase enzyme activity and increased activities of Ca(2+),Mg(2+)-ATPase and acetylcholine esterase in chronic alcoholics compared to controls. The in vitro studies of erythrocytes suggested that there is an increased uptake of glucose through chronic alcoholic red cells. However, glucose utilization by chronic alcoholic red cells was decreased. An increased sensitivity of ouabain for its binding site on Na(+), K(+)-ATPase in chronic alcoholic erythrocyte membrane was evident from this study. Though there appears to be an increased Na(+) influx in chronic alcoholic cells, the status of Na(+) transport is not altered much. However, ouabain caused slight disturbances in the transport of sodium, similar disturbances in the potassium transport resulting in much accumulation of potassium in red cells. It was concluded that chronic alcohol consumption modified certain membrane bound proteins, enzymes and transport mechanisms in chronic alcoholics.

Characterization of a mutated Geobacillus stearothermophilus L-arabinose isomerase that increases the production rate of D-tagatose.

PubMed

Kim, H-J; Kim, J-H; Oh, H-J; Oh, D-K

2006-07-01

Characterization of a mutated Geobacillus stearothermophilus L-arabinose isomerase used to increase the production rate of D-tagatose. A mutated gene was obtained by an error-prone polymerase chain reaction using L-arabinose isomerase gene from G. stearothermophilus as a template and the gene was expressed in Escherichia coli. The expressed mutated L-arabinose isomerase exhibited the change of three amino acids (Met322-->Val, Ser393-->Thr, and Val408-->Ala), compared with the wild-type enzyme and was then purified to homogeneity. The mutated enzyme had a maximum galactose isomerization activity at pH 8.0, 65 degrees C, and 1.0 mM Co2+, while the wild-type enzyme had a maximum activity at pH 8.0, 60 degrees C, and 1.0-mM Mn2+. The mutated L-arabinose isomerase exhibited increases in D-galactose isomerization activity, optimum temperature, catalytic efficiency (kcat/Km) for D-galactose, and the production rate of D-tagatose from D-galactose. The mutated L-arabinose isomerase from G. stearothermophilus is valuable for the commercial production of D-tagatose. This work contributes knowledge on the characterization of a mutated L-arabinose isomerase, and allows an increased production rate for D-tagatose from D-galactose using the mutated enzyme.

Presynaptic transmitters and depolarizing influences regulate development of the substantia nigra in culture.

PubMed

Friedman, W J; Dreyfus, C F; McEwen, B; Black, I B

1988-10-01

Recent evidence suggests that extracellular signals regulate neurotransmitter traits in brain catecholaminergic (CA) neurons as in the periphery. Development of the dopaminergic phenotype in the mouse substantia nigra (SN) was studied by monitoring tyrosine hydroxylase (TH), the rate-limiting enzyme in CA biosynthesis in vivo and in culture. Explants of SN were dissected from embryonic day 15 embryos and grown in culture for a week. To define the influence of depolarizing signals on central dopaminergic neurons, cultures were grown with the pharmacologic depolarizing agent veratridine. This treatment elicited a significant increase in TH enzyme activity, accompanied by elevated levels of enzyme protein. The increase in activity was prevented by TTX, suggesting that transmembrane Na+ influx was necessary for the rise in TH. A physiologic presynaptic agonist, substance P, also evoked a significant increase in TH activity; however, the coproduced tachykinin peptide, substance K (SK, neurokinin A) elicited a more dramatic rise. The SK effect was blocked by TTX, suggesting that the physiologic agonist was acting through the same mechanism as the pharmacologic agent veratridine. Immunoblot analysis revealed that SK elicited a parallel increase in TH enzyme protein. Our observations suggest that the novel peptide, SK, serves a physiological role in the regulation of TH in the striatonigral pathway.

Mechanical stimulation of skeletal muscle increases prostaglandin F2(alpha) synthesis and cyclooxygenase activity by a pertussis toxin sensitive mechanism

NASA Technical Reports Server (NTRS)

Vandenburgh, Herman H.; Shansky, Janet; Solerssi, Rosa; Chromiak, Joseph

1992-01-01

Repetitive mechanical stimulation of differentiated skeletal muscle in tissue culture increases the production of prostaglandin F(sub 2(alpha)), an anabolic stimulator of myofiber growth. Within 4 h of initiating mechanical activity, the activity of cyclooxygenase, a regulatory enzyme in prostaglandin synthesis, was increased 82% (P is less than .005), and this increase was maintained for at least 24 h. Kinetic analysis of the stretch-activated cyclooxygenase indicated a two to three-fold decrease in the enzyme's K(sub m) with no change in V(sub max). The stretch-induced increase in enzymatic activity was not inhibited by cycloheximide, was independent of cellular electrical activity (tetrodotoxin-insensitive), but was prevented by the G protein inhibitor pertussis toxin. Pertussis toxin also inhibited the stretch-induced increases in PGF(sub 2(alpha)) production, and cell growth. It is concluded that stretch of skeletal muscle increases the synthesis of the anabolic modulator PGF(sub 2(alpha)) by a G protein-dependent process which involves activation of cyclooxygenase by a posttranslational mechanism.

Covalent immobilization of β-glucosidase on magnetic particles for lignocellulose hydrolysis.

PubMed

Alftrén, Johan; Hobley, Timothy John

2013-04-01

β-Glucosidase hydrolyzes cellobiose to glucose and is an important enzyme in the consortium used for hydrolysis of cellulosic and lignocellulosic feedstocks. In the present work, β-glucosidase was covalently immobilized on non-porous magnetic particles to enable re-use of the enzyme. It was found that particles activated with cyanuric chloride and polyglutaraldehyde gave the highest bead-related immobilized enzyme activity when tested with p-nitrophenyl-β-D-glucopyranoside (104.7 and 82.2 U/g particles, respectively). Furthermore, the purified β-glucosidase preparation from Megazyme gave higher bead-related enzyme activities compared to Novozym 188 (79.0 and 9.8 U/g particles, respectively). A significant improvement in thermal stability was observed for immobilized enzyme compared to free enzyme; after 5 h (at 65 °C), 36 % of activity remained for the former, while there was no activity in the latter. The performance and recyclability of immobilized β-glucosidase on more complex substrate (pretreated spruce) was also studied. It was shown that adding immobilized β-glucosidase (16 U/g dry matter) to free cellulases (8 FPU/g dry matter) increased the hydrolysis yield of pretreated spruce from ca. 44 % to ca. 65 %. In addition, it was possible to re-use the immobilized β-glucosidase in the spruce and retain activity for at least four cycles. The immobilized enzyme thus shows promise for lignocellulose hydrolysis.

Purification and characterization of a fibrinolytic enzyme from tempeh bongkrek as an alternative of thrombolytic agents

NASA Astrophysics Data System (ADS)

Sasmita, I. R. A.; Sutrisno, A.; Zubaidah, E.; Wardani, A. K.

2018-03-01

Tempeh is one of Indonesia’s traditional foods that contain fibrinolytic enzymes. Tempeh bongkrek shows very strong activity among various tempeh. The fibrinolytic enzymes of bongkrek tempeh are obtained by steps of purification i.e, ammonium sulphate precipitation, ion exchange chromatography and gel filtration chromatography. The fibrinolytic enzymes has been successfully purified with a yield of 4.37%, specific activity of 3,361 U / mg and purification fold of 44.02. SDS PAGE analysis showed that the enzyme was purified in to single band with estimated molecular mass of 75.82 kDa. The purified enzyme has optimum pH of 7 and optimum temperature of 50°C and pH stability between pH 4 - 7 with temperature stability from 30°-50°C. The fibrinolytic activity is increased with addition of CaCl2 but inhibited with CuSO4, phenylmethylsulfonyl fluoride (PMSF), sodium dodecyl sulfate (SDS), and ethylenediaminetetraacetic acid (EDTA).

Effects of the naturally occurring alkenylbenzenes eugenol and trans-anethole on drug-metabolizing enzymes in the rat liver.

PubMed

Rompelberg, C J; Verhagen, H; van Bladeren, P J

1993-09-01

In order to study the effects of trans-anethole and eugenol on drug-metabolizing enzyme activities in vivo, male Wistar rats were treated by gavage with trans-anethole (125 or 250 mg/kg body weight) or eugenol (250, 500 or 1000 mg/kg body weight) daily for 10 days. In liver microsomes and cytosol various phase-I and phase-II biotransformation enzyme activities were determined. No effect on total cytochrome P-450 content in liver microsomes from rats treated with eugenol or trans-anethole was observed. Administration of 1000 mg eugenol/kg body weight, but not the lower doses, significantly increased cytochrome P-450-dependent 7-ethoxy-resorufin O-deethylation (EROD) and 7-pentoxyresorufin O-depentylation (PROD); administration of trans-anethole (125 or 250 mg/kg body weight) did not alter EROD and PROD activities. In rat liver cytosol, UDP-glucuronyl transferase (GT) activity towards the substrate 4-chlorophenol was significantly increased in all treated rats, and activity towards 4-hydroxybiphenyl as substrate was significantly increased in rats treated with 250 mg trans-anethole/kg or with 500 or 1000 mg eugenol/kg. DT-diaphorase (DTD) activity was only significantly enhanced in the liver cytosol of rats treated with trans-anethole at 250 mg/kg body weight. Enhancement of cytosolic glutathione S-transferase (GST) activity towards 1-chloro-2,4-dinitrobenzene was found for all eugenol- and trans-anethole-treated rats. In addition, significantly increased levels of GST subunit 2 were measured by HPLC in the liver cytosol of rats treated with eugenol (500 or 1000 mg/kg body eight) or trans-anethole (250 mg/kg body weight). It is concluded that both eugenol and trans-anethole preferentially induced phase II biotransformation enzymes in rat liver in vivo.

Cranberry extract-enriched diets increase NAD(P)H:quinone oxidoreductase and catalase activities in obese but not in nonobese mice.

PubMed

Boušová, Iva; Bártíková, Hana; Matoušková, Petra; Lněničková, Kateřina; Zappe, Lukáš; Valentová, Kateřina; Szotáková, Barbora; Martin, Jan; Skálová, Lenka

2015-10-01

Consumption of antioxidant-enriched diets is 1 method of addressing obesity, which is associated with chronic oxidative stress and changes in the activity/expression of various enzymes. In this study, we hypothesized that the modulation of antioxidant enzymes and redox status through a cranberry extract (CBE)-enriched diet would differ between obese and nonobese mice. The CBE used in this study was obtained from the American cranberry (Vaccinium macrocarpon, Ericaceae), a popular constituent of dietary supplements that is a particularly rich source of (poly)phenols and has strong antioxidant properties. The present study was designed to test and compare the in vivo effects of 28-day consumption of a CBE-enriched diet (2%) on the antioxidant status of nonobese mice and mice with monosodium glutamate-induced obesity. Plasma, erythrocytes, liver, and small intestine were studied concurrently to obtain more complex information. The specific activities, protein, and messenger RNA expression levels of antioxidant enzymes as well as the levels of malondialdehyde and thiol (SH) groups were analyzed. Cranberry extract treatment increased the SH group content in plasma and the glutathione S-transferase activity in the erythrocytes of the obese and nonobese mice. In addition, in the obese animals, the CBE treatment reduced the malondialdehyde content in erythrocytes and increased quinone oxidoreductase (liver) and catalase (erythrocytes and small intestine) activities. The elevation of hepatic quinone oxidoreductase activity was accompanied by an increase in the corresponding messenger RNA levels. The effects of CBE on the activity of antioxidant enzymes and redox status were more pronounced in the obese mice compared with the nonobese mice. Copyright © 2015 Elsevier Inc. All rights reserved.

Chapter 8: Selective Stoichiometric and Catalytic Reactivity in the Confines of a Chiral Supramolecular Assembly

DOE Office of Scientific and Technical Information (OSTI.GOV)

University of California, Berkeley; Lawrence Berkeley National Laboratory; Raymond, Kenneth

2007-09-27

Nature uses enzymes to activate otherwise unreactive compounds in remarkable ways. For example, DNases are capable of hydrolyzing phosphate diester bonds in DNA within seconds,[1-3]--a reaction with an estimated half-life of 200 million years without an enzyme.[4] The fundamental features of enzyme catalysis have been much discussed over the last sixty years in an effort to explain the dramatic rate increases and high selectivities of enzymes. As early as 1946, Linus Pauling suggested that enzymes must preferentially recognize and stabilize the transition state over the ground state of a substrate.[5] Despite the intense study of enzymatic selectivity and ability tomore » catalyze chemical reactions, the entire nature of enzyme-based catalysis is still poorly understood. For example, Houk and co-workers recently reported a survey of binding affinities in a wide variety of enzyme-ligand, enzyme-transition-state, and synthetic host-guest complexes and found that the average binding affinities were insufficient to generate many of the rate accelerations observed in biological systems.[6] Therefore, transition-state stabilization cannot be the sole contributor to the high reactivity and selectivity of enzymes, but rather, other forces must contribute to the activation of substrate molecules. Inspired by the efficiency and selectivity of Nature, synthetic chemists have admired the ability of enzymes to activate otherwise unreactive molecules in the confines of an active site. Although much less complex than the evolved active sites of enzymes, synthetic host molecules have been developed that can carry out complex reactions with their cavities. While progress has been made toward highly efficient and selective reactivity inside of synthetic hosts, the lofty goal of duplicating enzymes specificity remains.[7-9] Pioneered by Lehn, Cram, Pedersen, and Breslow, supramolecular chemistry has evolved well beyond the crown ethers and cryptands originally studied.[10-12] Despite the increased complexity of synthetic host molecules, most assembly conditions utilize self-assembly to form complex highly-symmetric structures from relatively simple subunits. For supramolecular assemblies able to encapsulate guest molecules, the chemical environment in each assembly--defined by the size, shape, charge, and functional group availability--greatly influences the guest-binding characteristics.[6, 13-17]« less

Changes in nitrogen metabolism and antioxidant enzyme activities of maize tassel in black soils region of northeast China

PubMed Central

Xu, Hongwen; Lu, Yan; Xie, Zhiming; Song, Fengbin

2014-01-01

Two varieties of maize (Zea mays L.) grown in fields in black soils of northeast China were tested to study the dynamic changes of nitrogen metabolism and antioxidant enzyme activity in tassels of maize. Results showed that antioxidant enzyme activity in tassels of maize increased first and then decreased with the growing of maize, and reached peak value at shedding period. Pattern of proline was consistent with antioxidant enzyme activity, showing that osmotic adjustment could protect many enzymes, which are important for cell metabolism. Continuous reduction of soluble protein content along with the growing of maize was observed in the study, which indicated that quantitative material and energy were provided for pollen formation. Besides, another major cause was that a large proportion of nitrogen was used for the composition of structural protein. Nitrate nitrogen concentrations of tassels were more variable than ammonium nitrogen, which showed that nitrate nitrogen was the favored nitrogen source for maize. PMID:25324855

Cloning, sequencing, and expression of the gene encoding amylopullulanase from Pyrococcus furiosus and biochemical characterization of the recombinant enzyme.

PubMed Central

Dong, G; Vieille, C; Zeikus, J G

1997-01-01

The gene encoding the Pyrococcus furiosus hyperthermophilic amylopullulanase (APU) was cloned, sequenced, and expressed in Escherichia coli. The gene encoded a single 827-residue polypeptide with a 26-residue signal peptide. The protein sequence had very low homology (17 to 21% identity) with other APUs and enzymes of the alpha-amylase family. In particular, none of the consensus regions present in the alpha-amylase family could be identified. P. furiosus APU showed similarity to three proteins, including the P. furiosus intracellular alpha-amylase and Dictyoglomus thermophilum alpha-amylase A. The mature protein had a molecular weight of 89,000. The recombinant P. furiosus APU remained folded after denaturation at temperatures of < or = 70 degrees C and showed an apparent molecular weight of 50,000 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Denaturating temperatures of above 100 degrees C were required for complete unfolding. The enzyme was extremely thermostable, with an optimal activity at 105 degrees C and pH 5.5. Ca2+ increased the enzyme activity, thermostability, and substrate affinity. The enzyme was highly resistant to chemical denaturing reagents, and its activity increased up to twofold in the presence of surfactants. PMID:9293009

Expression, Purification, and Characterization of a Carbohydrate-Active Enzyme: A Research-Inspired Methods Optimization Experiment for the Biochemistry Laboratory

ERIC Educational Resources Information Center

Willbur, Jaime F.; Vail, Justin D.; Mitchell, Lindsey N.; Jakeman, David L.; Timmons, Shannon C.

2016-01-01

The development and implementation of research-inspired, discovery-based experiences into science laboratory curricula is a proven strategy for increasing student engagement and ownership of experiments. In the novel laboratory module described herein, students learn to express, purify, and characterize a carbohydrate-active enzyme using modern…

Short term total sleep deprivation in the rat increases antioxidant responses in multiple brain regions without impairing spontaneous alternation behavior

PubMed Central

Ramanathan, Lalini; Hu, Shuxin; Frautschy, Sally A.; Siegel, Jerome M.

2009-01-01

Total sleep deprivation (TSD) induces a broad spectrum of cognitive, behavioral and cellular changes. We previously reported that long term (5–11 days) TSD in the rat, by the disk-over-water method, decreases the activity of the antioxidant enzyme superoxide dismutase (SOD) in the brainstem and hippocampus. To gain insight into the mechanisms causing cognitive impairment, here we explore the early associations between metabolic activity, antioxidant responses and working memory (one form of cognitive impairment). Specifically we investigated the impact of short term (6 h) TSD, by gentle handling, on the levels of the endogenous antioxidant, total glutathione (GSHt), and the activities of the antioxidative enzymes, SOD and glutathione peroxidase (GPx). Short term TSD had no significant impact on SOD activity, but increased GSHt levels in the rat cortex, brainstem and basal forebrain, and GPx activity in the rat hippocampus and cerebellum. We also observed increased activity of hexokinase, (HK), the rate limiting enzyme of glucose metabolism, in the rat cortex and hypothalamus. We further showed that 6h of TSD leads to increased exploratory behavior to a new environment, without impairing spontaneous alternation behavior (SAB) in the Y maze. We conclude that acute (6h) sleep loss may trigger compensatory mechanisms (like increased antioxidant responses) that prevent initial deterioration in working memory. PMID:19850085

Actions of p-synephrine on hepatic enzyme activities linked to carbohydrate metabolism and ATP levels in vivo and in the perfused rat liver.

PubMed

Maldonado, Marcos Rodrigues; Bracht, Lívia; de Sá-Nakanishi, Anacharis Babeto; Corrêa, Rúbia Carvalho Gomes; Comar, Jurandir Fernando; Peralta, Rosane Marina; Bracht, Adelar

2018-01-01

p-Synephrine is one of the main active components of the fruit of Citrus aurantium (bitter orange). Extracts of the bitter orange and other preparations containing p-synephrine have been used worldwide to promote weight loss and for sports performance. The purpose of the study was to measure the action of p-synephrine on hepatic enzyme activities linked to carbohydrate and energy metabolism and the levels of adenine mononucleotides. Enzymes and adenine mononucleotides were measured in the isolated perfused rat liver and in vivo after oral administration of the drug (50 and 300 mg/kg) by using standard techniques. p-Synephrine increased the activity of glycogen phosphorylase in vivo and in the perfused liver. It decreased, however, the activities of pyruvate kinase and pyruvate dehydrogenase also in vivo and in the perfused liver. p-Synephrine increased the hepatic pools of adenosine diphosphate and adenosine triphosphate. Stimulation of glycogen phosphorylase is consistent with the reported increased glycogenolysis in the perfused liver and increased glycemia in rats. The decrease in the pyruvate dehydrogenase activity indicates that p-synephrine is potentially capable of inhibiting the transformation of carbohydrates into lipids. The capability of increasing the adenosine triphosphate-adenosine diphosphate pool indicates a beneficial effect of p-synephrine on the cellular energetics. Copyright © 2017 John Wiley & Sons, Ltd.

Enhancing saccharification of wheat straw by mixing enzymes from genetically-modified Trichoderma reesei and Aspergillus niger.

PubMed

Jiang, Yanping; Duarte, Alexandra Vivas; van den Brink, Joost; Wiebenga, Ad; Zou, Gen; Wang, Chengshu; de Vries, Ronald P; Zhou, Zhihua; Benoit, Isabelle

2016-01-01

To increase the efficiency of enzymatic hydrolysis for plant biomass conversion into renewable biofuel and chemicals. By overexpressing the point mutation A824 V transcriptional activator Xyr1 in Trichoderma reesei, carboxymethyl cellulase, cellobiosidase and β-D-glucosidase activities of the best mutant were increased from 1.8 IU/ml, 0.1 IU/ml and 0.05 IU/ml to 4.8 IU/ml, 0.4 IU/ml and 0.3 IU/ml, respectively. The sugar yield of wheat straw saccharification by combining enzymes from this mutant and the Aspergillus niger genetically modified strain ΔcreA/xlnR c/araR c was improved up to 7.5 mg/ml, a 229 % increase compared to the combination of wild type strains. Mixing enzymes from T. reesei and A. niger combined with the genetic modification of transcription factors is a promising strategy to increase saccharification efficiency.

Investigation of repressive and enhancive effects of fruit extracts on the activity of glucose-6-phophatase.

PubMed

Zahoor, Muhammad; Jan, Muhammad Rasul; Naz, Sumaira

2016-11-01

Glucose-6-phosphatase is a key enzyme of glucose metabolic pathways. Deficiency of this enzyme leads to glycogen storage disease. This enzyme also plays a negative role in diabetes mellitus disorder in which the catalytic activity of this enzyme increases. Thus there is need for activators to enhance the activity of glucose-6-phosphatase in glycogen storage disease of type 1b while in diabetes mellitus repressors are needed to reduce its activity. Crude extracts of apricot, fig, mulberry and apple fruits were investigated for their repressive/enhancive effects on glucose-6-phosphatase in vivo. Albino mice were used as experimental animal. All the selected extracts showed depressive effects on glucose-6-phosphatase, which shows that all these extracts can be used as antidiabetic supplement of food. The inhibitory pattern was competitive one, which was evident from the effect of increasing dose from 1g/Kg body weight to 3g/Kg body weight for all the selected fruit extracts. However fig and apple fruit extracts showed high repressive effects for high doses as compared to apricot and mulberry fruit extracts. None of these selected fruit extracts showed enhancive effect on glucose-6-phosphatase activity. All these fruits or their extracts can be used as antidiabetic dietary supplement for diabetes mellitus.

Co-occurring increases of calcium and organellar reactive oxygen species determine differential activation of antioxidant and defense enzymes in Ulva compressa (Chlorophyta) exposed to copper excess.

PubMed

Gonzalez, Alberto; Vera, Jeannette; Castro, Jorge; Dennett, Geraldine; Mellado, Macarena; Morales, Bernardo; Correa, Juan A; Moenne, Alejandra

2010-10-01

In order to analyse copper-induced calcium release and (reactive oxygen species) ROS accumulation and their role in antioxidant and defense enzymes activation, the marine alga Ulva compressa was exposed to 10 µM copper for 7 d. The level of calcium, extracellular hydrogen peroxide (eHP), intracellular hydrogen peroxide (iHP) and superoxide anions (SA) as well as the activities of ascorbate peroxidase (AP), glutathione reductase (GR), glutathione-S-transferase (GST), phenylalanine ammonia lyase (PAL) and lipoxygenase (LOX) were determined. Calcium release showed a triphasic pattern with peaks at 2, 3 and 12 h. The second peak was coincident with increases in eHP and iHP and the third peak with the second increase of iHP. A delayed wave of SA occurred after day 3 and was not accompanied by calcium release. The accumulation of iHP and SA was mainly inhibited by organellar electron transport chains inhibitors (OETCI), whereas calcium release was inhibited by ryanodine. AP activation ceased almost completely after the use of OETCI. On the other hand, GR and GST activities were partially inhibited, whereas defense enzymes were not inhibited. In contrast, PAL and LOX were inhibited by ryanodine, whereas AP was not inhibited. Thus, copper stress induces calcium release and organellar ROS accumulation that determine the differential activation of antioxidant and defense enzymes. © 2010 Blackwell Publishing Ltd.

Active-site copper reduction promotes substrate binding of fungal lytic polysaccharide monooxygenase and reduces stability.

PubMed

Kracher, Daniel; Andlar, Martina; Furtmüller, Paul G; Ludwig, Roland

2018-02-02

Lytic polysaccharide monooxygenases (LPMOs) are a class of copper-containing enzymes that oxidatively degrade insoluble plant polysaccharides and soluble oligosaccharides. Upon reductive activation, they cleave the substrate and promote biomass degradation by hydrolytic enzymes. In this study, we employed LPMO9C from Neurospora crassa , which is active toward cellulose and soluble β-glucans, to study the enzyme-substrate interaction and thermal stability. Binding studies showed that the reduction of the mononuclear active-site copper by ascorbic acid increased the affinity and the maximum binding capacity of LPMO for cellulose. The reduced redox state of the active-site copper and not the subsequent formation of the activated oxygen species increased the affinity toward cellulose. The lower affinity of oxidized LPMO could support its desorption after catalysis and allow hydrolases to access the cleavage site. It also suggests that the copper reduction is not necessarily performed in the substrate-bound state of LPMO. Differential scanning fluorimetry showed a stabilizing effect of the substrates cellulose and xyloglucan on the apparent transition midpoint temperature of the reduced, catalytically active enzyme. Oxidative auto-inactivation and destabilization were observed in the absence of a suitable substrate. Our data reveal the determinants of LPMO stability under turnover and non-turnover conditions and indicate that the reduction of the active-site copper initiates substrate binding. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

Cholinesterase and Paraoxonase (PON1) enzyme activities in Mexican-American Mothers and Children from an Agricultural Community

PubMed Central

Gonzalez, V.; Huen, K.; Venkat, S.; Pratt, K.; Xiang, P.; Harley, K.G.; Kogut, K.; Trujillo, C.M.; Bradman, A.; Eskenazi, B.; Holland, N.T.

2014-01-01

Exposure to organophosphate and carbamate pesticides can lead to neurotoxic effects through inhibition of cholinesterase enzymes. The paraoxonase (PON1) enzyme can detoxify oxon derivatives of some organophosphates. Lower PON1, acetylcholinesterase, and butyrylcholinesterase activities have been reported in newborns relative to adults, suggesting increased susceptibility to organophosphate exposure in young children. We determined PON1, acetylcholinesterase, and butyrylcholinesterase activities in Mexican-American mothers and their 9-year-old children (n=202 pairs) living in an agricultural community in California. We used paired t-tests to compare enzymatic activities among mothers and their children and analysis of variance to determine which factors are associated with enzyme activities. Substrate-specific PON1 activities were slightly lower in children than their mothers; however, these differences were not statistically significant. We observed significantly lower acetylcholinesterase but higher butyrylcholinesterase levels in children compared to their mothers. Mean butyrylcholinesterase levels were strongly associated with child obesity status (BMI Z scores >95%). We observed highly significant correlations among mother-child pairs for each of the enzymatic activities analyzed; however, PON1 activities did not correlate with acetylcholinesterase or butyrylcholinesterase activities. Our findings suggest that by age nine, PON1 activities approach adult levels and host factors including sex and obesity may affect key enzymes involved in pesticide metabolism. PMID:22760442

Effect of Reduction of Redox Modifications of Cys-Residues in the Na,K-ATPase α1-Subunit on Its Activity

PubMed Central

Dergousova, Elena A.; Petrushanko, Irina Yu.; Klimanova, Elizaveta A.; Mitkevich, Vladimir A.; Ziganshin, Rustam H.; Lopina, Olga D.; Makarov, Alexander A.

2017-01-01

Sodium-potassium adenosine triphosphatase (Na,K-ATPase) creates a gradient of sodium and potassium ions necessary for the viability of animal cells, and it is extremely sensitive to intracellular redox status. Earlier we found that regulatory glutathionylation determines Na,K-ATPase redox sensitivity but the role of basal glutathionylation and other redox modifications of cysteine residues is not clear. The purpose of this study was to detect oxidized, nitrosylated, or glutathionylated cysteine residues in Na,K-ATPase, evaluate the possibility of removing these modifications and assess their influence on the enzyme activity. To this aim, we have detected such modifications in the Na,K-ATPase α1-subunit purified from duck salt glands and tried to eliminate them by chemical reducing agents and the glutaredoxin1/glutathione reductase enzyme system. Detection of cysteine modifications was performed using mass spectrometry and Western blot analysis. We have found that purified Na,K-ATPase α1-subunit contains glutathionylated, nitrosylated, and oxidized cysteines. Chemical reducing agents partially eliminate these modifications that leads to the slight increase of the enzyme activity. Enzyme system glutaredoxin/glutathione reductase, unlike chemical reducing agents, produces significant increase of the enzyme activity. At the same time, the enzyme system deglutathionylates native Na,K-ATPase to a lesser degree than chemical reducing agents. This suggests that the enzymatic reducing system glutaredoxin/glutathione reductase specifically affects glutathionylation of the regulatory cysteine residues of Na,K-ATPase α1-subunit. PMID:28230807

Profiling of Volatile Compounds and Associated Gene Expression and Enzyme Activity during Fruit Development in Two Cucumber Cultivars

PubMed Central

Chen, Shuxia; Zhang, Ranran; Hao, Lining; Chen, Weifeng; Cheng, Siqiong

2015-01-01

Changes in volatile content, as well as associated gene expression and enzyme activity in developing cucumber fruits were investigated in two Cucumis sativus L. lines (No. 26 and No. 14) that differ significantly in fruit flavor. Total volatile, six-carbon (C6) aldehyde, linolenic and linoleic acid content were higher during the early stages, whereas the nine-carbon (C9) aldehyde content was higher during the latter stages in both lines. Expression of C. sativus hydroperoxide lyase (CsHPL) mirrored 13-hydroperoxide lyase (13-HPL) enzyme activity in variety No. 26, whereas CsHPL expression was correlated with 9-hydroperoxide lyase (9-HPL) enzyme activity in cultivar No. 14. 13-HPL activity decreased significantly, while LOX (lipoxygenase) and 9-HPL activity increased along with fruit ripening in both lines, which accounted for the higher C6 and C9 aldehyde content at 0-6 day post anthesis (dpa) and 9-12 dpa, respectively. Volatile compounds from fruits at five developmental stages were analyzed by principal component analysis (PCA), and heatmaps of volatile content, gene expression and enzyme activity were constructed. PMID:25799542

Increasing the thermal stability of cellulase C using rules learned from thermophilic proteins: a pilot study.

PubMed

Németh, Attila; Kamondi, Szilárd; Szilágyi, András; Magyar, Csaba; Kovári, Zoltán; Závodszky, Péter

2002-05-02

Some structural features underlying the increased thermostability of enzymes from thermophilic organisms relative to their homologues from mesophiles are known from earlier studies. We used cellulase C from Clostridium thermocellum to test whether thermostability can be increased by mutations designed using rules learned from thermophilic proteins. Cellulase C has a TIM barrel fold with an additional helical subdomain. We designed and produced a number of mutants with the aim to increase its thermostability. Five mutants were designed to create new electrostatic interactions. They all retained catalytic activity but exhibited decreased thermostability relative to the wild-type enzyme. Here, the stabilizing contributions are obviously smaller than the destabilization caused by the introduction of the new side chains. In another mutant, the small helical subdomain was deleted. This mutant lost activity but its melting point was only 3 degrees C lower than that of the wild-type enzyme, which suggests that the subdomain is an independent folding unit and is important for catalytic function. A double mutant was designed to introduce a new disulfide bridge into the enzyme. This mutant is active and has an increased stability (deltaT(m)=3 degrees C, delta(deltaG(u))=1.73 kcal/mol) relative to the wild-type enzyme. Reduction of the disulfide bridge results in destabilization and an altered thermal denaturation behavior. We conclude that rules learned from thermophilic proteins cannot be used in a straightforward way to increase the thermostability of a protein. Creating a crosslink such as a disulfide bond is a relatively sure-fire method but the stabilization may be smaller than calculated due to coupled destabilizing effects.

Using soil enzymes to explain observed differences in the response of soil decomposition to nitrogen fertilization

NASA Astrophysics Data System (ADS)

Stone, M.; Weiss, M.; Goodale, C. L.

2010-12-01

Soil microbes produce extracellular enzymes that degrade a variety of carbon-rich polymers contained within soil organic matter (SOM). These enzymes are key regulators of the terrestrial carbon cycle. However, basic information about the kinetics of extracellular enzymes and key environmental variables that regulate their catalytic ability is lacking. This study aims to clarify the mechanisms by which microbial carbon-degrading enzymes drive different responses to nitrogen (N) fertilization in soil decomposition at two sites with long-term N fertilization experiments, the Bear Brook (BB) forest in Maine and Fernow Forest (FF) in West Virginia. We examined a suite of cellulolytic and lignolytic enzymes that break down common SOM constituents. We hypothesized that enzymes derived from the site with a higher mean annual temperature (FF) would be more heat-tolerant, and retain their catalytic efficiency (Km) as temperature rises, relative to enzymes from the colder environment (BB). We further hypothesized that cellulolytic enzyme activity would be unaffected by N, while oxidative enzyme activity would be suppressed in N-fertilized soils. To test these hypotheses and examine the interactive effects of temperature and N, we measured enzyme activity in unfertilized and N-fertilized soils under a range of laboratory temperature manipulations. Preliminary results show a significant decrease in cellulolytic enzyme efficiency with temperature at the colder site (BB), as well as a significant increase in efficiency due to N-fertilization for two cellulolytic enzymes. Oxidative enzyme activity shows a marginally significant reduction due to N-fertilization at BB. These results suggest that soil warming may produce a negative feedback on carbon turnover in certain climates, while N-fertilization may alter the relative decomposition rates of different soil organic matter constituents. FF activity will be analyzed in a similar manner and the two sites will be compared in order to fully assess our hypotheses.

Oxidation of monohydric phenol substrates by tyrosinase: effect of dithiothreitol on kinetics.

PubMed

Naish-Byfield, S; Cooksey, C J; Riley, P A

1994-11-15

The effect of thiol compounds on the monophenolase activity of tyrosinase was investigated using 4-hydroxyanisole as the substrate and dithiothreitol (DTT) as the model thiol compound. We have demonstrated three actions of DTT on tyrosinase-catalysed reactions: (1) direct reduction of the copper at the active site of the enzyme; (2) generation of secondary, oxidizable species by adduct formation with the o-quinone reaction product, 4-MOB, which leads to an increase in the total oxygen utilization by the reaction system; and (3) reversible inhibition of the enzyme. We confirm our previous observation that, at approx. 10 mol of DTT/mol of enzyme, the lag phase associated with monohydric phenol oxidation by tyrosinase is abolished. We suggest that this is due to reduction of the copper at the active site of the enzyme by DTT, since (a) reduction of active-site copper in situ by DTT was demonstrated by [Cu(I)]2-carbon monoxide complex formation and (b) abolition of the lag at low DTT concentration occurs without effect on the maximum rate of reaction or on the total amount of oxygen utilized. At concentrations of DTT above that required to abolish the lag, we found that the initial velocity of the reaction increased with increasing DTT, with a concomitant increase in the total oxygen utilization. This is due to the formation of DTT-4-methoxy-o-benzoquinone (4-MOB) adducts which provide additional dihydric phenol substrate either directly or by reducing nascent 4-MOB. We present n.m.r. evidence for the formation of mono- and di-aromatic DTT adducts with 4-MOB, consistent with a suggested reoxidation scheme in the presence of tyrosinase. Inhibition of the enzyme at concentrations of DTT above 300 pmol/unit of enzyme was released on exhaustion of DTT by adduct formation with 4-MOB as it was generated.

Ex vivo effects of ibogaine on the activity of antioxidative enzymes in human erythrocytes.

PubMed

Nikolić-Kokić, Aleksandra; Oreščanin-Dušić, Zorana; Spasojević, Ivan; Slavić, Marija; Mijušković, Ana; Paškulin, Roman; Miljević, Čedo; Spasić, Mihajlo B; Blagojević, Duško P

2015-04-22

Ibogaine is a naturally occurring alkaloid with psychotropic and metabotropic effects, derived from the bark of the root of the West African Tabernanthe iboga plant. The tribes of Kongo basin have been using iboga as a stimulant, for medicinal purposes, and in rite of passage ceremonies, for centuries. Besides, it has been found that this drug has anti-addictive effects. Previous studies have demonstrated that ibogaine changed the quantity of ATP and energy related enzymes as well as the activity of antioxidant enzymes in cells thus altering redox equilibrium in a time manner. In this work, the mechanism of its action was further studied by measuring the effects of ibogaine in human erythrocytes in vitro on ATP liberation, membrane fluidity and antioxidant enzymes activity. Heparinized human blood samples were incubated with ibogaine (10 and 20 μM) at 37°C for 1h. Blood plasma was separated by centrifugation and the levels of ATP and uric acid were measured 10 min after the addition of ibogaine using standard kits. The activity of copper-zinc superoxide dismutase (SOD1), catalase (CAT), glutathione peroxidase (GSH-Px) and glutathione reductase (GR) were measured in erythrocytes after incubation period. The stability of SOD1 activity was further tested through in vitro incubation with H2O2 and scanning of its electrophoretic profiles. Membrane fluidity was determined using an electron paramagnetic resonance spin-labelling method. Results showed that ibogaine treatment of erythrocytes in vitro increased ATP concentration in the blood plasma without changes in neither erythrocytes membrane fluidity nor uric acid concentration. Ibogaine also increased SOD1 activity in erythrocytes at both doses applied here. Treatment with 20 μM also elevated GR activity after in vitro incubation at 37°C. Electrophoretic profiles revealed that incubation with ibogaine mitigates H2O2 mediated suppression of SOD1 activity. Some of the effects of ibogaine seem to be mediated through its influence on energy metabolism, redox active processes and the effects of discrete fluctuations of individual reactive oxygen species on different levels of enzyme activities. Overall, ibogaine acts as a pro-antioxidant by increasing activity of antioxidative enzymes and as an adaptagene in oxidative distress. Copyright © 2015. Published by Elsevier Ireland Ltd.

Effect of saposins on acid sphingomyelinase.

PubMed Central

Tayama, M; Soeda, S; Kishimoto, Y; Martin, B M; Callahan, J W; Hiraiwa, M; O'Brien, J S

1993-01-01

The effect of saposins (A, B, C and D) on acid sphingomyelinase activity was determined using a crude human kidney sphingomyelinase preparation and a purified sphingomyelinase preparation from human placenta. Saposin D stimulated the activity of the crude enzyme by increasing its apparent Km and Vmax. values for sphingomyelin hydrolysis. Unlike the crude enzyme, the activity of the purified enzyme was strongly inhibited by saposin D as well as other saposins. Saposin D decreased the apparent Km and Vmax values of purified sphingomyelinase activity. The effects of saposin D on the activity of different sphingomyelinase preparations appear to depend on Triton X-100, which is present in the crude enzyme but not in the purified enzyme. When the detergent was removed from the crude preparation, the effect of saposin D changed from being stimulatory to inhibitory. Conversely, when the detergent is added to the purified enzyme, the effect of saposin D on sphingomyelinase activity changed from being inhibitory to stimulatory. While other saposins were inhibitory or had no effect on sphingomyelinase activity in the above assay system, not only saposin D but also saposins A and C exhibited a stimulatory effect upon purified sphingomyelinase activity when the substrate, sphingomyelin, was added in the form of liposomes without detergent. Saposin B was not only inhibitory in the liposome system, but also reduced the stimulatory effect of saposins A, C and D. These observations indicate that the stimulatory effect of saposins A, C and D on acid sphingomyelinase activity is greatly influenced by the physical environment of the enzyme and suggest that similar effects by saposins may be exerted in lysosomal membranes. PMID:8452527

The Different Physiological and Antioxidative Responses of Zucchini and Cucumber to Sewage Sludge Application.

PubMed

Wyrwicka, Anna; Urbaniak, Magdalena

2016-01-01

The present study investigates the effect of soil amended with sewage sludge on oxidative changes in zucchini and cucumber plants (Cucurbitaceae) and the consequent activation of their antioxidative systems and detoxification mechanisms. The plants were grown in pots containing soil amended with three concentrations of sewage sludge (1.8 g, 5.4 g and 10.8 g per pot), while controls were potted with vegetable soil. The activities of three antioxidative enzymes, ascorbate peroxidase (APx), catalase (CAT) and guaiacol peroxidase (POx), were assessed, as well as of the detoxifying enzyme S-glutathione transferase (GST). Lipid peroxidation was evaluated by measuring the extent of oxidative damage; α-tocopherol content, the main lipophilic antioxidant, was also measured. Visible symptoms of leaf blade damage after sewage sludge application occurred only on the zucchini plants. The zucchini and cucumber plants showed a range of enzymatic antioxidant responses to sewage sludge application. While APx and POx activities increased significantly with increasing sludge concentration in the zucchini plants, they decreased in the cucumber plants. Moreover, although the activity of these enzymes increased gradually with increasing doses of sewage sludge, these levels fell at the highest dose. An inverse relationship between peroxidases activity and CAT activity was observed in both investigated plant species. In contrast, although GST activity increased progressively with sludge concentration in both the zucchini and cucumber leaves, the increase in GST activity was greater in the zucchini plants, being visible at the lowest dose used. The results indicate that signs of sewage sludge toxicity were greater in zucchini than cucumber, and its defense reactions were mainly associated with increases in APx, POx and GST activity.

The Different Physiological and Antioxidative Responses of Zucchini and Cucumber to Sewage Sludge Application

PubMed Central

Wyrwicka, Anna; Urbaniak, Magdalena

2016-01-01

The present study investigates the effect of soil amended with sewage sludge on oxidative changes in zucchini and cucumber plants (Cucurbitaceae) and the consequent activation of their antioxidative systems and detoxification mechanisms. The plants were grown in pots containing soil amended with three concentrations of sewage sludge (1.8 g, 5.4 g and 10.8 g per pot), while controls were potted with vegetable soil. The activities of three antioxidative enzymes, ascorbate peroxidase (APx), catalase (CAT) and guaiacol peroxidase (POx), were assessed, as well as of the detoxifying enzyme S-glutathione transferase (GST). Lipid peroxidation was evaluated by measuring the extent of oxidative damage; α-tocopherol content, the main lipophilic antioxidant, was also measured. Visible symptoms of leaf blade damage after sewage sludge application occurred only on the zucchini plants. The zucchini and cucumber plants showed a range of enzymatic antioxidant responses to sewage sludge application. While APx and POx activities increased significantly with increasing sludge concentration in the zucchini plants, they decreased in the cucumber plants. Moreover, although the activity of these enzymes increased gradually with increasing doses of sewage sludge, these levels fell at the highest dose. An inverse relationship between peroxidases activity and CAT activity was observed in both investigated plant species. In contrast, although GST activity increased progressively with sludge concentration in both the zucchini and cucumber leaves, the increase in GST activity was greater in the zucchini plants, being visible at the lowest dose used. The results indicate that signs of sewage sludge toxicity were greater in zucchini than cucumber, and its defense reactions were mainly associated with increases in APx, POx and GST activity. PMID:27327659

Using the β-glucosidase catalyzed reaction product glucose to improve the ionic liquid tolerance of β-glucosidases.

PubMed

Goswami, Shubhasish; Gupta, Neha; Datta, Supratim

2016-01-01

Pretreating biomass with ionic liquids (IL) increases enzyme accessibility and cellulose is typically recovered through precipitation with an anti-solvent. An industrially feasible pretreatment and hydrolysis process requires robust cellulases that are stable and active in the presence of either small amounts of ILs co-precipitated with recovered cellulose or for saccharifications in the presence of IL. β-glucosidase (BG) hydrolyzes cellobiose into two molecules of glucose (Glc) and is the last step of biomass hydrolysis. These enzymes are prone not only to product inhibition by glucose but also to inactivation by ILs. With increasing interest in IL-based pretreatment methods, there is increasing focus toward a search for Glc-tolerant and IL-tolerant BG. We identified a BG belonging to the GH1 family, H0HC94, encoded in Agrobacterium tumefaciens 5A, and cloned and overexpressed the protein in Escherichia coli. H0HC94 exhibited high enzymatic activity with β-glycosidic substrates (248 µmol/min/mg on pNPGlc and 262 µmol/min/mg on cellobiose) and tolerant to Glc (apparent K i = 686 mM). Further evidence of Glc-based stabilization came from the increase in melting temperature of H0HC94, with increasing Glc concentrations. The half-life of H0HC94 also increased between 2- and 20-fold in the presence of increasing concentrations of Glc. In the presence of 0.9 M of different [C2mim]-based ionic liquids, the specific activity of H0HC94 decreased by around 20-30 %. However, the addition of 100 mM glucose to the IL-enzyme mix resulted in a more stable enzyme as evidenced by the slight recovery of H0HC94 melting temperature and up to tenfold increase in half-life. This higher stability came at a cost of 2-10 % decrease in specific activity. The steady-state kinetic analyses for a subset of the ionic liquids tested indicate that the enzyme undergoes uncompetitive inhibition by glucose and ionic liquid, indicating the possibility of binding of the ionic liquid and glucose to the enzyme-substrate complex. H0HC94 is a Glc-stabilized BG that is also tolerant up to 0.9 M concentrations of different IL's and indicates the possibilities of using an IL-Glc-based cellulose solvent that displays enzyme-compatibility.

Prolonged fasting does not increase oxidative damage or inflammation in postweaned northern elephant seal pups

PubMed Central

Vázquez-Medina, José Pablo; Crocker, Daniel E.; Forman, Henry Jay; Ortiz, Rudy M.

2010-01-01

Elephant seals are naturally adapted to survive up to three months of absolute food and water deprivation (fasting). Prolonged food deprivation in terrestrial mammals increases reactive oxygen species (ROS) production, oxidative damage and inflammation that can be induced by an increase in the renin–angiotensin system (RAS). To test the hypothesis that prolonged fasting in elephant seals is not associated with increased oxidative stress or inflammation, blood samples and muscle biopsies were collected from early (2–3 weeks post-weaning) and late (7–8 weeks post-weaning) fasted seals. Plasma levels of oxidative damage, inflammatory markers and plasma renin activity (PRA), along with muscle levels of lipid and protein oxidation, were compared between early and late fasting periods. Protein expression of angiotensin receptor 1 (AT1), pro-oxidant (Nox4) and antioxidant enzymes (CuZn- and Mn-superoxide dismutases, glutathione peroxidase and catalase) was analyzed in muscle. Fasting induced a 2.5-fold increase in PRA, a 50% increase in AT1, a twofold increase in Nox4 and a 70% increase in NADPH oxidase activity. By contrast, neither tissue nor systemic indices of oxidative damage or inflammation increased with fasting. Furthermore, muscle antioxidant enzymes increased 40–60% with fasting in parallel with an increase in muscle and red blood cell antioxidant enzyme activities. These data suggest that, despite the observed increases in RAS and Nox4, an increase in antioxidant enzymes appears to be sufficient to suppress systemic and tissue indices of oxidative damage and inflammation in seals that have fasted for a prolonged period. The present study highlights the importance of antioxidant capacity in mammals during chronic periods of stress to help avoid deleterious systemic consequences. PMID:20581282

Prolonged fasting does not increase oxidative damage or inflammation in postweaned northern elephant seal pups.

PubMed

Vázquez-Medina, José Pablo; Crocker, Daniel E; Forman, Henry Jay; Ortiz, Rudy M

2010-07-15

Elephant seals are naturally adapted to survive up to three months of absolute food and water deprivation (fasting). Prolonged food deprivation in terrestrial mammals increases reactive oxygen species (ROS) production, oxidative damage and inflammation that can be induced by an increase in the renin-angiotensin system (RAS). To test the hypothesis that prolonged fasting in elephant seals is not associated with increased oxidative stress or inflammation, blood samples and muscle biopsies were collected from early (2-3 weeks post-weaning) and late (7-8 weeks post-weaning) fasted seals. Plasma levels of oxidative damage, inflammatory markers and plasma renin activity (PRA), along with muscle levels of lipid and protein oxidation, were compared between early and late fasting periods. Protein expression of angiotensin receptor 1 (AT(1)), pro-oxidant (Nox4) and antioxidant enzymes (CuZn- and Mn-superoxide dismutases, glutathione peroxidase and catalase) was analyzed in muscle. Fasting induced a 2.5-fold increase in PRA, a 50% increase in AT(1), a twofold increase in Nox4 and a 70% increase in NADPH oxidase activity. By contrast, neither tissue nor systemic indices of oxidative damage or inflammation increased with fasting. Furthermore, muscle antioxidant enzymes increased 40-60% with fasting in parallel with an increase in muscle and red blood cell antioxidant enzyme activities. These data suggest that, despite the observed increases in RAS and Nox4, an increase in antioxidant enzymes appears to be sufficient to suppress systemic and tissue indices of oxidative damage and inflammation in seals that have fasted for a prolonged period. The present study highlights the importance of antioxidant capacity in mammals during chronic periods of stress to help avoid deleterious systemic consequences.

Altered small intestinal absorptive enzyme activities in leptin-deficient obese mice: influence of bowel resection.

PubMed

Kiely, James M; Noh, Jae H; Svatek, Carol L; Pitt, Henry A; Swartz-Basile, Deborah A

2006-07-01

Residual bowel increases absorption after massive small bowel resection. Leptin affects intestinal adaptation, carbohydrate, peptide, and lipid handling. Sucrase, peptidase, and acyl coenzyme A:monoacylglycerol acyltransferase (MGAT) are involved in carbohydrate, protein, and lipid absorption. We hypothesized that leptin-deficient obese mice would have altered absorptive enzymes compared with controls before and after small bowel resection. Sucrase, peptidase (aminopeptidase N [ApN], dipeptidyl peptidase IV [DPPIV]), and MGAT activities were determined from lean control (C57BL/6J, n = 16) and leptin-deficient (Lep(ob), n = 16) mice small bowel before and after 50% resection. Ileal sucrase activity was greater in obese mice before and after resection. Jejunal ApN and DPPIV activities were lower for obese mice before resection; ileal ApN activity was unaltered after resection for both strains. Resection increased DPPIV activity in both strains. Jejunal MGAT in obese mice decreased postresection. In both strains, ileal MGAT activity decreased after resection, and obese mice had greater activity in remnant ileum. After small bowel resection, leptin-deficient mice have increased sucrase activity and diminished ileal ApN, DPPIV, and MGAT activity compared with controls. Therefore, we conclude that leptin deficiency alters intestinal enzyme activity in unresected animals and after small bowel resection. Altered handling of carbohydrate, protein, and lipid may contribute to obesity and diabetes in leptin-deficient mice.

Effect of Sulfated Chitooligosaccharides on Wheat Seedlings (Triticum aestivum L.) under Salt Stress.

PubMed

Zou, Ping; Li, Kecheng; Liu, Song; He, Xiaofei; Zhang, Xiaoqian; Xing, Ronge; Li, Pengcheng

2016-04-13

In this study, sulfated chitooligosaccharide (SCOS) was applied to wheat seedlings to investigate its effect on the plants' defense response under salt stress. The antioxidant enzyme activities, chlorophyll contents, and fluorescence characters of wheat seedlings were determined at a certain time. The results showed that treatment with exogenous SCOS could decrease the content of malondialdehyde, increase the chlorophyll contents, and modulate fluorescence characters in wheat seedlings under salt stress. In addition, SCOS was able to regulate the activities of antioxidant enzymes containing superoxide dismutase, catalase, peroxidase, ascorbate peroxidase, glutathione reductase, and dehydroascorbate reductase. Similarly, the mRNA expression levels of several antioxidant enzymes were efficiently modulated by SCOS. The results indicated that SCOS could alleviate the damage of salt stress by adjusting the antioxidant enzyme activities of plant. The effect of SCOS on the photochemical efficiency of wheat seedlings was associated with its enhanced capacity for antioxidant enzymes, which prevented structure degradation of the photosynthetic apparatus under NaCl stress. Furthermore, the effective activities of alleviating salt stress indicated the activities of SCOS were closely related with the sulfate group.

Effect of mitoguazone on polyamine oxidase activity in rat liver.

PubMed

Ferioli, Maria Elena; Berselli, Debora; Caimi, Samuela

2004-12-01

Mitoguazone is a known inhibitor of polyamine biosynthesis through competitive inhibition of S-adenosylmethionine decarboxylase. A recent renewed interest in mitoguazone as an antineoplastic agent prompted us to investigate the effect of the drug on polyamine catabolism in rat liver, since the organ plays an important role in detoxification mechanisms. Thus, the purpose of this work was to evaluate the effect of in vivo mitoguazone administration on polyamine catabolic enzymes. In particular, our interest was directed to the changes in polyamine oxidase activity, since this enzyme has been recently confirmed to exert important functions that until now were underestimated. Mitoguazone administration induced hepatic polyamine oxidase activity starting at 4 h after administration, and the enzyme returned to basal levels 96 h after treatment. The changes in enzyme activity were accompanied by changes in putrescine concentrations, which increased starting at 4 h until 72 h after treatment. We also evaluated the activity of the newly identified spermine oxidase, which was not significantly changed by mitoguazone treatment. Therefore, we hypothesized that the enzyme involved in mitoguazone response of the liver is the polyamine oxidase, which acts on acetylated polyamines as substrate.

Curcumin regulates gene expression of insulin like growth factor, B-cell CLL/lymphoma 2 and antioxidant enzymes in streptozotocin induced diabetic rats

PubMed Central

2013-01-01

Background The effects of curcumin on the activities and gene expression of antioxidant enzymes, superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPX), glutathione-S-transferase (G-ST), B-cell CLL/lymphoma 2 (Bcl-2) and insulin like growth factor-1 (IGF-1) in diabetic rats were studied. Methods Twenty four rats were assigned to three groups (8 rats for each). Rats of first group were non diabetic and rats of the second group were rendered diabetic by streptozotocin (STZ). Both groups received vehicle, corn oil only (5 ml/kg body weight) and served as negative and positive controls, respectively. Rats of the third group were rendered diabetic and received oral curcumin dissolved in corn oil at a dose of 15 mg/5 ml/kg body weight for 6 weeks. Results Diabetic rats showed significant increase of blood glucose, thiobarbituric acid reactive substances (TBARS) and activities of all antioxidant enzymes with significant reduction of reduced glutathione (GSH) compare to the control non diabetic group. Gene expression of Bcl2, SOD, CAT, GPX and GST was increased significantly in diabetic untreated rats compare to the control non diabetic group. The administration of curcumin to diabetic rats normalized significantly their blood sugar level and TBARS values and increased the activities of all antioxidant enzymes and GSH concentration. In addition, curcumin treated rats showed significant increase in gene expression of IGF-1, Bcl2, SOD and GST compare to non diabetic and diabetic untreated rats. Conclusion Curcumin was antidiabetic therapy, induced hypoglycemia by up-regulation of IGF-1 gene and ameliorate the diabetes induced oxidative stress via increasing the availability of GSH, increasing the activities and gene expression of antioxidant enzymes and Bcl2. Further studies are required to investigate the actual mechanism of action of curcumin regarding the up regulation of gene expression of examined parameters. PMID:24364912

Independent modulation of the activity of alpha-ketoglutarate dehydrogenase complex by Ca2+ and Mg2+.

PubMed

Panov, A; Scarpa, A

1996-01-16

The activity of alpha-ketoglutarate dehydrogenase complex (KGDHC), an important enzyme regulating several metabolic pathways, could be regulated by changes in the environment within the mitochondrial matrix. It has been postulated that the activity of this and other dehydrogenases in vivo could be modulated by changes in the intramitochondrial concentrations of Ca2+ or Mg2+. Using a purified alpha-ketoglutarate dehydrogenase from pig hearts, the effect of Ca2+ and/or Mg2+ on the enzyme activity was investigated. Either Ca2+ or Mg2+ increased enzyme activity, and the effects were additive if the concentrations of free divalent cations were below 0.1 and 1 mM for Ca2+ and Mg2+, respectively. In the presence of 1 mM alpha-ketoglutarate and other cofactors, the KM for Mg2+ was 25 microM and less than 1 microM for Ca2+. The KM for alpha-ketoglutarate was a function of the divalent cation(s) present: 4 +/- 1.1 mM in the absence of Ca2+, with or without Mg2+; 2.2 mM in the presence of 1.8 microM Ca2+ alone; and 0.3 mM in the presence of both Ca2+ and Mg2+. Mg2+ increased KGDHC activity only in the presence of thiamine pyrophosphate (TPP) indicating that KGDHC requires both TPP and Mg2+ for enzyme's maximal activity. The affinity of KGDHC for NAD+ is significantly changed by either Mg2+ or Ca2+. The conclusions are that changes in both Ca2+ and Mg2+, in concentrations possibly occurring within mitochondria, could control KGDHC activity and that thiamine pyrophosphate is required for maximal enzyme activity.

Effects of single-walled carbon nanotubes on soil microorganisms

NASA Astrophysics Data System (ADS)

Jin, L.; Chung, H.; Son, Y.

2011-12-01

Single-walled carbon nanotubes (SWCNTs) are novel materials that have the potential to be used in various commercial fields due to their unique physicochemical properties. As a result of commercial development of nanotechnology, SWCNTs may be discharged to the soil environment with unknown consequences. However, there are as yet no data in the scientific literature that demonstrate the effects of SWCNTs on microbial function in soils. Therefore, we aimed to determine the effects of SWCNTs on soil microbial activity through a 2-week incubation study on urban soils supplemented with different concentrations of SWCNTs ranging from 0 to 1000 μg CNT/g soil. Fluorometric test using fluorogenic substrates were employed for the measurement of several enzyme activities in soil samples. More specifically, we determined the changes in the activities of cellobiohydrolase, β-1,4-glucosidase, β-1,4-xylosidase, β-1,4-N-acetylglucosaminidase, L-leucine aminopeptidase and acid phosphatase which play important roles in the carbon, nitrogen, and phosphorus cycles in response to the addition of SWCNTs. We found that microbial enzyme activities decreased as the concentrations of SWCNT added increased. The lowest enzyme activities were observed under 1000 μg CNT/g soil. The overall pattern shows that enzyme activities decreased slightly in the first 2-3 days and increased in the later stage of the incubation. Our results suggest that relatively high concentrations of SWCNTs can inhibit microbial activities, and this may be due to microbial cell membrane damage caused by SWCNTs. However, further study needs to be conducted to determine the mechanism responsible for inhibitory effect of SWCNTs on soil microbial activity. It can be concluded that changes in the activities of extracellular enzymes can indicate the effect of SWCNTs on soil microorganisms and nutrient cycling.

Effects of obesity on liver cytochromes P450 in various animal models.

PubMed

Tomankova, Veronika; Anzenbacher, Pavel; Anzenbacherova, Eva

2017-06-01

The prevalence of obesity and other obesity-related diseases is increasing worldwide. Obesity is a disease characterized by increased body weight, or a condition resulting from excessive accumulation of body fat. Due to increased body fat deposits, obesity has also been associated with increased mortality resulting from higher incidence rates of hypertension, diabetes, or various types of cancer, such as breast, colorectal, cervical and prostate cancer. Physiological changes associated with obesity are likely to result in altered drug biotransformation. The main enzymes enabling the oxidative biotransformation of most drugs are cytochromes P450 (CYPs). The review summarizes how pathophysiological factors, especially obesity, affect properties (e.g. enzyme activity, protein expression, gene expression) of CYP enzymes in various experimental models of human obesity. Results reported by various authors suggest that obesity is associated with a decrease of CYP activities (except for the CYP2C and CYP2E1 enzymes). The only exception is mouse obesity induced by monosodium glutamate (administered to newborn mice) as it usually leads to increased CYP expression. Selecting an animal model that is as close as possible to the properties of human obesity is of paramount importance.

Delta 9 -tetrahydrocannabinol and ethanol: differential effects on sympathetic activity in differing environmental setting.

PubMed

Ng, L K; Lamprecht, F; Williams, R B; Kopin, I J

1973-06-29

Serum dopamine beta-hydroxylase activity, a useful biochemical index of peripheral sympathetic nervous activity, was measured in rats treated with Delta(9)-tetrahydrocannabinol or ethanol or both substances. After 7 days of treatment with either substance, serum dopamine beta-hydroxylase activity decreased significantly. Combined treatment with both agents enhanced the effects of each given alone. In rats subjected to immobilization stress, treatment with Delta(9)- tetrahydrocannabinol appeared to potentiate the stress-induced increase in serum enzyme activity. Treatment with ethanol, with or without Delta(9)-tetrahydrocannabinol, effectively blocked this increase in enzyme activity. These results show that both substances have significant effects on the sympathetic nervous system which are critically influenced by environmental setting.

Soy isoflavone supplementation elevates erythrocyte superoxide dismutase, but not plasma ceruloplasmin in postmenopausal breast cancer survivors.

PubMed

DiSilvestro, Robert A; Goodman, Jaime; Dy, Emily; Lavalle, Gregory

2005-02-01

Soy isoflavone antioxidant effects may help prevent breast cancer re-occurrence, but isoflavone estrogen-like actions may increase breast cancer risk. These isoflavone actions can be reflected by effects on two copper enzymes activities, superoxide dismutase 1 (SOD 1), which has antioxidant function relevant to breast cancer prevention, and ceruloplasmin, which has its synthesis up-regulated by estrogen, and for which high values are associated with high breast cancer risk. A soy isoflavone-rich concentrate supplement was examined for effects on these two copper enzyme activities in post-menopausal breast cancer survivors (n = 7) in a crossover design with a placebo (24 days on supplement or placebo; 14 day wash out). The soy concentrate, but not the placebo, increased erythrocyte SOD 1 activities, but not ceruloplasmin activities or protein. The effect on superoxide dismutase activities was not likely due to increased copper intake since analysis of the soy extract showed little copper. The effect on superoxide dismutase was not accompanied by a change in urinary contents of 8-deoxyhydroxyguanosine, a DNA oxidant product, though perhaps this would change with a longer intervention. In summary, in regard to two copper enzyme activities, an isoflavone-rich soy concentrate showed an antioxidant effect considered relevant to breast cancer, but not an effect associated with estrogenic activity and increased breast cancer risk.

Stepwise Loop Insertion Strategy for Active Site Remodeling to Generate Novel Enzyme Functions.

PubMed

Hoque, Md Anarul; Zhang, Yong; Chen, Liuqing; Yang, Guangyu; Khatun, Mst Afroza; Chen, Haifeng; Hao, Liu; Feng, Yan

2017-05-19

The remodeling of active sites to generate novel biocatalysts is an attractive and challenging task. We developed a stepwise loop insertion strategy (StLois), in which randomized residue pairs are inserted into active site loops. The phosphotriesterase-like lactonase from Geobacillus kaustophilus (GkaP-PLL) was used to investigate StLois's potential for changing enzyme function. By inserting six residues into active site loop 7, the best variant ML7-B6 demonstrated a 16-fold further increase in catalytic efficiency toward ethyl-paraoxon compared with its initial template, that is a 609-fold higher, >10 7 fold substrate specificity shift relative to that of wild-type lactonase. The remodeled variants displayed 760-fold greater organophosphate hydrolysis activity toward the organophosphates parathion, diazinon, and chlorpyrifos. Structure and docking computations support the source of notably inverted enzyme specificity. Considering the fundamental importance of active site loops, the strategy has potential for the rapid generation of novel enzyme functions by loop remodeling.

A saposin-like domain influences the intracellular localization, stability, and catalytic activity of human acyloxyacyl hydrolase.

PubMed

Staab, J F; Ginkel, D L; Rosenberg, G B; Munford, R S

1994-09-23

Acyloxyacyl hydrolase, a leukocyte enzyme that acts on bacterial lipopolysaccharides (LPSs) and many glycerolipids, is synthesized as a precursor polypeptide that undergoes internal disulfide linkage before being proteolytically processed into two subunits. The larger subunit contains an amino acid sequence (Gly-X-Ser-X-Gly) that is found at the active sites of many lipases, while the smaller subunit has amino acid sequence similarity to saposins (sphingolipid activator proteins), cofactors for sphingolipid glycohydrolases. We show here that both acyloxyacyl hydrolase subunits are required for catalytic activity toward LPS and glycerophosphatidylcholine. In addition, mutations that truncate or delete the small subunit have profound effects on the intracellular localization, proteolytic processing, and stability of the enzyme in baby hamster kidney cells. Remarkably, proteolytic cleavage of the precursor protein increases the activity of the enzyme toward LPS by 10-20-fold without altering its activity toward glycerophosphatidylcholine. Proper orientation of the two subunits thus seems very important for the substrate specificity of this unusual enzyme.

Effect of Chokeberry (Aronia melanocarpa) juice on the metabolic activation and detoxication of carcinogenic N-nitrosodiethylamine in rat liver.

PubMed

Krajka-Kuźniak, Violetta; Szaefer, Hanna; Ignatowicz, Ewa; Adamska, Teresa; Oszmiański, Jan; Baer-Dubowska, Wanda

2009-06-10

Chokeberry is a rich source of polyphenols, which may counteract the action of chemical carcinogens. The aim of this study was to examine the effect of chokeberry juice alone or in combination with N-nitrosodiethylamine (NDEA) on phase I and phase II enzymes and DNA damage in rat liver. The forced feeding with chokeberry juice alone decreased the activities of enzymatic markers of cytochrome P450, CYP1A1 and 1A2. NDEA treatment also decreased the activity of CYP2E1 but enhanced the activity of CYP2B. Pretreatment with chokeberry juice further reduced the activity of these enzymes. Modulation of P450 enzyme activities was accompanied by the changes in the relevant proteins levels. Phase II enzymes were increased in all groups of animals tested. Chokeberry juice augmented DNA damage and aggravated the effect of NDEA. These results indicate that chokeberry may protect against liver damage; however, in combination with chemical carcinogens it might enhance their effect.

Functionalized Anodic Aluminum Oxide Membrane–Electrode System for Enzyme Immobilization

PubMed Central

2015-01-01

A nanoporous membrane system with directed flow carrying reagents to sequentially attached enzymes to mimic nature’s enzyme complex system was demonstrated. Genetically modified glycosylation enzyme, OleD Loki variant, was immobilized onto nanometer-scale electrodes at the pore entrances/exits of anodic aluminum oxide membranes through His6-tag affinity binding. The enzyme activity was assessed in two reactions—a one-step “reverse” sugar nucleotide formation reaction (UDP-Glc) and a two-step sequential sugar nucleotide formation and sugar nucleotide-based glycosylation reaction. For the one-step reaction, enzyme specific activity of 6–20 min–1 on membrane supports was seen to be comparable to solution enzyme specific activity of 10 min–1. UDP-Glc production efficiencies as high as 98% were observed at a flow rate of 0.5 mL/min, at which the substrate residence time over the electrode length down pore entrances was matched to the enzyme activity rate. This flow geometry also prevented an unwanted secondary product hydrolysis reaction, as observed in the test homogeneous solution. Enzyme utilization increased by a factor of 280 compared to test homogeneous conditions due to the continuous flow of fresh substrate over the enzyme. To mimic enzyme complex systems, a two-step sequential reaction using OleD Loki enzyme was performed at membrane pore entrances then exits. After UDP-Glc formation at the entrance electrode, aglycon 4-methylumbelliferone was supplied at the exit face of the reactor, affording overall 80% glycosylation efficiency. The membrane platform showed the ability to be regenerated with purified enzyme as well as directly from expression crude, thus demonstrating a single-step immobilization and purification process. PMID:25025628

Influence of altered gravity on brain cellular energy and plasma membrane metabolism of developing lower aquatic vertebrates

NASA Astrophysics Data System (ADS)

Slenzka, K.; Appel, R.; Kappel, Th.; Rahmann, H.

Biochemical analyses of the brain of cichlid fish larvae, exposed for 7 days to increased acceleration of 3g (hyper-g), revealed an increase in energy availability (succinate dehydrogenase activity, SDH), and in mitochondrial energy transformation (creatine kinase, Mi_a-CK), but no changes in an energy consumptive process (high-affinity Ca^2+-ATPase). Brain glucose-6-phosphate dehydrogenase (G6PDH) of developing fish was previously found to be increased after hyper-g exposure. Three respectively 5 hours thereafter dramatic fluctuations in enzyme activity were registered. Analysing the cytosolic or plasma membrane-located brain creatine kinase (BB-CK) of clawed toad larvae after long-term hyper-g exposure a significant increase in enzyme activity was demonstrated, whereas the activity of a high affinity Ca^2+-ATPase remained unaffected.

Improvement of activity, thermo-stability and fruit juice clarification characteristics of fungal exo-polygalacturonase.

PubMed

Amin, Faiza; Bhatti, Haq Nawaz; Bilal, Muhammad; Asgher, Muhammad

2017-02-01

An extracellular exo-polygalacturonase (exo-PG) from Penicillium notatum was immobilized in sodium-alginate matrix through two different protocols, viz. covalent bonding and adsorption to enhance its catalytic activity, thermal stability and life-time properties for industrial applications. Covalent immobilization was more efficient in terms of high relative activity (45.89%) and immobilization yield (71.6%) as compared to adsorption. Immobilized exo-PG derivatives displayed maximum activities at pH 5.5 and 55°C as compared to free enzyme which showed its optimum activity at pH 6.0 and 50°C. The affinity of enzyme towards its substrate (K m(app) ) was reduced after immobilization and V max of covalently immobilized exo-PG decreased to 66.7% while the V max value of adsorbed enzyme increased up to 150% as compared to free counterpart. Both immobilization techniques greatly enhanced the thermal stability profile of the enzyme. At 60°C, immobilized exo-PGs retained more than 90% of their residual activities after 60min of heating, while free enzyme did not show any activity at the same temperature. Thermodynamic properties (i.e., Ea, ΔH*, ΔS*and ΔG*) of the free and immobilized enzymes were also investigated. Sodium-alginate covalently immobilized and adsorbed enzymes showed excellent recycling efficiencies and retained 50.0% and 41.0% of original activities, respectively after seven consecutive batch reactions. Moreover, the immobilized enzymes treatment achieved promising results in turbidity and viscosity reduction as well as clarity amelioration in various fruit juices. Altogether catalytic, thermo-stability and fruit juices clarification characteristics of the immobilized ex-PGs suggest a high potential for biotechnological exploitability. Copyright © 2016 Elsevier B.V. All rights reserved.

Comparison of the effects of gemfibrozil and clofibric acid on peroxisomal enzymes and cholesterol synthesis of rat hepatocytes.

PubMed

Hashimoto, F; Taira, S; Hayashi, H

1998-11-01

We studied whether the peroxisomal proliferation, induction of 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-CoA reductase) and activation of cholesterol synthesis by gemfibrozil shown in whole body (Hashimoto F., Ishikawa T., Hamada S. and Hayashi H., Biochemical. Pharm., 49, 1213-1221 (1995)) is also detected at a culture cell level, and we made a comparative analysis of the effects of clofibric acid. Gemfibrozil at 0.25 mM increased the activity of some peroxisomal enzymes (catalase and the cyanide-insensitive fatty acyl-CoA oxidizing system) after incubation for 72 h. However, contrary to whole body experiments, gemfibrozil decreased the activity of HMG-CoA reductase and cholesterol synthesis from [14C]acetate. At 1 mM, gemfibrozil decreased not only the activity of HMG-CoA reductase and cholesterol synthesis, but also the protein content of the cells and peroxisomal enzyme activity, indicating nonspecific inhibition at this concentration. Clofibric acid (0.25 and 1 mM) increased the activity of peroxisomal enzymes, but decreased the activity of HMG-CoA reductase and cholesterol synthesis. With respect to the direct effect on HMG-CoA reductase in the cell homogenate, gemfibrozil at 0.25 mm did not affect the activity, but it clearly inhibited the activity at 2 mM and above. Clofibric acid at 2 mM hardly affected the activity, but it clearly decreased the activity at 5 mM and over. That is, gemfibrozil directly inhibited the activity more strongly than clofibric acid. The direct inhibition of the enzyme itself required higher concentrations of both agents than did inhibition at the culture cell level. These results suggest that the cytotoxicity of gemfibrozil is greater than that of clofibric acid, and that gemfibrozil, as well as clofibric acid, can induce peroxisomal enzymes in the culture cell level. In contrast to whole body results, gemfibrozil may suppress cholesterol synthesis from [14C]acetate through the inhibition of HMG-CoA reductase at the culture cell level. The decreases in the reductase activity caused by gemfibrozil and clofibric acid at the culture cell level may not be caused by the direct inhibition of the enzyme.

Cloning, Production and Characterization of a Glycoside Hydrolase Family 7 Enzyme from the Gut Microbiota of the Termite Coptotermes curvignathus.

PubMed

Woon, James Sy-Keen; King, Patricia Jie Hung; Mackeen, Mukram Mohamed; Mahadi, Nor Muhammad; Wan Seman, Wan Mohd Khairulikhsan; Broughton, William J; Abdul Murad, Abdul Munir; Abu Bakar, Farah Diba

2017-07-01

Coptotermes curvignathus is a termite that, owing to its ability to digest living trees, serves as a gold mine for robust industrial enzymes. This unique characteristic reflects the presence of very efficient hydrolytic enzyme systems including cellulases. Transcriptomic analyses of the gut of C. curvignathus revealed that carbohydrate-active enzymes (CAZy) were encoded by 3254 transcripts and that included 69 transcripts encoding glycoside hydrolase family 7 (GHF7) enzymes. Since GHF7 enzymes are useful to the biomass conversion industry, a gene encoding for a GHF7 enzyme (Gh1254) was synthesized, sub-cloned and expressed in the methylotrophic yeast Pichia pastoris. Expressed GH1254 had an apparent molecular mass of 42 kDa, but purification was hampered by its low expression levels in shaken flasks. To obtain more of the enzyme, GH1254 was produced in a bioreactor that resulted in a fourfold increase in crude enzyme levels. The purified enzyme was active towards soluble synthetic substrates such as 4-methylumbelliferyl-β-D-cellobioside, 4-nitrophenyl-β-D-cellobioside and 4-nitrophenyl-β-D-lactoside but was non-hydrolytic towards Avicel or carboxymethyl cellulose. GH1254 catalyzed optimally at 35 °C and maintained 70% of its activity at 25 °C. This enzyme is thus potentially useful in food industries employing low-temperature conditions.

Temperature Sensitivity as a Microbial Trait Using Parameters from Macromolecular Rate Theory

PubMed Central

Alster, Charlotte J.; Baas, Peter; Wallenstein, Matthew D.; Johnson, Nels G.; von Fischer, Joseph C.

2016-01-01

The activity of soil microbial extracellular enzymes is strongly controlled by temperature, yet the degree to which temperature sensitivity varies by microbe and enzyme type is unclear. Such information would allow soil microbial enzymes to be incorporated in a traits-based framework to improve prediction of ecosystem response to global change. If temperature sensitivity varies for specific soil enzymes, then determining the underlying causes of variation in temperature sensitivity of these enzymes will provide fundamental insights for predicting nutrient dynamics belowground. In this study, we characterized how both microbial taxonomic variation as well as substrate type affects temperature sensitivity. We measured β-glucosidase, leucine aminopeptidase, and phosphatase activities at six temperatures: 4, 11, 25, 35, 45, and 60°C, for seven different soil microbial isolates. To calculate temperature sensitivity, we employed two models, Arrhenius, which predicts an exponential increase in reaction rate with temperature, and Macromolecular Rate Theory (MMRT), which predicts rate to peak and then decline as temperature increases. We found MMRT provided a more accurate fit and allowed for more nuanced interpretation of temperature sensitivity in all of the enzyme × isolate combinations tested. Our results revealed that both the enzyme type and soil isolate type explain variation in parameters associated with temperature sensitivity. Because we found temperature sensitivity to be an inherent and variable property of an enzyme, we argue that it can be incorporated as a microbial functional trait, but only when using the MMRT definition of temperature sensitivity. We show that the Arrhenius metrics of temperature sensitivity are overly sensitive to test conditions, with activation energy changing depending on the temperature range it was calculated within. Thus, we propose the use of the MMRT definition of temperature sensitivity for accurate interpretation of temperature sensitivity of soil microbial enzymes. PMID:27909429

Reporter enzyme inhibitor study to aid assembly of orthogonal reporter gene assays.

PubMed

Ho, Pei-i; Yue, Kimberley; Pandey, Pramod; Breault, Lyne; Harbinski, Fred; McBride, Aaron J; Webb, Brian; Narahari, Janaki; Karassina, Natasha; Wood, Keith V; Hill, Adam; Auld, Douglas S

2013-05-17

Reporter gene assays (RGAs) are commonly used to measure biological pathway modulation by small molecules. Understanding how such compounds interact with the reporter enzyme is critical to accurately interpret RGA results. To improve our understanding of reporter enzymes and to develop optimal RGA systems, we investigated eight reporter enzymes differing in brightness, emission spectrum, stability, and substrate requirements. These included common reporter enzymes such as firefly luciferase (Photinus pyralis), Renilla reniformis luciferase, and β-lactamase, as well as mutated forms of R. reniformis luciferase emitting either blue- or green-shifted luminescence, a red-light emitting form of Luciola cruciata firefly luciferase, a mutated form of Gaussia princeps luciferase, and a proprietary luciferase termed "NanoLuc" derived from the luminescent sea shrimp Oplophorus gracilirostris. To determine hit rates and structure-activity relationships, we screened a collection of 42,460 PubChem compounds at 10 μM using purified enzyme preparations. We then compared hit rates and chemotypes of actives for each enzyme. The hit rates ranged from <0.1% for β-lactamase to as high as 10% for mutated forms of Renilla luciferase. Related luciferases such as Renilla luciferase mutants showed high degrees of inhibitor overlap (40-70%), while unrelated luciferases such as firefly luciferases, Gaussia luciferase, and NanoLuc showed <10% overlap. Examination of representative inhibitors in cell-based assays revealed that inhibitor-based enzyme stabilization can lead to increases in bioluminescent signal for firefly luciferase, Renilla luciferase, and NanoLuc, with shorter half-life reporters showing increased activation responses. From this study we suggest strategies to improve the construction and interpretation of assays employing these reporter enzymes.

Mechanism of Calcium Lactate Facilitating Phytic Acid Degradation in Soybean during Germination.

PubMed

Hui, Qianru; Yang, Runqiang; Shen, Chang; Zhou, Yulin; Gu, Zhenxin

2016-07-13

Calcium lactate facilitates the growth and phytic acid degradation of soybean sprouts, but the mechanism is unclear. In this study, calcium lactate (Ca) and calcium lactate with lanthanum chloride (Ca+La) were used to treat soybean sprouts to reveal the relevant mechanism. Results showed that the phytic acid content decreased and the availability of phosphorus increased under Ca treatment. This must be due to the enhancement of enzyme activity related to phytic acid degradation. In addition, the energy metabolism was accelerated by Ca treatment. The energy status and energy metabolism-associated enzyme activity also increased. However, the transmembrane transport of calcium was inhibited by La(3+) and concentrated in intercellular space or between the cell wall and cell membrane; thus, Ca+La treatment showed reverse results compared with those of Ca treatment. Interestingly, gene expression did not vary in accordance with their enzyme activity. These results demonstrated that calcium lactate increased the rate of phytic acid degradation by enhancing growth, phosphorus metabolism, and energy metabolism.

Probiotic activity of lignocellulosic enzyme as bioactivator for rice husk degradation

NASA Astrophysics Data System (ADS)

Lamid, Mirni; Al-Arif, Anam; Warsito, Sunaryo Hadi

2017-02-01

The utilization of lignocellulosic enzyme will increase nutritional value of rice husk. Cellulase consists of C1 (β-1, 4-glucan cellobiohydrolase or exo-β-1,4glucanase), Cc (endo-β-1,4-glucanase) and component and cellobiose (β-glucocidase). Hemicellulase enzyme consists of endo-β-1,4-xilanase, β-xilosidase, α-L arabinofuranosidase, α-D-glukuronidaseand asetil xilan esterase. This research aimed to study the activity of lignocellulosic enzyme, produced by cows in their rumen, which can be used as a bioactivator in rice husk degradation. This research resulted G6 and G7 bacteria, producing xylanase and cellulase with the activity of 0.004 U mL-1 and 0.021 U mL-1; 0.003 ( U mL-1) and 0.026 (U mL-1) respectively.

Activated fibrinolytic enzymes in the synovial fluid during acute arthritis induced by urate crystal injection in dogs.

PubMed

Morimoto, N; Sumi, H; Tsushima, H; Etou, Y; Yoshida, E; Mihara, H

1991-10-01

To identify the relationship of the severity of inflammation and fibrinolytic activity in arthritis, the fibrinolytic activity of synovial fluid was studied in acute experimental arthritis induced by injecting monosodium urate crystals into dogs' knee joints. The maximum activity in the synovial fluid was observed 6 h after crystal injection. It was inferred that the fibrinolytic activity was mainly due to plasminogen activator based on fibrin plate assays, substrate specificity, inhibitor effects and zymography. On the other hand, the activity of lysosomal enzymes (beta-glucuronidase and cathepsin G) reached a peak in the synovia after 12 h. Histological examination of the synovial membrane after 12 h also showed greater inflammation than at 6 h. The peak in fibrinolytic activity preceded the peak of lysosomal enzymes and histological changes. These results suggest that an increase in fibrinolytic activity by plasminogen activator may contribute to the development of an acute inflammatory response.

A fluorogenic near-infrared imaging agent for quantifying plasma and local tissue renin activity in vivo and ex vivo

PubMed Central

Zhang, Jun; Preda, Dorin V.; Vasquez, Kristine O.; Morin, Jeff; Delaney, Jeannine; Bao, Bagna; Percival, M. David; Xu, Daigen; McKay, Dan; Klimas, Michael; Bednar, Bohumil; Sur, Cyrille; Gao, David Z.; Madden, Karen; Yared, Wael; Rajopadhye, Milind

2012-01-01

The renin-angiotensin system (RAS) is well studied for its regulation of blood pressure and fluid homeostasis, as well as for increased activity associated with a variety of diseases and conditions, including cardiovascular disease, diabetes, and kidney disease. The enzyme renin cleaves angiotensinogen to form angiotensin I (ANG I), which is further cleaved by angiotensin-converting enzyme to produce ANG II. Although ANG II is the main effector molecule of the RAS, renin is the rate-limiting enzyme, thus playing a pivotal role in regulating RAS activity in hypertension and organ injury processes. Our objective was to develop a near-infrared fluorescent (NIRF) renin-imaging agent for noninvasive in vivo detection of renin activity as a measure of tissue RAS and in vitro plasma renin activity. We synthesized a renin-activatable agent, ReninSense 680 FAST (ReninSense), using a NIRF-quenched substrate derived from angiotensinogen that is cleaved specifically by purified mouse and rat renin enzymes to generate a fluorescent signal. This agent was assessed in vitro, in vivo, and ex vivo to detect and quantify increases in plasma and kidney renin activity in sodium-sensitive inbred C57BL/6 mice maintained on a low dietary sodium and diuretic regimen. Noninvasive in vivo fluorescence molecular tomographic imaging of the ReninSense signal in the kidney detected increased renin activity in the kidneys of hyperreninemic C57BL/6 mice. The agent also effectively detected renin activity in ex vivo kidneys, kidney tissue sections, and plasma samples. This approach could provide a new tool for assessing disorders linked to altered tissue and plasma renin activity and to monitor the efficacy of therapeutic treatments. PMID:22674025

Regulation of 11 beta-hydroxysteroid dehydrogenase enzymes in the rat kidney by estradiol.

PubMed

Gomez-Sanchez, Elise P; Ganjam, Venkataseshu; Chen, Yuan Jian; Liu, Ying; Zhou, Ming Yi; Toroslu, Cigdem; Romero, Damian G; Hughson, Michael D; de Rodriguez, Angela; Gomez-Sanchez, Celso E

2003-08-01

The 11beta-hydroxysteroid dehydrogenase (11betaHSD) type 1 (11betaHSD1) enzyme is an NADP+-dependent oxidoreductase, usually reductase, of major glucocorticoids. The NAD+-dependent type 2 (11betaHSD2) enzyme is an oxidase that inactivates cortisol and corticosterone, conferring extrinsic specificity of the mineralocorticoid receptor for aldosterone. We reported that addition of a reducing agent to renal homogenates results in the monomerization of 11betaHSD2 dimers and a significant increase in NAD+-dependent corticosterone conversion. Estrogenic effects on expression, dimerization, and activity of the kidney 11betaHSD1 and -2 enzymes are described herein. Renal 11betaHSD1 mRNA and protein expressions were decreased to very low levels by estradiol (E2) treatment of both intact and castrated male rats; testosterone had no effect. NADP+-dependent enzymatic activity of renal homogenates from E2-treated rats measured under nonreducing conditions was less than that of homogenates from intact animals. Addition of 10 mM DTT to aliquots from these same homogenates abrogated the difference in NADP+-dependent activity between E2-treated and control rats. In contrast, 11betaHSD2 mRNA and protein expressions were significantly increased by E2 treatment. There was a marked increase in the number of juxtamedullary proximal tubules stained by the antibody against 11betaHSD2 after the administration of E2. Notwithstanding, neither the total corticosterone and 11-dehydrocorticosterone excreted in the urine nor their ratio differed between E2- and vehicle-treated rats. NAD+-dependent enzymatic activity in the absence or presence of a reducing agent demonstrated that the increase in 11betaHSD2 protein was not associated with an increase in in vitro activity unless the dimers were reduced to monomers.

Kinetic and biophysical investigation of the inhibitory effect of caffeine on human salivary aldehyde dehydrogenase: Implications in oral health and chemotherapy

NASA Astrophysics Data System (ADS)

Laskar, Amaj Ahmed; Alam, Md Fazle; Ahmad, Mohammad; Younus, Hina

2018-04-01

Human salivary aldehyde dehydrogenase (hsALDH) is primarily a class 3 ALDH (ALDH3A1), and is an important antioxidant enzyme present in the saliva which maintains healthy oral cavity. It detoxifies toxic aldehydes into non-toxic carboxylic acids in the oral cavity. Reduced level of hsALDH activity is a risk factor for oral cancer development. It is involved in the resistance of certain chemotherapeutic drugs. Coffee has been reported to affect the activity of salivary ALDH. In this study, the effect of caffeine on the activity (dehydrogenase and esterase) of hsALDH was investigated. The binding of caffeine to hsALDH was studied using different biophysical methods and molecular docking analysis. Caffeine was found to inhibit both crude and purified hsALDH. The Km increased and the Vmax decreased showing a mixed type of inhibition. Caffeine decreased the nucleophilicity of the catalytic cysteine residue. It binds to the active site of ALDH3A1 by forming a complex through non-covalent interactions with some highly conserved amino acid residues. It partially alters the secondary structure of the enzyme. Therefore, it is very likely that caffeine binds and inhibits the activity of hsALDH by decreasing substrate binding affinity and the catalytic efficiency of the enzyme. The study indicates that oral intake of caffeine may have a harmful effect on the oral health and may increase the risk of carcinogenesis through the inhibition of this important enzyme. Further, the inactivation of oxazaphosphorine based chemotherapeutic drugs by ALDH3A1 may be prevented by using caffeine as an adjuvant during medication which is expected to increase the sensitivity of these drugs through its inhibitory effect on the enzyme.

The dogfish shark (Squalus acanthias) increases both hepatic and extrahepatic ornithine urea cycle enzyme activities for nitrogen conservation after feeding.

PubMed

Kajimura, Makiko; Walsh, Patrick J; Mommsen, Thomas P; Wood, Chris M

2006-01-01

Urea not only is utilized as a major osmolyte in marine elasmobranchs but also constitutes their main nitrogenous waste. This study investigated the effect of feeding, and thus elevated nitrogen intake, on nitrogen metabolism in the Pacific spiny dogfish Squalus acanthias. We determined the activities of ornithine urea cycle (O-UC) and related enzymes in liver and nonhepatic tissues. Carbamoyl phosphate synthetase III (the rate-limiting enzyme of the O-UC) activity in muscle is high compared with liver, and the activities in both tissues increased after feeding. The contribution of muscle to urea synthesis in the dogfish body appears to be much larger than that of liver when body mass is considered. Furthermore, enhanced activities of the O-UC and related enzymes (glutamine synthetase, ornithine transcarbamoylase, arginase) were seen after feeding in both liver and muscle and were accompanied by delayed increases in plasma urea, trimethylamine oxide, total free amino acids, alanine, and chloride concentrations, as well as in total osmolality. The O-UC and related enzymes also occurred in the intestine but showed little change after feeding. Feeding did not change the rate of urea excretion, indicating strong N retention after feeding. Ammonia excretion, which constituted only a small percentage of total N excretion, was raised in fed fish, while plasma ammonia did not change, suggesting that excess ammonia in plasma is quickly ushered into synthesis of urea or protein. In conclusion, we suggest that N conservation is a high priority in this elasmobranch and that feeding promotes ureogenesis and growth. Furthermore, exogenous nitrogen from food is converted into urea not only by the liver but also by the muscle and to a small extent by the intestine.

Thermostable Fe/Mn superoxide dismutase from Bacillus licheniformis SPB-13 from thermal springs of Himalayan region: Purification, characterization and antioxidative potential.

PubMed

Thakur, Abhishek; Kumar, Pradeep; Lata, Jeevan; Devi, Neena; Chand, Duni

2018-05-01

Superoxide dismutase (SOD; EC 1.15.1.1) is an enzyme that scavenges free radicals and increases the longevity. In this study, a thermostable superoxide dismutase (SOD) from Bacillus licheniformis SPB-13, from Himalayan region was purified to homogeneity using ion exchange chromatography (DEAE-Sepharose). The SDS and native PAGE analysis showed that SOD is composed of two subunits of 32 kDa each and total molecular mass of the enzyme was estimated as 68 kDa. The specific activity of enzyme was 3965.51 U/mg and was purified to 16.17 folds. The SOD showed maximum activity with 60 mM Tris-HCl buffer at pH 8.0 for 2 min of incubation. Enzyme along with FeCl 3 as metal ion remained active till 70 °C. After reaction variables optimization, enzyme activity increased from 3965.51 to 4015.72 U/mg. Kinetic analysis of SOD showed k m of 1.4 mM of NADH and V max of 10000 U/mg of protein. Turnover number (k cat ) and catalytic efficiency (k cat /K m ) were found to be 11,333 s -1 and 7092.2 s -1 ·mM -1 NADH. The activation energy (E a ) was calculated as 2.67 kJ·mol -1 . After typing, it was found to be a member of Fe/Mn SOD family with IC 50 value of 25 μg/ml, prevented the cell death at a concentration of 30 μg/ml and it increased the cell viability by 30%. Copyright © 2018 Elsevier B.V. All rights reserved.

DYSFUNCTIONAL KYNURENINE PATHWAY METABOLISM IN THE R6/2 MOUSE MODEL OF HUNTINGTON’S DISEASE

PubMed Central

Sathyasaikumar, Korrapati V.; Stachowski, Erin K.; Amori, Laura; Guidetti, Paolo; Muchowski, Paul J.; Schwarcz, Robert

2013-01-01

Elevated concentrations of neurotoxic metabolites of the kynurenine pathway (KP) of tryptophan degradation may play a causative role in Huntington’s disease (HD). The brain levels of one of these compounds, 3-hydroxykynurenine (3-HK), are increased in both HD and several mouse models of the disease. In the present study, we examined this impairment in greater detail using the R6/2 mouse, a well-established animal model of HD. Initially, mutant and age-matched wild-type mice received an intrastriatal injection of 3H-tryptophan to assess the acute, local de novo production of kynurenine, the immediate bioprecursor of 3-HK, in vivo. No effect of genotype was observed between 4 and 12 weeks of age. In contrast, intrastriatally applied 3H-kynurenine resulted in significantly increased neosynthesis of 3H-3-HK, but not other tritiated KP metabolites, in the R6/2 striatum. Subsequent ex vivo studies in striatal, cortical and cerebellar tissue revealed substantial increases in the activity of the biosynthetic enzyme of 3-HK, kynurenine 3-monooxygenase (KMO) and significant reductions in the activity of its degradative enzyme, kynureninase, in HD mice starting at 4 weeks of age. Decreased kynureninase activity was most evident in the cortex and preceded the increase in KMO activity. The activity of other KP enzymes showed no consistent brain abnormalities in the mutant mice. These findings suggest that impairments in its immediate metabolic enzymes jointly account for the abnormally high brain levels of 3-HK in the R6/2 model of HD. PMID:20236387

Hydrolytic and ligninolytic enzyme activities in the Pb contaminated soil inoculated with litter-decomposing fungi.

PubMed

Kähkönen, Mika A; Lankinen, Pauliina; Hatakka, Annele

2008-06-01

The impact of Pb contamination was tested to five hydrolytic (beta-glucosidase, beta-xylosidase, beta-cellobiosidase, alpha-glucosidase and sulphatase) and two ligninolytic (manganese peroxidase, MnP and laccase) enzyme activities in the humus layer in the forest soil. The ability of eight selected litter-degrading fungi to grow and produce extracellular enzymes in the heavily Pb (40 g Pb of kg ww soil(-1)) contaminated and non-contaminated soil in the non-sterile conditions was also studied. The Pb content in the test soil was close to that of the shooting range at Hälvälä (37 g Pb of kg ww soil(-1)) in Southern Finland. The fungi were Agaricus bisporus, Agrocybe praecox, Gymnopus peronatus, Gymnopilus sapineus, Mycena galericulata, Gymnopilus luteofolius, Stropharia aeruginosa and Stropharia rugosoannulata. The Pb contamination (40 g Pb of kg ww soil(-1)) was deleterious to all five studied hydrolytic enzyme activities after five weeks of incubation. All five hydrolytic enzyme activities were significantly higher in the soil than in the extract of the soil indicating that a considerable part of enzymes were particle bound in the soils. Hydrolytic enzyme activities were higher in the non-contaminated soil than in the Pb contaminated soil. Fungal inocula increased the hydrolytic enzyme activities beta-cellobiosidase and beta-glucosidase in non-contaminated soils. All five hydrolytic enzyme activities were similar with fungi and without fungi in the Pb contaminated soil. This was in line that Pb contamination (40 g Pb of kg ww soil(-1)) depressed the growth of all fungi compared to those grown without Pb in the soil. Laccase and MnP activities were low in both Pb contaminated and non-contaminated soil cultures. MnP activities were higher in soil cultures containing Pb than without Pb. Our results showed that Pb in the shooting ranges decreased fungal growth and microbial functioning in the soil.

Effects of Elevated CO2 on Levels of Primary Metabolites and Transcripts of Genes Encoding Respiratory Enzymes and Their Diurnal Patterns in Arabidopsis thaliana: Possible Relationships with Respiratory Rates

PubMed Central

Watanabe, Chihiro K.; Sato, Shigeru; Yanagisawa, Shuichi; Uesono, Yukifumi; Terashima, Ichiro; Noguchi, Ko

2014-01-01

Elevated CO2 affects plant growth and photosynthesis, which results in changes in plant respiration. However, the mechanisms underlying the responses of plant respiration to elevated CO2 are poorly understood. In this study, we measured diurnal changes in the transcript levels of genes encoding respiratory enzymes, the maximal activities of the enzymes and primary metabolite levels in shoots of Arabidopsis thaliana grown under moderate or elevated CO2 conditions (390 or 780 parts per million by volume CO2, respectively). We examined the relationships between these changes and respiratory rates. Under elevated CO2, the transcript levels of several genes encoding respiratory enzymes increased at the end of the light period, but these increases did not result in changes in the maximal activities of the corresponding enzymes. The levels of some primary metabolites such as starch and sugar phosphates increased under elevated CO2, particularly at the end of the light period. The O2 uptake rate at the end of the dark period was higher under elevated CO2 than under moderate CO2, but higher under moderate CO2 than under elevated CO2 at the end of the light period. These results indicate that the changes in O2 uptake rates are not directly related to changes in maximal enzyme activities and primary metabolite levels. Instead, elevated CO2 may affect anabolic processes that consume respiratory ATP, thereby affecting O2 uptake rates. PMID:24319073

Regional variation in muscle metabolic enzymes in individual American shad (Alosa sapidissima)

USGS Publications Warehouse

Leonard, J.B.K.

1999-01-01

Evaluation of the activity of metabolic enzymes is often used to asses metabolic capacity at the tissue level, but the amount of regional variability within a tissue in an individual fish of a given species is frequently unknown. The activities of four enzymes (citrate synthase (CS), phosphofructokinase, lactate dehydrogenase (LDH), and ??-hydroxyacyl coenzyme A dehydrogenase (HOAD) were assayed in red and white muscle at 10 sites along the body of adult American shad (Alosa sapidissima). Red and white muscle HOAD and white muscle CS and LDH varied significantly, generally increasing posteriorly. Maximal variation occurs in red muscle HOAD (~450%) and white muscle LDH (~60%) activity. Differences between the sexes also vary with sampling location. This study suggests that the variability in enzyme activity may be linked to functional differences in the muscle at different locations, and also provides guidelines for sample collection in this species.

Optimal immobilization of β-galactosidase onto κ-carrageenan gel beads using response surface methodology and its applications.

PubMed

Elnashar, Magdy M; Awad, Ghada E; Hassan, Mohamed E; Mohy Eldin, Mohamed S; Haroun, Bakry M; El-Diwany, Ahmed I

2014-01-01

β-Galactosidase (β-gal) was immobilized by covalent binding on novel κ-carrageenan gel beads activated by two-step method; the gel beads were soaked in polyethyleneimine followed by glutaraldehyde. 2(2) full-factorial central composite experiment designs were employed to optimize the conditions for the maximum enzyme loading efficiency. 11.443 U of enzyme/g gel beads was achieved by soaking 40 units of enzyme with the gel beads for eight hours. Immobilization process increased the pH from 4.5 to 5.5 and operational temperature from 50 to 55 °C compared to the free enzyme. The apparent K(m) after immobilization was 61.6 mM compared to 22.9 mM for free enzyme. Maximum velocity Vmax was 131.2 μ mol · min(-1) while it was 177.1 μ mol · min(-1) for free enzyme. The full conversion experiment showed that the immobilized enzyme form is active as that of the free enzyme as both of them reached their maximum 100% relative hydrolysis at 4 h. The reusability test proved the durability of the κ-carrageenan beads loaded with β -galactosidase for 20 cycles with retention of 60% of the immobilized enzyme activity to be more convenient for industrial uses.

Active Site Flexibility as a Hallmark for Efficient PET Degradation by I. sakaiensis PETase.

PubMed

Fecker, Tobias; Galaz-Davison, Pablo; Engelberger, Felipe; Narui, Yoshie; Sotomayor, Marcos; Parra, Loreto P; Ramírez-Sarmiento, César A

2018-03-27

Polyethylene terephthalate (PET) is one of the most-consumed synthetic polymers, with an annual production of 50 million tons. Unfortunately, PET accumulates as waste and is highly resistant to biodegradation. Recently, fungal and bacterial thermophilic hydrolases were found to catalyze PET hydrolysis with optimal activities at high temperatures. Strikingly, an enzyme from Ideonella sakaiensis, termed PETase, was described to efficiently degrade PET at room temperature, but the molecular basis of its activity is not currently understood. Here, a crystal structure of PETase was determined at 2.02 Å resolution and employed in molecular dynamics simulations showing that the active site of PETase has higher flexibility at room temperature than its thermophilic counterparts. This flexibility is controlled by a novel disulfide bond in its active site, with its removal leading to destabilization of the catalytic triad and reduction of the hydrolase activity. Molecular docking of a model substrate predicts that PET binds to PETase in a unique and energetically favorable conformation facilitated by several residue substitutions within its active site when compared to other enzymes. These computational predictions are in excellent agreement with recent mutagenesis and PET film degradation analyses. Finally, we rationalize the increased catalytic activity of PETase at room temperature through molecular dynamics simulations of enzyme-ligand complexes for PETase and other thermophilic PET-degrading enzymes at 298, 323, and 353 K. Our results reveal that both the binding pose and residue substitutions within PETase favor proximity between the catalytic residues and the labile carbonyl of the substrate at room temperature, suggesting a more favorable hydrolytic reaction. These results are valuable for enabling detailed evolutionary analysis of PET-degrading enzymes and for rational design endeavors aiming at increasing the efficiency of PETase and similar enzymes toward plastic degradation. Copyright © 2018 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Molecular Basis of Impaired Glycogen Metabolism during Ischemic Stroke and Hypoxia

PubMed Central

Hossain, Mohammed Iqbal; Roulston, Carli Lorraine; Stapleton, David Ian

2014-01-01

Background Ischemic stroke is the combinatorial effect of many pathological processes including the loss of energy supplies, excessive intracellular calcium accumulation, oxidative stress, and inflammatory responses. The brain's ability to maintain energy demand through this process involves metabolism of glycogen, which is critical for release of stored glucose. However, regulation of glycogen metabolism in ischemic stroke remains unknown. In the present study, we investigate the role and regulation of glycogen metabolizing enzymes and their effects on the fate of glycogen during ischemic stroke. Results Ischemic stroke was induced in rats by peri-vascular application of the vasoconstrictor endothelin-1 and forebrains were collected at 1, 3, 6 and 24 hours post-stroke. Glycogen levels and the expression and activity of enzymes involved in glycogen metabolism were analyzed. We found elevated glycogen levels in the ipsilateral hemispheres compared with contralateral hemispheres at 6 and 24 hours (25% and 39% increase respectively; P<0.05). Glycogen synthase activity and glycogen branching enzyme expression were found to be similar between the ipsilateral, contralateral, and sham control hemispheres. In contrast, the rate-limiting enzyme for glycogen breakdown, glycogen phosphorylase, had 58% lower activity (P<0.01) in the ipsilateral hemisphere (24 hours post-stroke), which corresponded with a 48% reduction in cAMP-dependent protein kinase A (PKA) activity (P<0.01). In addition, glycogen debranching enzyme expression 24 hours post-stroke was 77% (P<0.01) and 72% lower (P<0.01) at the protein and mRNA level, respectively. In cultured rat primary cerebellar astrocytes, hypoxia and inhibition of PKA activity significantly reduced glycogen phosphorylase activity and increased glycogen accumulation but did not alter glycogen synthase activity. Furthermore, elevated glycogen levels provided metabolic support to astrocytes during hypoxia. Conclusion Our study has identified that glycogen breakdown is impaired during ischemic stroke, the molecular basis of which includes reduced glycogen debranching enzyme expression level together with reduced glycogen phosphorylase and PKA activity. PMID:24858129

The inhibitory effect of convulsant agents on the enzyme in brain which inactivates nerveside.

PubMed

Toh, C C

1969-07-01

1. An enzyme which can be extracted from brain inactivates nerveside in the optimum pH range 5.8-7.0.2. The polybasic acids trypan blue and its analogue trypan red, bromphenol blue and its analogue bromthymol blue at concentrations of 0.22 mM and ethylenediaminetetra-acetic acid (EDTA) at a concentration of 1 mM are strong inhibitors of the enzyme.3. Penicillin which is a monobasic carboxylic acid also inhibits the enzyme but only if concentrations as high as 3.6 mM are used. The antibiotic streptomycin which is a basic substance does not inhibit the enzyme.4. Caffeine at a concentration of 7.2 mM only weakly inhibits the enzyme.5. Chymotrypsin and wheat germ acid phosphatase also inactivate nerveside at pH 5.9 and are inhibited by the acidic dyes and penicillin. EDTA inhibits wheat germ phosphatase but activates chymotrypsin.6. Inactivation of nerveside by the brain enzyme and by wheat germ phosphatase is different from the action of chymotrypsin. Nerveside solutions incubated with chymotrypsin completely lose all biological activity whereas if incubation is carried out with either the brain enzyme or wheat germ acid phosphatase a residual biological activity remains even when the concentration of these two enzymes is increased. This residual biological activity is due to a peptide as it is destroyed by chymotrypsin.7. The manner in which nerveside is inactivated by the brain enzyme is uncertain as the preparation of the latter contained phosphodiesterase and protease activities which were similarly inhibited by the acid dyes, penicillin and EDTA.8. Pentylenetetrazole, picrotoxin, strychnine and tetanus toxin do not inhibit the brain enzyme.9. The nerveside-inactivating enzyme is not identical with the Substance P-inactivating enzyme in brain as the former is inhibited by EDTA while the latter is not.

Hypoglycemic and Hypolipidemic Effects of the Cracked-Cap Medicinal Mushroom Phellinus rimosus (Higher Basidiomycetes) in Streptozotocin-Induced Diabetic Rats.

PubMed

Rony, Kuttikkadan A; Ajith, Thekkuttuparambil A; Janardhanan, Kainoor K

2015-01-01

Phellinus rimosus is a parasitic host specific polypore mushroom with profound antioxidant, antihepatotoxic, anti-inflammatory, antitumor, and antimutagenic activities. This study investigated the hypoglycemic and hypolipidemic activities of the wood-inhabiting polypore mushroom Ph. Rimosus in streptozotocin (STZ)-induced diabetic rats. Diabetes was induced by single intraperitoneal injection of STZ (45 mg/kg) to Wistar rats. The effects of 30 days treatment with Ph. Rimosus (50 and 250 mg/ kg) and glibenclamide (0.65 mg/kg) on blood glucose level, serum insulin, serum lipid profile, liver glycogen, liver function enzymes, and non-enzymic and enzymic antioxidants activities in pancreas, liver, and kidney were evaluated in STZ-induced diabetic rats. Oral administration of Ph. Rimosus extract exhibited a significant reduction in blood glucose, triacylglycerol, total cholesterol, LDL-cholesterol, and liver function enzymes, and increased serum insulin, liver glycogen, and HDL-cholesterol levels in STZ-induced diabetic rats. Furthermore, Ph. Rimosus treatment increased antioxidant status in pancreas, liver, and kidney tissues with concomitant decreases in levels of thiobarbituric acid- reactive substances. Results of this study indicated that Ph. Rimosus possessed significant hypoglycemic and hypolipidemic activities and this effect may be related to its insulinogenic and antioxidant effect.

A multipurpose immobilized biocatalyst with pectinase, xylanase and cellulase activities

PubMed Central

Dalal, Sohel; Sharma, Aparna; Gupta, Munishwar Nath

2007-01-01

Background The use of immobilized enzymes for catalyzing various biotransformations is now a widely used approach. In recent years, cross-linked enzyme aggregates (CLEAs) have emerged as a novel and versatile biocatalyst design. The present work deals with the preparation of a CLEA from a commercial preparation, Pectinex™ Ultra SP-L, which contains pectinase, xylanase and cellulase activities. The CLEA obtained could be used for any of the enzyme activities. The CLEA was characterized in terms of kinetic parameters, thermal stability and reusability in the context of all the three enzyme activities. Results Complete precipitation of the three enzyme activities was obtained with n-propanol. When resulting precipitates were subjected to cross-linking with 5 mM glutaraldehyde, the three activities initially present (pectinase, xylanase and cellulase) were completely retained after cross-linking. The Vmax/Km values were increased from 11, 75 and 16 to 14, 80 and 19 in case of pectinase, xylanase and cellulase activities respectively. The thermal stability was studied at 50°C, 60°C and 70°C for pectinase, xylanase and cellulase respectively. Half-lives were improved from 17, 22 and 32 minutes to 180, 82 and 91 minutes for pectinase, xylanase and cellulase respectively. All three of the enzymes in CLEA could be reused three times without any loss of activity. Conclusion A single multipurpose biocatalyst has been designed which can be used for carrying out three different and independent reactions; 1) hydrolysis of pectin, 2) hydrolysis of xylan and 3) hydrolysis of cellulose. The preparation is more stable at higher temperatures as compared to the free enzymes. PMID:17880745

A multipurpose immobilized biocatalyst with pectinase, xylanase and cellulase activities.

PubMed

Dalal, Sohel; Sharma, Aparna; Gupta, Munishwar Nath

2007-06-08

The use of immobilized enzymes for catalyzing various biotransformations is now a widely used approach. In recent years, cross-linked enzyme aggregates (CLEAs) have emerged as a novel and versatile biocatalyst design. The present work deals with the preparation of a CLEA from a commercial preparation, Pectinex Ultra SP-L, which contains pectinase, xylanase and cellulase activities. The CLEA obtained could be used for any of the enzyme activities. The CLEA was characterized in terms of kinetic parameters, thermal stability and reusability in the context of all the three enzyme activities. Complete precipitation of the three enzyme activities was obtained with n-propanol. When resulting precipitates were subjected to cross-linking with 5 mM glutaraldehyde, the three activities initially present (pectinase, xylanase and cellulase) were completely retained after cross-linking. The V(max)/K(m) values were increased from 11, 75 and 16 to 14, 80 and 19 in case of pectinase, xylanase and cellulase activities respectively. The thermal stability was studied at 50 degrees C, 60 degrees C and 70 degrees C for pectinase, xylanase and cellulase respectively. Half-lives were improved from 17, 22 and 32 minutes to 180, 82 and 91 minutes for pectinase, xylanase and cellulase respectively. All three of the enzymes in CLEA could be reused three times without any loss of activity. A single multipurpose biocatalyst has been designed which can be used for carrying out three different and independent reactions; 1) hydrolysis of pectin, 2) hydrolysis of xylan and 3) hydrolysis of cellulose. The preparation is more stable at higher temperatures as compared to the free enzymes.

Enzymatic processing of pigmented and non pigmented rice bran on changes in oryzanol, polyphenols and antioxidant activity.

PubMed

Prabhu, Ashish A; Jayadeep, A

2015-10-01

Bran from different rice varieties is a treasure of nutrients and nutraceuticals, and its use is limited due to the poor sensory and functional properties. Application of enzymes can alter the functional and phytochemical properties. So the effect of endo-xylanase, cellulase and their combination on microstructural, nutraceutical and antioxidant properties of pigmented (Jyothi) and non-pigmented (IR64) rice bran were investigated. Scanning electron micrograph revealed micro structural changes in fibre structures on processing. All the enzymatic processing methods resulted in an increase in the content of oryzanol, soluble, bound and total polyphenols, flavonoid and tannin. It also showed an increase in the bioactivity with respect to free radical scavenging activity and total antioxidant activity. However, extent of the increase in bio-actives varied with the type of bran and enzyme application method. Endo-xylanase showed higher percentage difference compared to controls of Jyothi and IR64 bran extracts respectively in the content of the bound (10 & 19 %) and total (20 & 14 %) polyphenols. Combination of both the enzymes resulted in higher percentage increase of bioactive components and properties. It resulted in greater percentage difference compared to controls of Jyothi and IR64 extracts respectively in the content of soluble (58 & 17 %) and total (21 & 14 %) polyphenols, flavonoids (12 & 38 %), γ-oryzanol (10 & 12 %), free radical scavenging activity (64 & 30 %) and total antioxidant activity (82 & 136 %). It may be concluded that enzymatic bio-processing of bran with cellulose and hemicellulose degrading enzymes can improve its nutraceutical properties, and it may be used for development of functional foods.

Albumin Stimulates the Activity of the Human UDP-Glucuronosyltransferases 1A7, 1A8, 1A10, 2A1 and 2B15, but the Effects Are Enzyme and Substrate Dependent

PubMed Central

Svaluto-Moreolo, Paolo; Dziedzic, Klaudyna; Yli-Kauhaluoma, Jari; Finel, Moshe

2013-01-01

Human UDP-glucuronosyltransferases (UGTs) are important enzymes in metabolic elimination of endo- and xenobiotics. It was recently shown that addition of fatty acid free bovine serum albumin (BSA) significantly enhances in vitro activities of UGTs, a limiting factor in in vitro–in vivo extrapolation. Nevertheless, since only few human UGT enzymes were tested for this phenomenon, we have now performed detailed enzyme kinetic analysis on the BSA effects in six previously untested UGTs, using 2–4 suitable substrates for each enzyme. We also examined some of the previously tested UGTs, but using additional substrates and a lower BSA concentration, only 0.1%. The latter concentration allows the use of important but more lipophilic substrates, such as estradiol and 17-epiestradiol. In five newly tested UGTs, 1A7, 1A8, 1A10, 2A1, and 2B15, the addition of BSA enhanced, to a different degree, the in vitro activity by either decreasing reaction’s K m, increasing its V max, or both. In contrast, the activities of UGT2B17, another previously untested enzyme, were almost unaffected. The results of the assays with the previously tested UGTs, 1A1, 1A6, 2B4, and 2B7, were similar to the published BSA only as far as the BSA effects on the reactions’ K m are concerned. In the cases of V max values, however, our results differ significantly from the previously published ones, at least with some of the substrates. Hence, the magnitude of the BSA effects appears to be substrate dependent, especially with respect to V max increases. Additionally, the BSA effects may be UGT subfamily dependent since K m decreases were observed in members of subfamilies 1A, 2A and 2B, whereas large V max increases were only found in several UGT1A members. The results shed new light on the complexity of the BSA effects on the activity and enzyme kinetics of the human UGTs. PMID:23372764

A macro-enzyme cause of an isolated increase of alkaline phosphatase.

PubMed

Cervinski, Mark A; Lee, Hong Kee; Martin, Isabella W; Gavrilov, Dimitar K

2015-02-02

Macroenzyme complexes of serum enzymes and antibody can increase the circulating enzymatic activity and may lead to unnecessary additional testing and procedures. Laboratory physicians and scientists need to be aware of techniques to identify macroenzyme complexes when suspected. To investigate the possibility of a macro-alkaline phosphatase in the serum of a 74 year old male with persistently increased alkaline phosphatase we coupled a protein A/G agarose affinity chromatography technique with isoenzyme electrophoresis to look for the presence of macro-alkaline phosphatase. The majority of the alkaline phosphatase activity in the patient's serum sample was bound to the column and only a minor fraction (25%) of alkaline phosphatase activity was present in the column flow-through. The alkaline phosphatase activity was also found to co-elute with the immunoglobulins in the patient sample. The alkaline phosphatase activity in a control serum sample concurrently treated in the same manner did not bind to the column and was found in the column flow-through. The use of protein A/G agarose affinity chromatography is a rapid and simple method that can be applied to the investigation of other macro-enzyme complexes. Copyright © 2014 Elsevier B.V. All rights reserved.

Phosphorylation and mutations of Ser(16) in human phenylalanine hydroxylase. Kinetic and structural effects.

PubMed

Miranda, Frederico Faria; Teigen, Knut; Thórólfsson, Matthías; Svebak, Randi M; Knappskog, Per M; Flatmark, Torgeir; Martínez, Aurora

2002-10-25

Phosphorylation of phenylalanine hydroxylase (PAH) at Ser(16) by cyclic AMP-dependent protein kinase is a post-translational modification that increases its basal activity and facilitates its activation by the substrate l-Phe. So far there is no structural information on the flexible N-terminal tail (residues 1-18), including the phosphorylation site. To get further insight into the molecular basis for the effects of phosphorylation on the catalytic efficiency and enzyme stability, molecular modeling was performed using the crystal structure of the recombinant rat enzyme. The most probable conformation and orientation of the N-terminal tail thus obtained indicates that phosphorylation of Ser(16) induces a local conformational change as a result of an electrostatic interaction between the phosphate group and Arg(13) as well as a repulsion by Glu(280) in the loop at the entrance of the active site crevice structure. The modeled reorientation of the N-terminal tail residues (Met(1)-Leu(15)) on phosphorylation is in agreement with the observed conformational change and increased accessibility of the substrate to the active site, as indicated by circular dichroism spectroscopy and the enzyme kinetic data for the full-length phosphorylated and nonphosphorylated human PAH. To further validate the model we have prepared and characterized mutants substituting Ser(16) with a negatively charged residue and found that S16E largely mimics the effects of phosphorylation of human PAH. Both the phosphorylated enzyme and the mutants with acidic side chains instead of Ser(16) revealed an increased resistance toward limited tryptic proteolysis and, as indicated by circular dichroism spectroscopy, an increased content of alpha-helical structure. In agreement with the modeled structure, the formation of an Arg(13) to Ser(16) phosphate salt bridge and the conformational change of the N-terminal tail also explain the higher stability toward limited tryptic proteolysis of the phosphorylated enzyme. The results obtained with the mutant R13A and E381A further support the model proposed for the molecular mechanism for the activation of the enzyme by phosphorylation.

A designed bifunctional laccase/β-1,3-1,4-glucanase enzyme shows synergistic sugar release from milled sugarcane bagasse.

PubMed

Furtado, G P; Ribeiro, L F; Lourenzoni, M R; Ward, R J

2013-01-01

A bifunctional enzyme has been created by fusing two Bacillus subtilis enzymes: the β-1,3-1,4-glucanase (BglS, EC 3.2.1.73) that hydrolyzes plant cell wall β-glucans and the copper-dependent oxidase laccase (CotA, EC 1.10.3.2) that catalyzes the oxidation of aromatic compounds with simultaneous reduction of oxygen to water. The chimeric laccase/β-1,3-1,4-glucanase was created by insertion fusion of the bglS and cotA genes, and expressed in Escherichia coli. The affinity-purified recombinant chimeric enzyme showed both laccase and glucanase activities, with a maximum laccase activity at pH 4.5 and 75°C that showed a V(max) 30% higher than observed for the parental laccase. The maximum glucanase activity in the chimeric enzyme was at pH 6.0 and 50°C, with a slight reduction in V(max) by ∼10% compared with the parental glucanase. A decreased K(M) resulted in an overall increase in the K(cat)/K(M) value for the glucanase activity of the chimeric enzyme. The hydrolytic activity of the chimera was 20% higher against natural milled sugarcane bagasse as compared with equimolar mixtures of the separate parental enzymes. Molecular dynamics simulations indicated the approximation of the two catalytic domains in the chimeric enzyme, and the formation of an inter-domain interface may underlie the improved catalytic function.

6-shogaol, a major compound in ginger, induces aryl hydrocarbon receptor-mediated transcriptional activity and gene expression.

PubMed

Yoshida, Kazutaka; Satsu, Hideo; Mikubo, Ayano; Ogiwara, Haru; Yakabe, Takafumi; Inakuma, Takahiro; Shimizu, Makoto

2014-06-18

Xenobiotics are usually detoxified by drug-metabolizing enzymes and excreted from the body. The expression of many of drug-metabolizing enzymes is regulated by the aryl hydrocarbon receptor (AHR). Some substances in vegetables have the potential to be AHR ligands. To search for vegetable components that exhibit AHR-mediated transcriptional activity, we assessed the activity of vegetable extracts and identified the active compounds using the previously established stable AHR-responsive HepG2 cell line. Among the hot water extracts of vegetables, the highest activity was found in ginger. The ethyl acetate fraction of the ginger hot water extract remarkably induced AHR-mediated transcriptional activity, and the major active compound was found to be 6-shogaol. Subsequently, the mRNA levels of AHR-targeting drug-metabolizing enzymes (CYP1A1, UGT1A1, and ABCG 2) and the protein level of CYP1A1 in HepG2 cells were shown to be increased by 6-shogaol. This is the first report that 6-shogaol can regulate the expression of detoxification enzymes by AHR activation.

The use of enzymopathic human red cells in the study of malarial parasite glucose metabolism.

PubMed

Roth, E; Joulin, V; Miwa, S; Yoshida, A; Akatsuka, J; Cohen-Solal, M; Rosa, R

1988-05-01

The in vitro growth of Plasmodium falciparum malaria parasites was assayed in mutant red cells deficient in either diphosphoglycerate mutase (DPGM) or phosphoglycerate kinase (PGK). In addition, cDNA probes developed for human DNA sequences coding for these enzymes were used to examine the parasite genome by means of restriction endonuclease digestion and Southern blot analysis of parasite DNA. In both types of enzymopathic red cells, parasite growth was normal. In infected DPGM deficient red cells, no DPGM activity could be detected, and in normal red cells, DPGM activity declined slightly in a manner suggestive of parasite catabolism of host protein. However, in infected PGK deficient red cells, there was a 100-fold increase in PGK activity, and in normal red cells, a threefold increase in PGK activity was observed. Parasite PGK could be recovered from isolated parasites, and a marked increase in heat instability of parasite PGK as compared with the host cell enzyme was noted. Neither cDNA probe was found to cross-react with DNA sequences in the parasite genome. It is concluded that the parasite has no requirement for DPGM, and probably has no gene for this enzyme. On the other hand, the parasite does require PGK, (an adenosine triphosphate [ATP] generating enzyme) and synthesizes its own enzyme, which must have been encoded in the parasite genome. The parasite PGK gene most likely lacks sufficient homology to be detected by a human cDNA probe. Enzymopathic red cells are useful tools for elucidating the glycolytic enzymology of parasites and their co-evolution with their human hosts.

Mechanisms of the antihypertensive effects of Nigella sativa oil in L-NAME-induced hypertensive rats

PubMed Central

Jaarin, Kamsiah; Foong, Wai Dic; Yeoh, Min Hui; Kamarul, Zaman Yusoff Nik; Qodriyah, Haji Mohd Saad; Azman, Abdullah; Zuhair, Japar Sidik Fadhlullah; Juliana, Abdul Hamid; Kamisah, Yusof

2015-01-01

OBJECTIVES This study was conducted to determine whether the blood pressure-lowering effect of Nigella sativa might be mediated by its effects on nitric oxide, angiotensin-converting enzyme, heme oxygenase and oxidative stress markers. METHODS: Twenty-four adult male Sprague-Dawley rats were divided equally into 4 groups. One group served as the control (group 1), whereas the other three groups (groups 2-4) were administered L-NAME (25 mg/kg, intraperitoneally). Groups 3 and 4 were given oral nicardipine daily at a dose of 3 mg/kg and Nigella sativa oil at a dose of 2.5 mg/kg for 8 weeks, respectively, concomitantly with L-NAME administration. RESULTS Nigella sativa oil prevented the increase in systolic blood pressure in the L-NAME-treated rats. The blood pressure reduction was associated with a reduction in cardiac lipid peroxidation product, NADPH oxidase, angiotensin-converting enzyme activity and plasma nitric oxide, as well as with an increase in heme oxygenase-1 activity in the heart. The effects of Nigella sativa on blood pressure, lipid peroxidation product, nicotinamide adenine dinucleotide phosphate oxidase and angiotensin-converting enzyme were similar to those of nicardipine. In contrast, L-NAME had opposite effects on lipid peroxidation, angiotensin-converting enzyme and NO. CONCLUSION: The antihypertensive effect of Nigella sativa oil appears to be mediated by a reduction in cardiac oxidative stress and angiotensin-converting enzyme activity, an increase in cardiac heme oxygenase-1 activity and a prevention of plasma nitric oxide loss. Thus, Nigella sativa oil might be beneficial for controlling hypertension. PMID:26602523

Molecular engineering of industrial enzymes: recent advances and future prospects.

PubMed

Yang, Haiquan; Li, Jianghua; Shin, Hyun-Dong; Du, Guocheng; Liu, Long; Chen, Jian

2014-01-01

Many enzymes are efficiently produced by microbes. However, the use of natural enzymes as biocatalysts has limitations such as low catalytic efficiency, low activity, and low stability, especially under industrial conditions. Many protein engineering technologies have been developed to modify natural enzymes and eliminate these limitations. Commonly used protein engineering strategies include directed evolution, site-directed mutagenesis, truncation, and terminal fusion. This review summarizes recent advances in the molecular engineering of industrial enzymes and discusses future prospects in this field. We expect this review to increase interest in and advance the molecular engineering of industrial enzymes.

Physiochemical Studies of Sodium Chloride on Mungbean (Vigna radiata L. Wilczek) and Its Possible Recovery with Spermine and Gibberellic Acid

PubMed Central

Mitra, Sanglap; Paul, Atreyee

2015-01-01

The physiological and biochemical responses to increasing NaCl concentrations, along with low concentrations of gibberellic acid or spermine, either alone or in their combination, were studied in mungbean seedlings. In the test seedlings, the root-shoot elongation, biomass production, and the chlorophyll content were significantly decreased with increasing NaCl concentrations. Salt toxicity severely affected activities of different antioxidant enzymes and oxidative stress markers. Activities of antioxidant enzymes, superoxide dismutase (SOD), and catalase (CAT) increased significantly over water control. Similarly, oxidative stress markers such as proline, malondialdehyde (MDA), and hydrogen peroxide (H2O2) contents also increased as a result of progressive increase in salt stress. Combined application of NaCl along with low concentrations of either gibberellic acid (5 µM) or spermine (50 µM) in the test seedlings showed significant alterations, that is, drastic increase in seedling elongation, increased biomass production, increased chlorophyll content, and significant lowering in all the antioxidant enzyme activities as well as oxidative stress marker contents in comparison to salt treated test seedlings, leading to better growth and metabolism. Our study shows that low concentrations of either gibberellic acid or spermine will be able to overcome the toxic effects of NaCl stress in mungbean seedlings. PMID:25734186

Developmental changes in digestive enzyme activity in American shad, Alosa sapidissima, during early ontogeny.

PubMed

Gao, Xiao-Qiang; Liu, Zhi-Feng; Guan, Chang-Tao; Huang, Bin; Lei, Ji-Lin; Li, Juan; Guo, Zheng-Long; Wang, Yao-Hui; Hong, Lei

2017-04-01

In order to assess the digestive physiological capacity of the American shad Alosa sapidissima and to establish feeding protocols that match larval nutritional requirements, we investigated the ontogenesis of digestive enzymes (trypsin, amylase, lipase, pepsin, alkaline phosphatase, and leucine aminopeptidase) in larvae, from hatching to 45 days after hatching (DAH). We found that all of the target enzymes were present at hatching, except pepsin, which indicated an initial ability to digest nutrients and precocious digestive system development. Trypsin rapidly increased to a maximum at 14 DAH. Amylase sharply increased until 10 DAH and exhibited a second increase at 33 DAH, which coincided with the introduction of microdiet at 30 DAH, thereby suggesting that the increase was associated with the microdiet carbohydrate content. Lipase increased until 14 DAH, decreased until 27 DAH, and then increased until 45 DAH. Pepsin was first detected at 27 DAH and then sharply increased until 45 DAH, which suggested the formation of a functional stomach. Both alkaline phosphatase and leucine aminopeptidase markedly increased until 18 DAH, which indicated intestinal maturation. According to our results, we conclude that American shad larvae possess the functional digestive system before mouth opening, and the significant increases in lipase, amylase, pepsin, and intestinal enzyme activities between 27 and 33 DAH suggest that larvae can be successfully weaned onto microdiets around this age.

Effect of Simulated Microgravity on the Activity of Regulatory Enzymes of Glycolysis and Gluconeogenesis in Mice Liver

NASA Astrophysics Data System (ADS)

Ramirez, Joaquin; Periyakaruppan, Adaikkappan; Sarkar, Shubhashish; Ramesh, Govindarajan T.; Sharma, S. Chidananda

2014-02-01

Gravity supports all the life activities present on earth. Microgravity environments have effect on the biological functions and physiological status of an individual. The present study was undertaken to investigate the effect of simulated microgravity on important regulatory enzymes of carbohydrate metabolism in liver using HLS mice model. Following hind limb unloading of mice for 11 days the animal's average body weights were found to be not different, while the liver weights were decreased and found to be significantly different ( p < 0.05) from control mice. Further, in liver the specific activity of hexokinase enzyme was reduced ( p < 0.02) and the phosphoenolpyruvate carboxykinase activity was significantly increased in simulated microgravity subjected mice compared to control ( p < 0.003). Immunoblot analysis show decreased phosphofructokinase-2 activity in HLS mice compared to control. Liver lactate dehydrogenase activity significantly reduced in simulated microgravity subjected mice ( p < 0.005). Thus in our study the rodents have adapted to simulated microgravity conditions, with decreased glycolysis and increased gluconeogenesis in liver and reciprocally regulated.

Effects of gas periodic stimulation on key enzyme activity in gas double-dynamic solid state fermentation (GDD-SSF).

PubMed

Chen, Hongzhang; Shao, Meixue; Li, Hongqiang

2014-03-05

The heat and mass transfer have been proved to be the important factors in air pressure pulsation for cellulase production. However, as process of enzyme secretion, the cellulase formation has not been studied in the view of microorganism metabolism and metabolic key enzyme activity under air pressure pulsation condition. Two fermentation methods in ATPase activity, cellulase productivity, weight lose rate and membrane permeability were systematically compared. Results indicated that gas double-dynamic solid state fermentation had no obviously effect on cell membrane permeability. However, the relation between ATPase activity and weight loss rate was linearly dependent with r=0.9784. Meanwhile, the results also implied that gas periodic stimulation had apparently strengthened microbial metabolism through increasing ATPase activity during gas double-dynamic solid state fermentation, resulting in motivating the production of cellulase by Trichoderma reesei YG3. Therefore, the increase of ATPase activity would be another crucial factor to strengthen fermentation process for cellulase production under gas double-dynamic solid state fermentation. Copyright © 2013 Elsevier Inc. All rights reserved.

[Effects of Hippophae rhamnoides L. subsp. chinensis Rousi polysaccharide on alpha-glucosidase enzyme activity and level of blood glucose].

PubMed

Guo, Feng-Xia; Zeng, Yang; Li, Jin-Ping; Chen, Zhen-Ning; Ma, Ji-Xiong

2013-04-01

The enzyme-inhibitor model and the sugar tolerance mouse model were used to evaluate the relationship between the inhibition rate of enzyme activity and concentration of Hippophae rhamnoides L. subsp. chinensis Rousi polysaccharide (HRP). The inhibitory patterns of enzyme and dose-dependent effects of HRP's effect on blood glucose using acarbose tablets as control were also examined. The mechanism underlying hypoglycemic effects of HRP was discussed. The results showed: in the enzyme-inhibitor model, the inhibitory activity of different concentrations of HRP (9.80, 19.60, 39.20, 78.40, 156.80 and 312.50 mg x L(-1)) on alpha-glucosaminidase (AG) inhibitory activity were 6.62%, 18.02%, 33.26%, 48.23%, 62.11%, 76.31%, 90.12%, IC50 was 31.59 mg x L(-1). The inhibitory rate of 25.00 x 10(3) mg x L(-1) acarbose tablets was only 64.87%, and IC50 was 10.75 x 10(3) mg x L(-1). In the sugar tolerance mouse model, different doses of HRP (240, 480, 960 mg x kg(-1)) tended to decrease levels of blood glucose compared with control group (acarbose tablets 375 mg x kg(-1)) at 15, 30, 60 and 120 min. It's further confirmed that HRP is a kind of competitive inhibitor of AG activity. Its inhibition rate increases with the increase of concentration in normal mice, and it subsequently improves the sugar tolerance showing the effect of reducing blood sugar.

Flexibility and Stability Trade-Off in Active Site of Cold-Adapted Pseudomonas mandelii Esterase EstK.

PubMed

Truongvan, Ngoc; Jang, Sei-Heon; Lee, ChangWoo

2016-06-28

Cold-adapted enzymes exhibit enhanced conformational flexibility, especially in their active sites, as compared with their warmer-temperature counterparts. However, the mechanism by which cold-adapted enzymes maintain their active site stability is largely unknown. In this study, we investigated the role of conserved D308-Y309 residues located in the same loop as the catalytic H307 residue in the cold-adapted esterase EstK from Pseudomonas mandelii. Mutation of D308 and/or Y309 to Ala or deletion resulted in increased conformational flexibility. Particularly, the D308A or Y309A mutant showed enhanced substrate affinity and catalytic rate, as compared with wild-type EstK, via enlargement of the active site. However, all mutant EstK enzymes exhibited reduced thermal stability. The effect of mutation was greater for D308 than Y309. These results indicate that D308 is not preferable for substrate selection and catalytic activity, whereas hydrogen bond formation involving D308 is critical for active site stabilization. Taken together, conformation of the EstK active site is constrained via flexibility-stability trade-off for enzyme catalysis and thermal stability. Our study provides further insights into active site stabilization of cold-adapted enzymes.

'Enzyme Test Bench': A biochemical application of the multi-rate modeling

NASA Astrophysics Data System (ADS)

Rachinskiy, K.; Schultze, H.; Boy, M.; Büchs, J.

2008-11-01

In the expanding field of 'white biotechnology' enzymes are frequently applied to catalyze the biochemical reaction from a resource material to a valuable product. Evolutionary designed to catalyze the metabolism in any life form, they selectively accelerate complex reactions under physiological conditions. Modern techniques, such as directed evolution, have been developed to satisfy the increasing demand on enzymes. Applying these techniques together with rational protein design, we aim at improving of enzymes' activity, selectivity and stability. To tap the full potential of these techniques, it is essential to combine them with adequate screening methods. Nowadays a great number of high throughput colorimetric and fluorescent enzyme assays are applied to measure the initial enzyme activity with high throughput. However, the prediction of enzyme long term stability within short experiments is still a challenge. A new high throughput technique for enzyme characterization with specific attention to the long term stability, called 'Enzyme Test Bench', is presented. The concept of the Enzyme Test Bench consists of short term enzyme tests conducted under partly extreme conditions to predict the enzyme long term stability under moderate conditions. The technique is based on the mathematical modeling of temperature dependent enzyme activation and deactivation. Adapting the temperature profiles in sequential experiments by optimum non-linear experimental design, the long term deactivation effects can be purposefully accelerated and detected within hours. During the experiment the enzyme activity is measured online to estimate the model parameters from the obtained data. Thus, the enzyme activity and long term stability can be calculated as a function of temperature. The results of the characterization, based on micro liter format experiments of hours, are in good agreement with the results of long term experiments in 1L format. Thus, the new technique allows for both: the enzyme screening with regard to the long term stability and the choice of the optimal process temperature. The presented article gives a successful example for the application of multi-rate modeling, experimental design and parameter estimation within biochemical engineering. At the same time, it shows the limitations of the methods at the state of the art and addresses the current problems to the applied mathematics community.

Effects of plant lectin from cobra lily, Arisaema curvatum Kunth on development of melon fruit fly, Bactrocera cucurbitae (Coq.).

PubMed

Singh, Kuljinder; Kaur, Manpreet; Rup, Pushpinder J; Singh, Jatinder

2008-11-01

The lectin from tubers of cobra lily, Arisaema curvatum Kunth was purified by affinity chromatography using asialofetuin-linked amino activated porous silica beads. The concentration dependent effect of lectin was studied on second instar larvae (64-72 hr) of Bactrocera cucurbitae (Coq.). The treatment not only resulted in a significant reduction in the percentage pupation and emergence of the adults from treated larvae but it also prolonged the remaining larval development period. A very low LC50 value, 39 mgl(-1) of lectin was obtained on the basis of adult emergence using probit analysis. The activity of three hydrolase enzymes (esterases, acid and alkaline phosphatases), one oxidoreductase (catalase) and one group transfer enzyme (GSTs: Glutathione S-transferases) was assayed in second instar larvae under the influence of the LC50 of lectin at increasing exposure intervals (0, 24, 48 and 72 hr). The Arisaema curvatum lectin significantly decreased the activity of all the enzymes except for esterases, where the activity increased as compared to control at all exposure intervals. The decrease in pupation and emergence as well as significant suppression in the activities of two hydrolases, one oxidoreductase and one GST enzyme in treated larvae of B. cucurbitae indicated that this lectin has anti-metabolic effect on the melon fruit fly larvae.

Delivery of Formulated Industrial Enzymes with Acoustic Technology.

PubMed

Hwang, Jennifer Dorcas; Ortiz-Maldonado, Mariliz; Paramonov, Sergey

2016-02-01

Industrial enzymes are instrumental in many applications, including carbohydrate processing, fabric and household care, biofuels, food, and animal nutrition, among others. Enzymes have to be active and stable not only in harsh application conditions, but also during shipment and storage. In protein stability studies, formulated concentrated enzyme solutions are frequently diluted gravimetrically prior to enzyme activity measurements, making it challenging to move toward more high-throughput techniques using conventional robotic equipment. Current assay methods pose difficulties when measuring highly concentrated proteins. For example, plastic pipette tips can introduce error because proteins adsorb to the tip surface, despite the presence of detergents, decreasing precision and overall efficiency of protein activity assays. Acoustic liquid handling technology, frequently used for various dilute small-molecule assays, may overcome such problems. Originally shown to effectively deliver dilute solutions of small molecules, this technology is used here as an effective alternative to the aforementioned challenge with viscous concentrated protein solutions. Because the acoustic liquid handler transfers nanoliter quantities of liquids without using pipette tips and without sample loss, it rapidly and uniformly prepares assay plates for enzyme activity measurements within minutes. This increased efficiency transforms the nature of enzyme stability studies toward high precision and throughput. © 2015 Society for Laboratory Automation and Screening.

Soil Minerals Affect Extracellular Enzyme Activities in Cold and Warm Environments

NASA Astrophysics Data System (ADS)

Yang, Z.; Morin, M. M.; Graham, D. E.; Wullschleger, S. D.; Gu, B.

2017-12-01

Extracellular enzymes are mainly responsible for degrading and cycling soil organic matter (SOM) in both cold and warm terrestrial ecosystems. Minerals can play important roles in affecting soil enzyme activities, however, the interactions between enzyme and soil minerals remain poorly understood. In this study, we developed a model soil-enzyme system to examine the mineral effects on a hydrolytic enzyme (i.e., β-glucosidase) under both cold (4°C) and relatively warm (20 and 30°C) conditions. Minerals including iron oxides and clays (e.g., kaolinite and montmorillonite) were used to mimic different types of soils, and enzyme adsorption experiments were conducted to determine the enzyme interactions with different mineral surfaces. Time-series experiments were also carried out to measure enzymatic degradation of the organic substrates, such as cellobiose and indican. We observed that fractions of adsorbed enzyme and the hydrolytic activity were higher on iron oxides (e.g., hematite) compared to kaolinite and montmorillonite at given experimental conditions. The degradation of cellobiose was significantly faster than that of indican in the presence of minerals. We also found that the adsorption of enzyme was not dependent on the mineral surface areas, but was controlled by the mineral surface charge. In addition, temperature increase from 4 to 30°C enhanced mineral-assisted glucosidase hydrolysis by 2 to 4 fold, suggesting greater degradation under warmer environments. The present work demonstrates that the enzyme activity is influenced not only by the soil temperature but also by the surface chemistry of soil minerals. Our results highlight the need to consider the physical and chemical properties of minerals in biogeochemical models, which could provide a better prediction for enzyme-facilitated SOM transformations in terrestrial ecosystems.

Improved enzymatic hydrolysis of microcrystalline cellulose (Avicel PH101) by polyethylene glycol addition.

PubMed

Ouyang, Jia; Dong, Zhenwei; Song, Xiangyang; Lee, Xin; Chen, Mu; Yong, Qiang

2010-09-01

The effects of additives on hydrolysis of microcrystalline cellulose (Avicel PH101) were examined using commercial cellulose-degrading enzymes (Celluclast 1.5L and Novozyme 188). Polyethylene glycol 4000 (PEG4000) was the most effective additive tested. When PEG4000 was added at 0.05 g/g glucan, the conversion of Avicel PH101 increased 91% (from 41.1% to 78.9%). The cellulase activity of Celluclast 1.5L increased 27.5% with PEG4000 addition. A positive effect on enzyme stabilities of Celluclast 1.5L and Novozyme 188 also occurred with PEG4000 addition. During hydrolysis process, significant changes in free protein concentration and cellulase activity were observed on Avicel PH101. More than 90% of the original enzyme activity remained in the solution after 48 h hydrolysis. Thus, PEG4000 addition is an efficient method to enhance digestibility of cellulosic materials and make enzyme recovery possible and valuable. This provides an opportunity of decreasing the operational cost of the hydrolysis process. (c) 2010 Elsevier Ltd. All rights reserved.

Screening and Characterization of Polygalacturonase as Potential Enzyme for Keprok Garut Orange (Citrus nobilis var. chrysocarpa) Juice Clarification

NASA Astrophysics Data System (ADS)

Widowati, E.; Utami, R.; Kalistyatika, K.

2017-11-01

Use of thermostable enzyme from bacilli for industrial application is significant. This research aimed to isolate thermophilic pectinolytic bacteria from orange peel and vegetable waste which produced thermostable polygalacturonase, to investigate the polygalacturonase ability in clarifying keprok Garut orange juice, and to characterize polygalacturonase based on pH optimum, temperature optimum, enzyme stability, enzyme kinetics KM, and Vmax. Obtained, 14 isolates that further selected to 4 best isolates based on highest polygalacturonase activity and keprok Garut orange juice clarification ability. Four selected enzyme isolates were AR 2, AR 4, KK 4, and KK 5 had ability to increase juice transmittance, decrease juice viscosity and also reduce total soluble solid. Furthermore 4 selected isolates were partially purified by ammonium sulphate precipitation and dialysis method. Four partially purified enzymes were known that enzyme character of AR 2 optimum at pH 6; AR 4 optimum at pH 5.5; KK 4 optimum at pH 6; and KK 5 optimum at pH 4.5. Four enzymes were optimum at temperature 60°C thus stable at temperature 50-60°C, this characteristic indicate that enzymes were thermostable. AR 2 showed active activity stable at pH 4-7; AR 4 showed active activity stable at pH 6-7; KK 4 showed active activity stable at pH 4-6; however KK 5 stable at pH 4-5. Enzyme AR 2 and KK 4 was getting inactive at pH 11, thus AR 4 and KK 5 inactive at pH 12. KM value of AR 2, AR 4, KK 4, and KK 5 was 0.0959; 0.0974; 0.0966; and 0.178 mg/ml respectively. Vmax of AR 2, AR 4, KK 4, and KK 5 was 0.0203; 0.0202; 0.0185; and 0.0229 U/ml respectively. Enzyme AR 2 was the most compatible enzyme to be applied in keprok Garut orange juice clarification for it had the lowest KM value.

Nitric Oxide Enhances Desiccation Tolerance of Recalcitrant Antiaris toxicaria Seeds via Protein S-Nitrosylation and Carbonylation

PubMed Central

Bai, Xuegui; Yang, Liming; Tian, Meihua; Chen, Jinhui; Shi, Jisen; Yang, Yongping; Hu, Xiangyang

2011-01-01

The viability of recalcitrant seeds is lost following stress from either drying or freezing. Reactive oxygen species (ROS) resulting from uncontrolled metabolic activity are likely responsible for seed sensitivity to drying. Nitric oxide (NO) and the ascorbate-glutathione cycle can be used for the detoxification of ROS, but their roles in the seed response to desiccation remain poorly understood. Here, we report that desiccation induces rapid accumulation of H2O2, which blocks recalcitrant Antiaris toxicaria seed germination; however, pretreatment with NO increases the activity of antioxidant ascorbate-glutathione pathway enzymes and metabolites, diminishes H2O2 production and assuages the inhibitory effects of desiccation on seed germination. Desiccation increases the protein carbonylation levels and reduces protein S-nitrosylation of these antioxidant enzymes; these effects can be reversed with NO treatment. Antioxidant protein S-nitrosylation levels can be further increased by the application of S-nitrosoglutathione reductase inhibitors, which further enhances NO-induced seed germination rates after desiccation and reduces desiccation-induced H2O2 accumulation. These findings suggest that NO reinforces recalcitrant seed desiccation tolerance by regulating antioxidant enzyme activities to stabilize H2O2 accumulation at an appropriate concentration. During this process, protein carbonylation and S-nitrosylation patterns are used as a specific molecular switch to control antioxidant enzyme activities. PMID:21674063

Phenylbutyrate Therapy for Pyruvate Dehydrogenase Complex Deficiency and Lactic Acidosis

PubMed Central

Ferriero, Rosa; Manco, Giuseppe; Lamantea, Eleonora; Nusco, Edoardo; Ferrante, Mariella I.; Sordino, Paolo; Stacpoole, Peter W.; Lee, Brendan; Zeviani, Massimo; Brunetti-Pierri, Nicola

2014-01-01

Lactic acidosis is a build-up of lactic acid in the blood and tissues, which can be due to several inborn errors of metabolism as well as nongenetic conditions. Deficiency of pyruvate dehydrogenase complex (PDHC) is the most common genetic disorder leading to lactic acidosis. Phosphorylation of specific serine residues of the E1α subunit of PDHC by pyruvate dehydrogenase kinase (PDK) inactivates the enzyme, whereas dephosphorylation restores PDHC activity. We found that phenylbutyrate enhances PDHC enzymatic activity in vitro and in vivo by increasing the proportion of unphosphorylated enzyme through inhibition of PDK. Phenylbutyrate given to C57B6/L wild-type mice results in a significant increase in PDHC enzyme activity and a reduction of phosphorylated E1α in brain, muscle, and liver compared to saline-treated mice. By means of recombinant enzymes, we showed that phenylbutyrate prevents phosphorylation of E1α through binding and inhibition of PDK, providing a molecular explanation for the effect of phenylbutyrate on PDHC activity. Phenylbutyrate increases PDHC activity in fibroblasts from PDHC-deficient patients harboring various molecular defects and corrects the morphological, locomotor, and biochemical abnormalities in the noam631 zebrafish model of PDHC deficiency. In mice, phenylbutyrate prevents systemic lactic acidosis induced by partial hepatectomy. Because phenylbutyrate is already approved for human use in other diseases, the findings of this study have the potential to be rapidly translated for treatment of patients with PDHC deficiency and other forms of primary and secondary lactic acidosis. PMID:23467562

Effect of immobilized polygalacturonase from Mucor circinelloides ITCC-6025 on wine fermentation.

PubMed

Sharma, Sakshi; Hiteshi, Kalpana; Gupta, Reena

2013-01-01

Pectinases are among the most widely distributed enzymes in bacteria, fungi, and plants. Almost all the commercial preparations of pectinases are produced from fungal sources. Mucor circinelloides ITCC-6025 produced polygalacturonase when grown in Riviere's medium containing pectin (methyl ester) as the sole source of carbon. Immobilization of purified polygalacturonase was done on silica gel with 86% efficiency. The enzyme took 60 Min to bind maximally on the support. The immobilized enzyme showed maximum activity at a temperature of 45°C (4.57 U/mg) and pH 5.4. The immobilized enzyme was reused for four cycles as it retained almost 55% of its activity. The immobilized enzyme treatment increased the formation of higher alcohols and phenolics during the course of wine formation from apple and plum juices, whereas there was a decrease in the amount of carbohydrates. The enzyme treatment also resulted in clarification of wine; there was an increase in transmittance at 650 nm (201.78% in the case of apple wine and 223.4% in the case of plum wine) as compared to the control (untreated wine). © 2013 International Union of Biochemistry and Molecular Biology, Inc.

Acetylation of mitochondrial proteins by GCN5L1 promotes enhanced fatty acid oxidation in the heart.

PubMed

Thapa, Dharendra; Zhang, Manling; Manning, Janet R; Guimarães, Danielle A; Stoner, Michael W; O'Doherty, Robert M; Shiva, Sruti; Scott, Iain

2017-08-01

Lysine acetylation is a reversible posttranslational modification and is particularly important in the regulation of mitochondrial metabolic enzymes. Acetylation uses acetyl-CoA derived from fuel metabolism as a cofactor, thereby linking nutrition to metabolic activity. In the present study, we investigated how mitochondrial acetylation status in the heart is controlled by food intake and how these changes affect mitochondrial metabolism. We found that there was a significant increase in cardiac mitochondrial protein acetylation in mice fed a long-term high-fat diet and that this change correlated with an increase in the abundance of the mitochondrial acetyltransferase-related protein GCN5L1. We showed that the acetylation status of several mitochondrial fatty acid oxidation enzymes (long-chain acyl-CoA dehydrogenase, short-chain acyl-CoA dehydrogenase, and hydroxyacyl-CoA dehydrogenase) and a pyruvate oxidation enzyme (pyruvate dehydrogenase) was significantly upregulated in high-fat diet-fed mice and that the increase in long-chain and short-chain acyl-CoA dehydrogenase acetylation correlated with increased enzymatic activity. Finally, we demonstrated that the acetylation of mitochondrial fatty acid oxidation proteins was decreased after GCN5L1 knockdown and that the reduced acetylation led to diminished fatty acid oxidation in cultured H9C2 cells. These data indicate that lysine acetylation promotes fatty acid oxidation in the heart and that this modification is regulated in part by the activity of GCN5L1. NEW & NOTEWORTHY Recent research has shown that acetylation of mitochondrial fatty acid oxidation enzymes has greatly contrasting effects on their activity in different tissues. Here, we provide new evidence that acetylation of cardiac mitochondrial fatty acid oxidation enzymes by GCN5L1 significantly upregulates their activity in diet-induced obese mice. Copyright © 2017 the American Physiological Society.

Protective effect of D-002, a mixture of beeswax alcohols, against indomethacin-induced gastric ulcers and mechanism of action.

PubMed

Pérez, Yohani; Oyárzabal, Ambar; Mas, Rosa; Molina, Vivian; Jiménez, Sonia

2013-01-01

D-002, a mixture of higher aliphatic beeswax alcohols, produces gastroprotective and antioxidant effects. To investigate the gastroprotective effect of D-002 against indomethacin-induced ulcers, oxidative variables and myeloperoxidase (MPO) activity in the rat gastric mucosa were examined. Rats were randomized into six groups: a negative vehicle control and five indomethacin (50 mg/kg) treated groups, comprising a positive control, three groups treated orally with D-002 (5, 25 and 100 mg/kg) and one group with omeprazole 20 mg/kg intraperitoneally (ip). The contents of malondialdehyde (MDA), protein carbonyl groups (PCG), hydroxyl radical generation and catalase (CAT), glutathione peroxidase (GSH-PX), superoxide dismutase (SOD) and MPO enzyme activities in the rat gastric mucosa were assessed. Indomethacin increased the content of MDA and PCG, the generation of *OH radical and MPO enzyme activity, while it decreased the CAT, GSH-PX and SOD activities as compared to the negative controls. D-002 (5-100 mg/kg) significantly and dose-dependently reduced indomethacin-induced ulceration to 75 %. Also, D-002 decreased the content of MDA and PCG, the generation of hydroxyl radicals and MPO activity as compared to the positive controls. The highest dose of D-002 (100 mg/kg) increased significantly GSH-PX and SOD activities, while all doses used increased CAT activities. Omeprazole 20 mg/kg, the reference drug, reduced significantly the ulcers (93 %), MDA and PCG, the generation of hydroxyl radicals and MPO activity, and increased the CAT, GSH-PX and SOD activities. D-002 treatment produced gastroprotective effects against indomethacin-induced gastric ulceration, which can be related to the reduction of hydroxyl radical generation, lipid peroxidation, protein oxidation and MPO activity, and to the increase of the antioxidant enzymes activities in the rat gastric mucosa.