Carotenoid-cleavage activities of crude enzymes from Pandanous amryllifolius.

PubMed

Ningrum, Andriati; Schreiner, Matthias

2014-11-01

Carotenoid degradation products, known as norisoprenoids, are aroma-impact compounds in several plants. Pandan wangi is a common name of the shrub Pandanus amaryllifolius. The genus name 'Pandanus' is derived from the Indonesian name of the tree, pandan. In Indonesia, the leaves from the plant are used for several purposes, e.g., as natural colorants and flavor, and as traditional treatments. The aim of this study was to determine the cleavage of β-carotene and β-apo-8'-carotenal by carotenoid-cleavage enzymes isolated from pandan leaves, to investigate dependencies of the enzymatic activities on temperature and pH, to determine the enzymatic reaction products by using Headspace Solid Phase Microextraction Gas Chromatography/Mass Spectrophotometry (HS-SPME GC/MS), and to investigate the influence of heat treatment and addition of crude enzyme on formation of norisoprenoids. Crude enzymes from pandan leaves showed higher activity against β-carotene than β-apo-8'-carotenal. The optimum temperature of crude enzymes was 70°, while the optimum pH value was 6. We identified β-ionone as the major volatile reaction product from the incubations of two different carotenoid substrates, β-carotene and β-apo-8'-carotenal. Several treatments, e.g., heat treatment and addition of crude enzymes in pandan leaves contributed to the norisoprenoid content. Our findings revealed that the crude enzymes from pandan leaves with carotenoid-cleavage activity might provide a potential application, especially for biocatalysis, in natural-flavor industry. Copyright © 2014 Verlag Helvetica Chimica Acta AG, Zürich.

Preparation of progenin III from total steroidal saponins of Dioscorea nipponica Makino using a crude enzyme from Aspergillus oryzae strain.

PubMed

Liu, Tingqiang; Yu, Hongshan; Liu, Chunying; Bao, Yongming; Hu, Xiangchun; Wang, Yuanhao; Liu, Bing; Fu, Yaoyao; Tang, Sihui; Jin, Fengxie

2013-05-01

Progenin III, one of the most active spirostanol saponins, is a potential candidate for anti-cancer therapy due to its strong antitumor activity and low hemolytic activity. However, the concentration of progenin III is extremely low in natural Dioscorea plants. In this paper, the progenin III production from total steroidal saponins of Dioscorea nipponica Makino was studied using the crude enzyme from Aspergillus oryzae DLFCC-38. The crude enzyme converting total steroidal saponins into progenin III was obtained from the A. oryzae DLFCC-38 culture. For enzyme production, the strain was cultured for 72 h at 30 °C with shaking at 150 rpm in 5 % (w/v) malt extract medium containing 2 % (v/v) extract of D. nipponica as the enzyme inducer. The crude enzyme converted total steroidal saponins into major progenin III with a high yield when the reaction was carried out for 9 h at 50 °C and pH 5.0 with the 20 mg/ml of substrate. In the preparation of progenin III, 117 g of crude progenin III was obtained from 160 g of substrate, and the crude product was purified with silica gel column to obtain 60.3 g progenin III of 93.4 % purity.

Production of xylanases by mangrove fungi from the Philippines and their application in enzymatic pretreatment of recycled paper pulps.

PubMed

Torres, Jeremy Martin O; Dela Cruz, Thomas Edison E

2013-04-01

Mangrove fungi are vastly unexplored for enzymes with industrial application. This study aimed to assess the biocatalytic activity of mangrove fungal xylanases on recycled paper pulp. Forty-four mangrove fungal (MF) isolates were initially screened for xylanolytic activity in minimal medium with corn cob xylan as the sole carbon source. Eight MF were further cultivated under submerged fermentation for the production of crude xylanases. These crude enzymes were then characterized and tested for the pretreatment of recycled paper pulps. Results showed that 93 % of the tested MF isolates exhibited xylanolytic activity in solid medium. In submerged fermentation, salinity improved the growth of the fungal isolates but did not influence xylanase production. The crude xylanases were mostly optimally active at 50 °C and pH 7. Changes in pH had a greater effect on xylanase stability than temperature. More than half of the activity was lost at pH 9 for majority of the crude enzymes. However, two thermophilic xylanases from Fusarium sp. KAWIT-A and Aureobasidium sp. 2LIPA-M and one alkaliphilic xylanase from Phomopsis sp. MACA-J were also produced. All crude enzymes exhibited cellulase activities ranging from 4 to 21 U/ml. Enzymatic pretreatment of recycled paper pulps with 5 % consistency produced 70-650 mg of reducing sugars per gram of pulp at 50 °C after 60 min. The release of high amounts of reducing sugars showed the potential of mangrove fungal crude xylanases in the local paper and pulp industry. The diverse properties shown by the tested crude enzymes also indicate its potential applications to other enzyme-requiring industries.

Production of Pectate Lyase by Penicillium viridicatum RFC3 in Solid-State and Submerged Fermentation.

PubMed

Ferreira, Viviani; da Silva, Roberto; Silva, Dênis; Gomes, Eleni

2010-01-01

Pectate lyase (PL) was produced by the filamentous fungus Penicillium viridicatum RFC3 in solid-state cultures of a mixture of orange bagasse and wheat bran (1 : 1 w/w), or orange bagasse, wheat bran and sugarcane bagasse (1 : 1 : 0.5 w/w), and in a submerged liquid culture with orange bagasse and wheat bran (3%) as the carbon source. PL production was highest (1,500 U mL(-1) or 300 Ug(-1) of substrate) in solid-state fermentation (SSF) on wheat bran and orange bagasse at 96 hours. PL production in submerged fermentation (SmF) was influenced by the initial pH of the medium. With the initial pH adjusted to 4.5, 5.0, and 5.5, the peak activity was observed after 72, 48, and 24 hours of fermentation, respectively, when the pH of the medium reached the value 5.0. PL from SSF and SmF were loaded on Sephadex-G75 columns and six activity peaks were obtained from crude enzyme from SSF and designated PL I, II, III, IV, V, and VI, while five peaks were obtained from crude enzyme from SmF and labeled PL I', II', III', IV', and VII'. Crude enzyme and fraction III from each fermentative process were tested further. The optimum pH for crude PL from either process was 5.5, while that for PL III was 8.0. The maximum activity of enzymes from SSF was observed at 35 degrees C, but crude enzyme was more thermotolerant than PL III, maintaining its maximum activity up to 45 degrees C. Crude enzyme from SmF and PL III' showed thermophilic profiles of activity, with maximum activity at 60 and 55 degrees C, respectively. In the absence of substrate, the crude enzyme from SSF was stable over the pH range 3.0-10.0 and PL III was most stable in the pH range 4.0-7.0. Crude enzyme from SmF retained 70%-80% of its maximum activity in the acid-neutral pH range (4.0-7.0), but PIII showed high stability at alkaline pH (7.5-9.5). PL from SSF was more thermolabile than that from SmF. The latter maintained 60% of its initial activity after 1 h at 55 degrees C. The differing behavior of the enzymes with respect to pH and temperature suggests that they are different isozymes.

Evaluation of Enzymatic Deinking of Non-impact Ink Laser-Printed Paper Using Crude Enzyme from Penicillium rolfsii c3-2(1) IBRL.

PubMed

Lee, Kok Chang; Tong, Woei Yenn; Ibrahim, Darah; Arai, Takamitsu; Murata, Yoshinori; Mori, Yutaka; Kosugi, Akihiko

2017-01-01

Application of microbial enzymes for paper deinking is getting tremendous attention due to the rapidly increasing of waste paper every year. This study reports the deinking efficiency of laser-printed paper by the lignocellulolytic enzyme from Penicillium rolfsii c3-2(1) IBRL strain compared to other enzyme sources as well as commercial available enzymes. High enzymatic deinking efficiency of approximately 82 % on laser-printed paper was obtained by pulp treatment with crude enzyme from P. rolfsii c3-2(1) IBRL. However, this crude enzyme was found to reduce the paper strength properties of the pulp based on the results of tensile, tear and burst indices, most probably due to the cellulose degradation. This was further proven by the low viscosity of paper pulp obtained after enzymatic treatment and increasing of sugar production during the treatment. Balancing to this detrimental effect on paper pulp, high deinking efficiency was achieved within a short period of time, in which the enzymatic treatment was conducted for 30 min that enabled contribution to higher brightness index obtained, thus promoting savings of time and energy consumption, therefore environmental sustainability. Extensive research should be conducted to understand the nature and mechanism of enzymatic deinking process by the crude enzyme from P. rolfsii c3-2(1) IBRL in order to improve paper strength properties.

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

Proteolytic and amylolytic enzymes from a newly isolated Bacillus mojavensis SA: Characterization and applications as laundry detergent additive and in leather processing.

PubMed

Hammami, Amal; Fakhfakh, Nahed; Abdelhedi, Ola; Nasri, Moncef; Bayoudh, Ahmed

2018-03-01

The present work aims to study the simultaneous production of highly alkaline proteases and thermostable α-amylases by a newly isolated bacterium Bacillus mojavensis SA. The optimum pH and temperature of amylase activity were 9.0 and 55°C, respectively, while those of the proteolytic activity were 12.0 and 60°C, respectively. Both α-amylase and protease enzymes showed a high stability towards a wide range of pH and temperature. Furthermore, SA crude enzymes were relatively stable towards non-ionic (Tween 20, Tween 80 and Triton X-100) and anionic (SDS) surfactants, as well as oxidizing agents. Both activities were improved by the presence of polyethylene glycol 4000 and glycerol. Additionally, the crude enzymes showed excellent stability against various solid and liquid detergents. Wash performance analysis revealed that the SA crude enzymes exhibited a remarkable efficiency in the removal of a variety type of stains, such as blood, chocolate, coffee and oil. On the other side, SA proteases revealed a potential dehairing activity of animal hide without chemical assistance or fibrous proteins hydrolysis. Thus, considering their promising properties, B. mojavensis SA crude enzymes could be used in several biotechnological bioprocesses. Copyright © 2017 Elsevier B.V. All rights reserved.

Production of Pectate Lyase by Penicillium viridicatum RFC3 in Solid-State and Submerged Fermentation

PubMed Central

Ferreira, Viviani; da Silva, Roberto; Silva, Dênis; Gomes, Eleni

2010-01-01

Pectate lyase (PL) was produced by the filamentous fungus Penicillium viridicatum RFC3 in solid-state cultures of a mixture of orange bagasse and wheat bran (1 : 1 w/w), or orange bagasse, wheat bran and sugarcane bagasse (1 : 1 : 0.5 w/w), and in a submerged liquid culture with orange bagasse and wheat bran (3%) as the carbon source. PL production was highest (1,500 U  mL−1 or 300 Ug−1 of substrate) in solid-state fermentation (SSF) on wheat bran and orange bagasse at 96 hours. PL production in submerged fermentation (SmF) was influenced by the initial pH of the medium. With the initial pH adjusted to 4.5, 5.0, and 5.5, the peak activity was observed after 72, 48, and 24 hours of fermentation, respectively, when the pH of the medium reached the value 5.0. PL from SSF and SmF were loaded on Sephadex-G75 columns and six activity peaks were obtained from crude enzyme from SSF and designated PL I, II, III, IV, V, and VI, while five peaks were obtained from crude enzyme from SmF and labeled PL  I′, II′, III′, IV′, and VII′. Crude enzyme and fraction III from each fermentative process were tested further. The optimum pH for crude PL from either process was 5.5, while that for PL III was 8.0. The maximum activity of enzymes from SSF was observed at 35°C, but crude enzyme was more thermotolerant than PL III, maintaining its maximum activity up to 45°C. Crude enzyme from SmF and PL III′ showed thermophilic profiles of activity, with maximum activity at 60 and 55°C, respectively. In the absence of substrate, the crude enzyme from SSF was stable over the pH range 3.0–10.0 and PL III was most stable in the pH range 4.0–7.0. Crude enzyme from SmF retained 70%–80% of its maximum activity in the acid-neutral pH range (4.0–7.0), but PIII showed high stability at alkaline pH (7.5–9.5). PL from SSF was more thermolabile than that from SmF. The latter maintained 60% of its initial activity after 1 h at 55°C. The differing behavior of the enzymes with respect to pH and temperature suggests that they are different isozymes. PMID:20689719

Biosynthesis of Oligomeric Anthocyanins from Grape Skin Extracts.

PubMed

Hwang, Jin-Woo; Natarajan, Sithranga Boopathy; Kim, Yon-Suk; Kim, Eun-Kyung; Lee, Jae Woong; Moon, Sang-Ho; Jeon, Byong-Tae; Park, Pyo-Jam

2017-03-21

We synthesized oligomeric anthocyanins from grape skin-derived monomeric anthocyanins such as anthocyanidin and proanthocyanidin by a fermentation technique using Aspergillus niger, crude enzymes and glucosidase. The biosyntheses of the oligomeric anthocyanins carried out by the conventional method using Aspergillus niger and crude enzymes were confirmed by ESI-MS. The molecular weight of the synthesized anthocyanin oligomers was determined using MALDI-MS. The yield of anthocyanin oligomers using crude enzymes was higher than that of the synthesis using Aspergillus fermentation. Several studies have been demonstrated that oligomeric anthocyanins have higher antioxidant activity than monomeric anthocyanins. Fermentation-based synthesis of oligomeric anthocyanins is an alternative way of producing useful anthocyanins that could support the food industry.

In vitro metabolic engineering for the salvage synthesis of NAD(.).

PubMed

Honda, Kohsuke; Hara, Naoya; Cheng, Maria; Nakamura, Anna; Mandai, Komako; Okano, Kenji; Ohtake, Hisao

2016-05-01

Excellent thermal and operational stabilities of thermophilic enzymes can greatly increase the applicability of biocatalysis in various industrial fields. However, thermophilic enzymes are generally incompatible with thermo-labile substrates, products, and cofactors, since they show the maximal activities at high temperatures. Despite their pivotal roles in a wide range of enzymatic redox reactions, NAD(P)(+) and NAD(P)H exhibit relatively low stabilities at high temperatures, tending to be a major obstacle in the long-term operation of biocatalytic chemical manufacturing with thermophilic enzymes. In this study, we constructed an in vitro artificial metabolic pathway for the salvage synthesis of NAD(+) from its degradation products by the combination of eight thermophilic enzymes. The enzymes were heterologously produced in recombinant Escherichia coli and the heat-treated crude extracts of the recombinant cells were directly used as enzyme solutions. When incubated with experimentally optimized concentrations of the enzymes at 60°C, the NAD(+) concentration could be kept almost constant for 15h. Copyright © 2016 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

Enhanced oxidation of benzo[a]pyrene by crude enzyme extracts produced during interspecific fungal interaction of Trametes versicolor and Phanerochaete chrysosporium.

PubMed

Qian, Linbo; Chen, Baoliang

2012-01-01

The effects of interspecific fungal interactions between Trametes versicolor and Phanerochaete chrysosporium on laccase activity and enzymatic oxidation of polycyclic aromatic hydrocarbons (PAHs) were investigated. A deadlock between the two mycelia rather than replacement of one fungus by another was observed on an agar medium. The laccase activity in crude enzyme extracts from interaction zones reached a maximum after a 5-day incubation, which was significantly higher than that from regions of T. versicolor or P. chrysosporium alone. The enhanced induction of laccase activity lasted longer in half nutrition than in normal nutrition. A higher potential to oxidize benzo[a]pyrene by a crude enzyme preparation extracted from the interaction zones was demonstrated. After a 48 hr incubation period, the oxidation of benzo[a]pyrene by crude enzyme extracts from interaction zones reached 26.2%, while only 9.5% of benzo[a]pyrene was oxidized by crude extracts from T. versicolor. The oxidation was promoted by the co-oxidant 2,2'-azinobis-3-ethylbenzthiazoline-6-sulphonate diammonium salt (ABTS). These findings indicate that the application of co-culturing of white-rot fungi in bioremediation is a potential ameliorating technique for the restoration of PAH-contaminated soil.

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

Effect of temperature and mixing speed on immobilization of crude enzyme from Aspergillus niger on chitosan for hydrolyzing cellulose

NASA Astrophysics Data System (ADS)

Hamzah, Afan; Gek Ela Kumala, P.; Ramadhani, Dwi; Maziyah, Nurul; Rahmah, Laila Nur; Soeprijanto, Widjaja, Arief

2017-05-01

Conversion of cellulose into reducing sugar through enzymatic hydrolysis has advantageous because it produces greater product yield, higher selectivity, require less energy, more moderate operating conditions and environment friendly. However, the nature of the enzyme that is difficult to separate and its expensive price become an obstacle. These obstacles can be overcome by immobilizing the enzyme on chitosan material so that the enzyme can be reused. Chitosan is chosen because it is cheap, inert, hydrophilic, and biocompatible. In this research, we use covalent attachment and combination between covalent attachment and cross-linking method for immobilizing crude enzyme. This research was focusing in study of Effect of temperature and mixing speed on Immobilization Enzyme From Aspergillus Niger on Chitosan For Hydrolyzing both soluble (Carboxymethylcellulose) and insoluble Cellulose (coconut husk). This Research was carried out by three main step. First, coconut husk was pre-treated mechanically and chemically, Second, Crude enzyme from Aspergillus niger strain was immobilized on chitosan in various immobilization condition. At last, the pre-treated coconut husk and Carboxymetylcellulose (CMC) were hydrolyzed by immobilized cellulose on chitosan for reducing sugar production. The result revealed that the most reducing sugar produced by immobilized enzyme on chitosan+GDA with immobilization condition at 30 °C and 125 rpm. Enzyme immobilized on chitosan cross-linked with GDA produced more reducing sugar from preteated coconut husk than enzyme immobilized on chitosan.

Extraction of Oil from Flaxseed (Linum usitatissimum L.) Using Enzyme-Assisted Three-Phase Partitioning.

PubMed

Tan, Zhi-Jian; Yang, Zi-Zhen; Yi, Yong-Jian; Wang, Hong-Ying; Zhou, Wan-Lai; Li, Fen-Fang; Wang, Chao-Yun

2016-08-01

In this study, enzyme-assisted three-phase partitioning (EATPP) was used to extract oil from flaxseed. The whole procedure is composed of two parts: the enzymolysis procedure in which the flaxseed was hydrolyzed using an enzyme solution (the influencing parameters such as the type and concentration of enzyme, temperature, and pH were optimized) and three-phase partitioning (TPP), which was conducted by adding salt and t-butanol to the crude flaxseed slurry, resulting in the extraction of flaxseed oil into alcohol-rich upper phase. The concentration of t-butanol, concentration of salt, and the temperature were optimized to maximize the extraction yield. Under optimized conditions of a 49.29 % t-butanol concentration, 30.43 % ammonium sulfate concentration, and 35 °C extraction temperature, a maximum extraction yield of 71.68 % was obtained. This simple and effective EATPP can be used to achieve high extraction yields and oil quality, and thus, it is potential for large-scale oil production.

[Decolorization of dyestuff and dying waste water by laccase solution with self-flocculent mycelial pellets of Coriolus versicolor].

PubMed

Wu, Mianbin; Xia, Liming

2002-06-01

Both laccase production by the white-rot fungus Coriolus versicolor and decolorization of dyestuff and dying waste water with crude solution of laccase were studied in this work. Laccase production meets the definition of secondary metabolism. For laccase production the optimum initial pH is 4.5. Addition of veratryl alcohol or elevated trace metals could both enhance the laccase activity, while Tween80 showed some inhibition. The immobilized mycelia of C. versicolor in polyurethane foam had less laccase production ability than mycelial pellets. A repeated batch cultivation process was found to be a very economical way for laccase harvest. The same pellets could be used for at least 14 times and average laccase activity of each batch could maintain 6.72 IU/mL. This method reduces the enzyme production course, medium consumption and the possibility of contamination, showing high efficient and great economic benefit. Good results were also obtained in decolorization experiments with the crude solution of laccase. With 3.3 IU/mL initial laccase activity, color removal of Acid Orange reached 98.5% after 24 h reaction. Also with 2.6 IU/mL initial laccase activity, color removal of dying waste water reached 93% after 24 h reaction.

Characterization and identification of the xylanolytic enzymes from Aspergillus fumigatus Z5.

PubMed

Miao, Youzhi; Li, Juan; Xiao, Zhizhuang; Shen, Qirong; Zhang, Ruifu

2015-06-23

Plant biomass, the most abundant natural material on earth, represents a vast source of food and energy in nature. As the main component of plant biomass, xylan is a complex polysaccharide comprising a linear β(1,4)-linked backbone of xylosyl residues substituted by acetyl, arabinosyl, glucuronysyl and 4-O-methylglucuronycyl residues. Aspergillus fumigatus Z5 is an efficient plant biomass depolymerization fungus. In this study, its crude xylanolytic enzymes were characterized and identified by two-dimensional gel electrophoresis (2-DE). The optimal temperature for the crude xylanases was close to 60 °C, the highest xylanase activity was achieved at pH ranged from 3 to 6, and the crude xylanases also showed a very broad region of pH (3-11) stability. The maximal xylanase activity of 21.45 U · ml(-1) was observed in the fourth day of cultivation at 50 °C and 150 rpm with 2 % xylan as the sole carbon source. Zymogram analysis indicated that there were more than seven secreted proteins with xylanase activity. In the crude enzyme, two major endoxylanases, five cellulases and several associated enzymes were identified to be involved in the hydrolysis of polysaccharides. Of the total 13 xylanase genes in the Z5 genome, 11 were observed using q-PCR to be induced by xylan, one of which, An endo-1,4-β-xylanase with a low secretion level, was also expressed and characterized. The final hydrolysis products of xylan by crude enzyme mainly consisted of xylobiose. This study provides a comprehensive understanding of the depolymerization of xylan by Z5 and will help to design enzymatic strategies for plant biomass utilization.

Impact of Extraction Parameters on the Recovery of Lipolytic Activity from Fermented Babassu Cake

PubMed Central

Silva, Jaqueline N.; Godoy, Mateus G.; Gutarra, Melissa L. E.; Freire, Denise M. G.

2014-01-01

Enzyme extraction from solid matrix is as important step in solid-state fermentation to obtain soluble enzymes for further immobilization and application in biocatalysis. A method for the recovery of a pool of lipases from Penicillium simplicissimum produced by solid-state fermentation was developed. For lipase recovery different extraction solution was used and phosphate buffer containing Tween 80 and NaCl showed the best results, yielding lipase activity of 85.7 U/g and 65.7 U/g, respectively. The parameters with great impacts on enzyme extraction detected by the Plackett-Burman analysis were studied by Central Composite Rotatable experimental designs where a quadratic model was built showing maximum predicted lipase activity (160 U/g) at 25°C, Tween 80 0.5% (w/v), pH 8.0 and extraction solution 7 mL/g, maintaining constant buffer molarity of 0.1 M and 200 rpm. After the optimization process a 2.5 fold increase in lipase activity in the crude extract was obtained, comparing the intial value (64 U/g) with the experimental design (160 U/g), thus improving the overall productivity of the process. PMID:25090644

Enzymes extracted from apple peels have activity in reducing higher alcohols in Chinese liquors.

PubMed

Han, Qi'an; Shi, Junling; Zhu, Jing; Lv, Hongliang; Du, Shuangkui

2014-10-01

As the unavoidable byproducts of alcoholic fermentation, higher alcohols are unhealthy compounds widespread in alcoholic drinks. To investigate the activity of apple crude enzymes toward higher alcohols in liquors, five kinds of apple peels, namely, Fuji, Gala, Golden Delicious, Red Star, and Jonagold, were chosen to prepare enzymes, and three kinds of Chinese liquors, namely, Xifeng (containing 45% ethanol), Taibai (containing 50% ethanol), and Erguotou (containing 56% ethanol), were tested. Enzymes were prepared in the forms of liquid solution, powder, and immobilized enzymes using sodium alginate (SA) and chitosan. The treatment was carried out at 37 °C for 1 h. The relative amounts of different alcohols (including ethanol, 1-propanol, isobutanol, 1-butanol, isoamylol, and 1-hexanol) were measured using gas chromatography (GC). Conditions for preparing SA-immobilized Fuji enzymes (SA-IEP) were optimized, and the obtained SA-IEP (containing 0.3 g of enzyme) was continuously used to treat Xifeng liquor eight times, 20 mL per time. Significant degradation rates (DRs) of higher alcohols were observed at different degrees, and it also showed enzyme specificity according to the apple varieties and enzyme preparations. After five repeated treatments, the DRs of the optimized Fuji SA-IEP remained 70% for 1-hexanol and >15% for other higher alcohols.

Preparation of minor ginsenosides C-Mc, C-Y, F2, and C-K from American ginseng PPD-ginsenoside using special ginsenosidase type-I from Aspergillus niger g.848.

PubMed

Liu, Chun-Ying; Zhou, Rui-Xin; Sun, Chang-Kai; Jin, Ying-Hua; Yu, Hong-Shan; Zhang, Tian-Yang; Xu, Long-Quan; Jin, Feng-Xie

2015-07-01

Minor ginsenosides, those having low content in ginseng, have higher pharmacological activities. To obtain minor ginsenosides, the biotransformation of American ginseng protopanaxadiol (PPD)-ginsenoside was studied using special ginsenosidase type-I from Aspergillus niger g.848. DEAE (diethylaminoethyl)-cellulose and polyacrylamide gel electrophoresis were used in enzyme purification, thin-layer chromatography and high performance liquid chromatography (HPLC) were used in enzyme hydrolysis and kinetics; crude enzyme was used in minor ginsenoside preparation from PPD-ginsenoside; the products were separated with silica-gel-column, and recognized by HPLC and NMR (Nuclear Magnetic Resonance). The enzyme molecular weight was 75 kDa; the enzyme firstly hydrolyzed the C-20 position 20-O-β-D-Glc of ginsenoside Rb1, then the C-3 position 3-O-β-D-Glc with the pathway Rb1→Rd→F2→C-K. However, the enzyme firstly hydrolyzed C-3 position 3-O-β-D-Glc of ginsenoside Rb2 and Rc, finally hydrolyzed 20-O-L-Ara with the pathway Rb2→C-O→C-Y→C-K, and Rc→C-Mc1→C-Mc→C-K. According to enzyme kinetics, K m and V max of Michaelis-Menten equation, the enzyme reaction velocities on ginsenosides were Rb1 > Rb2 > Rc > Rd. However, the pure enzyme yield was only 3.1%, so crude enzyme was used for minor ginsenoside preparation. When the crude enzyme was reacted in 3% American ginseng PPD-ginsenoside (containing Rb1, Rb2, Rc, and Rd) at 45°C and pH 5.0 for 18 h, the main products were minor ginsenosides C-Mc, C-Y, F2, and C-K; average molar yields were 43.7% for C-Mc from Rc, 42.4% for C-Y from Rb2, and 69.5% for F2 and C-K from Rb1 and Rd. Four monomer minor ginsenosides were successfully produced (at low-cost) from the PPD-ginsenosides using crude enzyme.

Biological indicators for steam sterilization: characterization of a rapid biological indicator utilizing Bacillus stearothermophilus spore-associated alpha-glucosidase enzyme.

PubMed

Albert, H; Davies, D J; Woodson, L P; Soper, C J

1998-11-01

The alpha-glucosidase enzyme was isolated from vegetative cells and spores of Bacillus stearothermophilus, ATCC 7953. Spore-associated enzyme had a molecular weight of approximately 92,700, a temperature optimum of 60 degrees C, and a pH optimum of 7.0-7.5. The enzyme in crude aqueous spore extract was stable for 30 min up to a temperature of 65 degrees C, above which the enzyme was rapidly denatured. The optimal pH for stability of the enzyme was approximately 7.2. The alpha-glucosidase in crude vegetative cell extract had similar characteristics to the spore-associated enzyme but its molecular weight was 86,700. The vegetative cell and spore-associated enzymes were cross-reactive. The enzymes are postulated to derive from a single gene product, which undergoes modification to produce the spore-associated form. The location of alpha-glucosidase in the spore coats (outside the spore protoplast) is consistent with the location of most enzymes involved in activation, germination and outgrowth.

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.

Characterization of co-products from producing ethanol by sequential extraction processing of corn

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

Hojilla-Evangelista, M.P.; Johnson, L.A.; Pometto, A.L. III

1996-12-31

Sequential Extraction Processing (SEP) is a new process for ethanol production that has potential to produce more valuable co-products than alternative processes. Previous work determined the yields of oil and protein and evaluated their chemical and functional properties. The properties of the crude fiber and spent solids, however, have yet to be studied. This research was conducted to evaluate the potential of SEP corn fiber to increase ethanol conversion and as replacement for gum arabic, and evaluate the potential of SEP starch and fiber to be fermented to ethanol. SEP hemicellulose from crude fiber was readily dispersible in water andmore » its solution (5%) gave low viscosity despite having high solids content. These properties indicated potential utilization as stabilizers, thickeners, and adhesive for coatings and batters in food and industrial products. Enzyme hydrolysis studies and batch fermentation of SEP starch/fiber indicated that SEP crude fiber was more readily accessible to the action of cellulases. More ethanol (about 10%) was produced from the fermentation of SEP starch/fiber than from undegermed or degermed soft dent corn, particularly when the hemicellulose fraction was absent from the SEP fiber.« less

Partial biochemical characterization of crude extract extracellular chitinase enzyme from Bacillus subtilis B 298

NASA Astrophysics Data System (ADS)

Lestari, P.; Prihatiningsih, N.; Djatmiko, H. A.

2017-02-01

Extraction and characterization of extracellular chitinase from Bacillus subtilis B 298 have been done. Growth curve determination of B. subtilis B 298, production curve determination of crude extract chitinase from B. subtilis B 298, and partial biochemical characterization of crude extract chitinase have been achieved in this study. Optimum growth of B. subtilis B 298 was achieved at logarithmic phase within 9 hours incubation time, so it was used as inoculum for enzyme production. According to production curve of the enzyme, it was known that incubation time which gave the highest chitinase activity of 15 hours with activity of 6.937 U/mL respectively. Effect of various temperatures on chitinase activity showed that optimum activity was achieved at 40°C with an activity of 5.764 U/mL respectively. Meanwhile, the optimum pH for chitinase activity was achieved at pH of 5.0 with an activity of 6.813 U/mL respectively. This enzyme was then classified as metalloenzyme due to the decline of the activity by EDTA addition. All divalent cations tested acted as inhibitors.

The Kinetics and Inhibition of the Enzyme Methemoglobin Reductase

ERIC Educational Resources Information Center

Splittgerber, A. G.; And Others

1975-01-01

Describes an undergraduate biochemistry experiment which involves the preparation and kinetics of an oxidation-reduction enzyme system, methemoglobin reductase. A crude enzyme extract is prepared and assayed spectrophotometrically. The enzyme system obeys Michaelis-Menton kinetics with respect to both substrate and the NADH cofactor. (MLH)

Extracellular esterases of phylloplane yeast Pseudozyma antarctica induce defect on cuticle layer structure and water-holding ability of plant leaves.

PubMed

Ueda, Hirokazu; Mitsuhara, Ichiro; Tabata, Jun; Kugimiya, Soichi; Watanabe, Takashi; Suzuki, Ken; Yoshida, Shigenobu; Kitamoto, Hiroko

2015-08-01

Aerial plant surface (phylloplane) is a primary key habitat for many microorganisms but is generally recognized as limited in nutrient resources. Pseudozyma antarctica, a nonpathogenic yeast, is commonly isolated from plant surfaces and characterized as an esterase producer with fatty acid assimilation ability. In order to elucidate the biological functions of these esterases, culture filtrate with high esterase activity (crude enzyme) of P. antarctica was applied onto leaves of tomato and Arabidopsis. These leaves showed a wilty phenotype, which is typically associated with water deficiency. Furthermore, we confirmed that crude enzyme-treated detached leaves clearly lost their water-holding ability. In treated leaves of both plants, genes associated to abscisic acid (ABA; a plant stress hormone responding osmotic stress) were activated and accumulation of ABA was confirmed in tomato plants. Microscopic observation of treated leaf surfaces revealed that cuticle layer covering the aerial epidermis of leaves became thinner. A gas chromatography-mass spectrometry (GC-MS) analysis exhibited that fatty acids with 16 and 18 carbon chains were released in larger amounts from treated leaf surfaces, indicating that the crude enzyme has ability to degrade lipid components of cuticle layer. Among the three esterases detected in the crude enzyme, lipase A, lipase B, and P. antarctica esterase (PaE), an in vitro enzyme assay using para-nitrophenyl palmitate as substrate demonstrated that PaE was the most responsible for the degradation. These results suggest that PaE has a potential role in the extraction of fatty acids from plant surfaces, making them available for the growth of phylloplane yeasts.

Multiple pharmacological targets, cytotoxicity, and phytochemical profile of Aphloia theiformis (Vahl.) Benn.

PubMed

Picot, Marie Carene Nancy; Bender, Onur; Atalay, Arzu; Zengin, Gokhan; Loffredo, Loïc; Hadji-Minaglou, Francis; Mahomoodally, Mohamad Fawzi

2017-05-01

Aphloia theiformis (Vahl.) Benn. (AT) is traditionally used in Sub-Saharan African countries including Mauritius as a biomedicine for the management of several diseases. However, there is a dearth of experimental studies to validate these claims. We endeavoured to evaluate the inhibitory effects of crude aqueous extract as traditionally used together with the crude methanol extracts of AT leaves on urease, angiotensin (I) converting enzyme (ACE), acetylcholinesterase (AChE), cholesterol esterase (CEase), glycogen phosphorylase a (GPa), and glycation in vitro. The crude extract showing potent activity against the studied enzymes was further partitioned using different solvents of increasing polarity. The enzyme inhibitory and antiglycation activities of each fraction was assessed. Kinetic of inhibition of the active crude extract/fractions on the aforementioned enzymes was consequently determined using Lineweaver-Burk plots. An ultra-high performance liquid chromatography (UHPLC-UV/MS) system was used to establish the phytochemical profile of AT. The real time cell analysis system (iCELLigence™) was used to monitor any cellular cytotoxicity of AT. Crude methanolextract (CME) was a potent inhibitor of the studied enzymes, with IC 50 ranging from 696.22 to 19.73μg/mL. CME (82.5%) significantly (p<0.05) inhibited glycation and was comparable to aminoguanidine (81.5%). Ethyl acetate and n-butanol fractions of CME showed non-competitive, competitive, and uncompetitive mode of inhibition against ACE, CEase, and AChE respectively. Mangiferin, a xanthone glucoside was present in CME, ethyl acetate, and n-butanol fractions. Active extract/fractions were found to be non-cytotoxic (IC 50 >20μg/mL) according to the U.S National Cancer Institute plant screening program. This study has established baseline data that tend to justify the traditional use of AT and open new avenues for future biomedicine development. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Studies on the "Aerobic" Acetyl-Coenzyme A Synthetase of Saccharomyces Cerevisiae: Purification, Crystallization, and Physical Properties of the Enzyme

NASA Technical Reports Server (NTRS)

Satyanarayana, T.; Klein, Harold P.

1976-01-01

A procedure for the purification of a stable acetyl-coenzyme A synthetase (ACS) from aerobic cells of Saccharomyces cerevisiae is presented. The steps include differential centrifugation, solubilization of the bound enzyme from the crude mitochondrial fraction, ammonium sulfate fractionation, crystallization to constant specific activity from ammonium sulfate solutions followed by Bio-Gel A-1.5 m column chromatography. The resulting enzyme preparation is homogeneous as judged by chromatography on Bio-Gel columns, QAE-Sephadex A-50 anion exchange columns, analytical ultracentrifugal studies, and polyacrylamide gel electrophoresis. Sedimentation velocity runs revealed a single symmetric peak with an s(sub (20,w)) value of 10.6. The molecular weight of the native enzyme, as determined by gel filtration and analytical ultracentrifugation, is 250,000 +/- 500. In polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, the molecular weight of the single polypeptide chain is 83,000 +/- 500. The purified enzyme is inhibited by palmityl-coenzyme A with a Hill interaction coefficient, n, of 2.88. These studies indicate that the ACS of aerobic Saccharomyces cerevisiae is composed of three subunits of identical or nearly identical size.

Pectinase production by Aspergillus niger using banana (Musa balbisiana) peel as substrate and its effect on clarification of banana juice.

PubMed

Barman, Sumi; Sit, Nandan; Badwaik, Laxmikant S; Deka, Sankar C

2015-06-01

Optimization of substrate concentration, time of incubation and temperature for crude pectinase production from A. niger was carried out using Bhimkol banana (Musa balbisiana) peel as substrate. The crude pectinase produced was partially purified using ethanol and effectiveness of crude and partially purified pectinase was studied for banana juice clarification. The optimum substrate concentration, incubation time and temperature of incubation were 8.07 %, 65.82 h and 32.37 °C respectively, and the polygalacturonase (PG) activity achieved was 6.6 U/ml for crude pectinase. The partially purified enzyme showed more than 3 times of polygalacturonase activity as compared to the crude enzyme. The SDS-PAGE profile showed that the molecular weight of proteins present in the different pectinases varied from 34 to 42 kDa. The study further revealed that highest clarification was achieved when raw banana juice was incubated for 60 min with 2 % concentration of partially purified pectinase and the absorbance obtained was 0.10.

Hydrolysis of various thai agricultural biomasses using the crude enzyme from Aspergillus aculeatus iizuka FR60 isolated from soil

PubMed Central

Boonmee, Atcha

2012-01-01

In this study, forty-two fungi from soil were isolated and tested for their carboxymethyl cellulase (CMCase) and xylanase activities. From all isolates, the fungal isolate FR60, which was identified as Aspergillus aculeatus Iizuka, showed high activities in both CMCase and xylanase with 517 mU/mg protein and 550 mU/mg protein, respectively. The crude enzyme from A. aculeatus Iizuka FR60 could hydrolyze several agricultural residues such as corncob, and sweet sorghum leaf and stalk at comparable rates with respect to the tested commercial enzymes and with a maximum rate in rice hull hydrolysis (29 μg sugar g-1 dry weight substrate mg-1 enzyme hr-1). The highest amount of glucose was obtained from corncob by using the crude enzyme from A. aculeatus Iizuka FR60 (10.1 g/100 g dry substrate). From overall enzymatic treatment results, the lowest sugar yield was from rice hulls treatment (1.6 g/100 g dry weight) and the highest amount of reducing sugar was obtained from rice straw treatment (15.3 g/100 g dry weight). Among tested agricultural wastes, rice hull could not be effectively hydrolyzed by enzymes, whereas sugarcane leaf and stalk, and peanut shell could be effectively hydrolyzed (30-31% total sugar comparing with total sugar yield from acid treatment). PMID:24031852

Production of L-lactic Acid from Biomass Wastes Using Scallop Crude Enzymes and Novel Lactic Acid Bacterium

NASA Astrophysics Data System (ADS)

Yanagisawa, Mitsunori; Nakamura, Kanami; Nakasaki, Kiyohiko

In the present study, biomass waste raw materials including paper mill sludge, bamboo, sea lettuce, and shochu residue (from a distiller) and crude enzymes derived from inedible and discarded scallop parts were used to produce L-lactic acid for the raw material of biodegradable plastic poly-lactic acid. The activities of cellulase and amylase in the crude enzymes were 22 and 170units/L, respectively, and L-lactic acid was produced from every of the above mentioned biomass wastes, by the method of liquid-state simultaneous saccharification and fermentation (SSF) . The L-lactic acid concentrations produced from sea lettuce and shochu residue, which contain high concentration of starch were 3.6 and 9.3g/L, respectively, and corresponded to greater than 25% of the conversion of glucans contained in these biomass wastes. Furthermore, using the solid state SSF method, concentrations as high as 13g/L of L-lactic acid were obtained from sea lettuce and 26g/L were obtained from shochu residue.

Production and Optimization of Physicochemical Parameters of Cellulase Using Untreated Orange Waste by Newly Isolated Emericella variecolor NS3.

PubMed

Srivastava, Neha; Srivastava, Manish; Manikanta, Ambepu; Singh, Pardeep; Ramteke, P W; Mishra, P K; Malhotra, Bansi D

2017-10-01

Cellulase enzymes have versatile industrial applications. This study was directed towards the isolation, production, and characterization of cellulase enzyme system. Among the five isolated fungal cultures, Emericella variecolor NS3 showed maximum cellulase production using untreated orange peel waste as substrate using solid-state fermentation (SSF). Maximum enzyme production of 31 IU/gds (per gram of dry substrate) was noticed at 6.0 g concentration of orange peel. Further, 50 °C was recorded as the optimum temperature for cellulase activity and the thermal stability for 240 min was observed at this temperature. In addition, the crude enzyme was stable at pH 5.0 and held its complete relative activity in presence of Mn 2+ and Fe 3+ . This study explored the production of crude enzyme system using biological waste with future potential for research and industrial applications.

Foam Properties and Detergent Abilities of the Saponins from Camellia oleifera

PubMed Central

Chen, Yu-Fen; Yang, Chao-Hsun; Chang, Ming-Shiang; Ciou, Yong-Ping; Huang, Yu-Chun

2010-01-01

The defatted seed meal of Camellia oleifera has been used as a natural detergent and its extract is commercially utilized as a foam-stabilizing and emulsifying agent. The goal of this study was to investigate the foam properties and detergent ability of the saponins from the defatted seed meal of C. oleifera. The crude saponin content in the defatted seed meal of C. oleifera was 8.34 and the total saponins content in the crude saponins extract was 39.5% (w/w). The foaming power of the 0.5 crude saponins extract solution from defatted seed meal of C. oleifera was 37.1 of 0.5 SLS solution and 51.3% to that of 0.5% Tween 80 solution. The R5 value of 86.0% represents good foam stability of the crude saponins extracted from the defatted seed meal of the plant. With the reduction of water surface tension from 72 mN/m to 50.0 mN/m, the 0.5% crude saponins extract solution has wetting ability. The sebum-removal experiment indicated that the crude saponins extract has moderate detergency. The detergent abilities of the saponins from C. oleifera and Sapindus mukorossi were also compared. PMID:21151446

Reduction of benzene and naphthalene mass transfer from crude oils by aging-induced interfacial films.

PubMed

Ghoshal, Subhasis; Pasion, Catherine; Alshafie, Mohammed

2004-04-01

Semi-rigid films or skins form at the interface of crude oil and water as a result of the accumulation of asphaltene and resin fractions when the water-immiscible crude oil is contacted with water for a period of time or "aged". The time varying patterns of area-independent mass transfer coefficients of two compounds, benzene and naphthalene, for dissolution from crude oil and gasoline were determined. Aqueous concentrations of the compounds were measured in the eluent from flow-through reactors, where a nondispersed oil phase and constant oil-water interfacial area were maintained. For Brent Blend crude oil and for gasoline amended with asphaltenes and resins, a rapid decrease in both benzene and naphthalene mass transfer coefficients over the first few days of aging was observed. The mass transfer coefficients of the two target solutes were reduced by up to 80% over 35 d although the equilibrium partition coefficients were unchanged. Aging of gasoline, which has negligible amounts of asphaltene and resin, did not result in a change in the solute mass transfer coefficients. The study demonstrates that formation of crude oil-water interfacial films comprised of asphaltenes and resins contribute to time-dependent decreases in rates of release of environmentally relevant solutes from crude oils and may contribute to the persistence of such solutes at crude oil-contaminated sites. It is estimated that the interfacial film has an extremely low film mass transfer coefficient in the range of 10(-6) cm/min.

Production of Fungal Amylases Using Cheap, Readily Available Agriresidues, for Potential Application in Textile Industry

PubMed Central

Singh, Sanamdeep; Bali, Vrinda; Mangla, Jyoti

2014-01-01

The study aimed at isolation and screening of fungal amylase producer, optimization of solid state fermentation conditions for maximum amylase production by the best amylase producer, and characterization of the crude amylases, so produced. Aspergillus fumigatus NTCC1222 showed the highest amylase activity (164.1 U/mL) in secondary screening under SSF conditions and was selected for further studies. The test strain showed maximum amylase production (341.7 U/mL) and supernatant protein concentration (9.7 mg/mL) for incubation period (6 days), temperature (35°C), initial pH (6.0), nutrient salt solution as moistening agent, and beef extract as nitrogen source. Pomegranate peel produced maximum amylase activity, but wheat bran (only slightly lesser amylase activity as compared to that of pomegranate peel) was chosen for further studies, keeping in mind the seasonal availability of pomegranate peel. TLC confirmed the amylase produced to be α-type and 60 kDa was the molecular weight of the partially purified amylase. The enzyme showed maximum enzyme activity at pH 6.0, temperature of 55°C, and incubation time of 60 minutes. UV (616.0 U/mL) and chemical (814.2 U/mL) mutation enhanced amylase activity as compared to wild test strain. The study indicates that Aspergillus fumigatus NTCC1222 can be an important source of amylase and the crude enzyme, hence obtained, can be cost effectively applied in multiple sections of textile wet processing. PMID:24527439

Cathepsin D immobilized capillary reactors for on-flow screening assays.

PubMed

Cornelio, Vivian Estevam; de Moraes, Marcela Cristina; Domingues, Vanessa de Cassia; Fernandes, João Batista; da Silva, Maria Fátima das Gracas Fernandes; Cass, Quezia Bezerra; Vieira, Paulo Cezar

2018-03-20

The treatment of diseases using enzymes as targets has called for the development of new and reliable methods for screening. The protease cathepsin D is one such target involved in several diseases such as tumors, degenerative processes, and vital processes of parasites causing schistosomiasis. Herein, we describe the preparation of a fused silica capillary, cathepsin D (CatD)-immobilized enzyme reactor (IMER) using in a multidimensional High Performance Liquid Chromatography-based method (2D-HPLC) and zonal affinity chromatography as an alternative in the search for new ligands. The activity and kinetic parameters of CatD-IMER were evaluated by monitoring the product MOCAc-Gly-Lys-Pro-Ile-Leu-Phe (P-MOCAc) (K M  = 81.9 ± 7.49 μmol/L) generated by cleavage of the fluorogenic substrate MOCAc-Gly-Lys-Pro-Ile-Leu-Phe-Phe-Arg-Leu-Lys(DNP)-d-Arg-NH2 (S-MOCAc). Stability studies have indicated that CatD-IMER retained 20% of activity after 5 months, a relevant result, because proteases are susceptible to autoproteolysis in solution assays with free enzyme. In the search for inhibitors, 12 crude natural product extracts were analyzed using CatD-IMER as the target, resulting in the isolation of different classes of natural products. In addition, 26 compounds obtained from different species of plants were also screened, demonstrating the efficiency and reproducibility of the herein reported assay even in the case of complex matrices such as plant crude extracts. Copyright © 2018 Elsevier B.V. All rights reserved.

Solubility of crude oil in methane as a function of pressure and temperature

USGS Publications Warehouse

Price, L.C.; Wenger, L.M.; Ging, T.; Blount, C.W.

1983-01-01

The solubility of a 44?? API (0.806 sp. gr.) whole crude oil has been measured in methane with water present at temperatures of 50 to 250??C and pressures of 740 to 14,852 psi, as have the solubilities of two high molecular weight petroleum distillation fractions at temperatures of 50 to 250??C and pressures of 4482 to 25,266 psi. Both increases in pressure and temperature increase the solubility of crude oil and petroleum distillation fractions in methane, the effect of pressure being greater than that of temperature. Unexpectedly high solubility levels (0.5-1.5 grams of oil per liter of methane-at laboratory temperature and pressure) were measured at moderate conditions (50-200??C and 5076-14504 psi). Similar results were found for the petroleum distillation fractions, one of which was the highest molecular weight material of petroleum (material boiling above 266??C at 6 microns pressure). Unexpectedly mild conditions (100??C and 15,200 psi; 200??C and 7513 psi) resulted in cosolubility of crude oil and methane. Under these conditions, samples of the gas-rich phase gave solubility values of 4 to 5 g/l, or greater. Qualitative analyses of the crude-oil solute samples showed that at low pressure and temperature equilibration conditions, the solute condensate would be enriched in C5-C15 range hydrocarbons and in saturated hydrocarbons in the C15+ fraction. With increases in temperature and especially pressure, these tendencies were reversed, and the solute condensate became identical to the starting crude oil. The data of this study, compared to that of previous studies, shows that methane, with water present, has a much greater carrying capacity for crude oil than in dry systems. The presence of water also drastically lowers the temperature and pressure conditions required for cosolubility. The data of this and/or previous studies demonstrate that the addition of carbon dioxide, ethane, propane, or butane to methane also has a strong positive effect on crude oil solubility, as does the presence of fine grained rocks. The n-paraffin distributions (as well as the overall composition) of the solute condensates are controlled by the temperature and pressure of solution and exsolution, as well as by the composition of the original starting material. It appears quite possible that primary migration by gaseous solution could 'strip' a source rock of crude-oil like components leaving behind a bitumen totally unlike the migrated crude oil. The data of this study demonstrate previous criticisms of primary petroleum migration by gas solution are invalid; that primary migration by gaseous solution cannot occur because methane cannot dissolve sufficient volumes of crude oil or cannot dissolve the highest molecular weight components of petroleum (tars and asphaltenes). ?? 1983.

Formate Dehydrogenase from Clostridium acidiurici

PubMed Central

Kearny, James J.; Sagers, Richard D.

1972-01-01

Partial purification of formate dehydrogenase from Clostridium acidiurici has been accomplished, and some properties of the enzyme have been determined. The molecular weight of the protein is at least 200,000 daltons. The enzyme showed marked instability to freezing and thawing and was inhibited strongly by oxygen and by light. Such inhibition was not reversed by incubation in the presence of thiol compounds. Cyanide inhibited the enzyme 90% at 0.1 mm concentrations, but ethylenediaminetetraacetate produced only slight inhibition at concentrations as high as 50 mm. The purified enzyme showed no ferredoxin activity in the Clostridium pasteurianum clastic system during pyruvate oxidation. Crude preparations of the enzyme could be coupled through ferredoxin to the reduction of nicotinamide adenine dinucleotide during formate oxidation, but the purified enzyme could not catalyze the reduction of pyridine nucleotides by formate in the presence of ferredoxin. Formate oxidation with the purified enzyme was readily coupled to benzyl viologen reduction, in which case ferredoxin was not required. An exchange between formate and bicarbonate was catalyzed by both crude and purified preparations of the enzyme, but the net synthesis of formate from CO2 was not accomplished. PMID:4333376

On-site production of crude glucoamylase for kitchen waste hydrolysis.

PubMed

Wang, Xiao Qiang; Wang, Qun Hui; Liu, Ying Ying; Ma, Hong Zhi

2010-06-01

Kitchen waste from dining rooms accounts for a considerable proportion of municipal solid garbage, and economical recycle ways are needed to be developed. This study investigated glucoamylase production from kitchen waste and the feasibility of kitchen waste hydrolysis by the crude enzymes produced. The key problems of high water content and poor porosity in kitchen waste for glucoamylase production under solid-state fermentation could be solved readily by the addition of corn stover or paddy husk. As a support medium, corn stover was better than paddy husk. Smashed kitchen waste (sKW) mixed with corn stover in the ratio of 3.75 : 1 (dry basis) produced 1838 U g(-1) of glucoamylase by Aspergillus niger UV-60 within 96 h. The enzyme productivity from kitchen waste was over two-fold higher than that from wheat bran with additional nutrients. Without any recovery treatment, the produced glucoamylase could be used directly to hydrolyse sKW slurry. The optimum enzyme dose 8% (crude enzyme/kichen waste, w/w) was not too big, and was sufficient to hydrolyse 10% (dry basis) sKW slurry to produce a maximum amount of reducing sugar of 55.4 g L(-1).

Enzymatic decolourisation of Methyl Orange and Bismarck Brown using crude peroxidase from Armoracia rusticana

NASA Astrophysics Data System (ADS)

Ambatkar, Mugdha; Mukundan, Usha

2015-12-01

The decolourisation of Methyl Orange (MO) and Bismarck Brown (BB) by crude peroxidase from Armoracia rusticana (Horseradish) was studied by varying different reaction parameters. The pH of the reaction mixture, initial dye concentration, amount of enzyme and hydrogen peroxide concentration were optimised for ambient temperatures (30 ± 2 °C). The optimum pH for decolourisation was 4.0 (72.95 %) and 3.0 (79.24 %) for MO and BB, respectively. Also it was found that the Chemical Oxygen Demand of the enzyme-treated sample was significantly lower than that of the untreated controls for both dyes. The addition of a complex iron salt like Ferric EDTA was found to enhance the decolourisation of both dyes at pH 6.0, showing an increase of 8.69 % and 14.17 % in the decolourisation of MO and of BB, respectively. The present study explores the potential of crude peroxidase from horseradish to decolourise representative monoazo and diazo dyes, MO and BB, respectively. An attempt has been made to utilise a crude enzyme with appreciable activity obtained after minimal processing for the decolourisation of the aforesaid dyes. The findings of this study would find application in the enzymatic treatment of wastewater containing azo dyes.

Response surface methodology optimization of partitioning of xylanase form Aspergillus Niger by metal affinity polymer-salt aqueous two-phase systems.

PubMed

Fakhari, Mohamad Ali; Rahimpour, Farshad; Taran, Mojtaba

2017-09-15

Aqueous two phase affinity partitioning system using metal ligands was applied for partitioning and purification of xylanase produced by Aspergillus Niger. To minimization the number of experiments for the design parameters and develop predictive models for optimization of the purification process, response surface methodology (RSM) with a face-centered central composite design (CCF) has been used. Polyethylene glycol (PEG) 6000 was activated using epichlorohydrin, covalently linked to iminodiacetic acid (IDA), and the specific metal ligand Cu was attached to the polyethylene glycol-iminodiacetic acid (PEG-IDA). The influence of some experimental variables such as PEG (10-18%w/w), sodium sulfate (8-12%), PEG-IDA-Cu 2+ concentration (0-50% w/w of total PEG), pH of system (4-8) and crude enzyme loading (6-18%w/w) on xylanase and total protein partitioning coefficient, enzyme yield and enzyme specific activity were systematically evaluated. Two optimal point with high enzyme partitioning factor 10.97 and yield 79.95 (including 10% PEG, 12% Na 2 SO 4 , 50% ligand, pH 8 and 6% crude enzyme loading) and high specific activity in top phase 42.21 (including 14.73% PEG, 8.02% Na 2 SO 4 , 28.43% ligand, pH 7.7 and 6.08% crude enzyme loading) were attained. The adequacy of the RSM models was verified by a good agreement between experimental and predicted results. Copyright © 2017 Elsevier B.V. All rights reserved.

Optimized preparation and characterization of CLEA-lipase from cocoa pod husk.

PubMed

Khanahmadi, Soofia; Yusof, Faridah; Amid, Azura; Mahmod, Safa Senan; Mahat, Mohd Khairizal

2015-05-20

Cross-linked enzyme aggregate (CLEA) is easily prepared from crude enzyme and has many advantages to the environment and it is considered as an economic method in the context of industrial biocatalysis compared to free enzyme. In this work, a highly active and stable CLEA-lipase from cocoa pod husk (CPH) which is a by-product after removal of cocoa beans, were assayed for their hydrolytic activity and characterized under the optimum condition successfully. Face centered central composite design (FCCCD) under response surface methodology (RSM) was used to get the optimal conditions of the three significant factors (concentration of ammonium sulfate, concentration of glutaraldehyde and concentration of additive) to achieve higher enzyme activity of CLEA. From 20 runs, the highest activity recorded was around 9.407U (83% recovered activity) under the condition of using 20% saturated ammonium sulfate, 60mM glutaraldehyde as cross-linker and 0.17mM bovine serum albumin as feeder. Moreover, the optimal reaction temperature and pH value in enzymatic reaction for both crude enzyme and immobilized were found to be 45°C at pH 8 and 60°C at pH 8.2, respectively. A systematic study of the stability of CLEA and crude enzyme was taken with regards to temperature (25-60°C) and pH (5-10) value and in both factors, CLEA-lipase showed more stability than free lipase. The Km value of CLEA was higher compared to free enzyme (0.55mM vs. 0.08mM). The CLEA retained more than 60% of the initial activity after six cycles of reuse compared to free enzyme. The high stability and recyclability of CLEA-lipase from CPH make it efficient for different industrial applications. Copyright © 2014 Elsevier B.V. All rights reserved.

Glutamine synthetase 2 is not essential for biosynthesis of compatible solutes in Halobacillus halophilus

PubMed Central

Shiyan, Anna; Thompson, Melanie; Köcher, Saskia; Tausendschön, Michaela; Santos, Helena; Hänelt, Inga; Müller, Volker

2014-01-01

Halobacillus halophilus, a moderately halophilic bacterium isolated from salt marshes, produces various compatible solutes to cope with osmotic stress. Glutamate and glutamine are dominant compatible solutes at mild salinities. Glutamine synthetase activity in cell suspensions of Halobacillus halophilus wild type was shown to be salt dependent and chloride modulated. A possible candidate to catalyze glutamine synthesis is glutamine synthetase A2, whose transcription is stimulated by chloride. To address the role of GlnA2 in the biosynthesis of the osmolytes glutamate and glutamine, a deletion mutant (ΔglnA2) was generated and characterized in detail. We compared the pool of compatible solutes and performed transcriptional analyses of the principal genes controlling the solute production in the wild type strain and the deletion mutant. These measurements did not confirm the hypothesized role of GlnA2 in the osmolyte production. Most likely the presence of another, yet to be identified enzyme has the main contribution in the measured activity in crude extracts and probably determines the total chloride-modulated profile. The role of GlnA2 remains to be elucidated. PMID:24782854

Production of plant cell wall degrading enzymes by monoculture and co-culture of Aspergillus niger and Aspergillus terreus under SSF of banana peels.

PubMed

Rehman, Shazia; Aslam, Hina; Ahmad, Aqeel; Khan, Shakeel Ahmed; Sohail, Muhammad

2014-01-01

Filamentous fungi are considered to be the most important group of microorganisms for the production of plant cell wall degrading enzymes (CWDE), in solid state fermentations. In this study, two fungal strains Aspergillus niger MS23 and Aspergillus terreus MS105 were screened for plant CWDE such as amylase, pectinase, xylanase and cellulases (β-glucosidase, endoglucanase and filterpaperase) using a novel substrate, Banana Peels (BP) for SSF process. This is the first study, to the best of our knowledge, to use BP as SSF substrate for plant CWDE production by co-culture of fungal strains. The titers of pectinase were significantly improved in co-culture compared to mono-culture. Furthermore, the enzyme preparations obtained from monoculture and co-culture were used to study the hydrolysis of BP along with some crude and purified substrates. It was observed that the enzymatic hydrolysis of different crude and purified substrates accomplished after 26 h of incubation, where pectin was maximally hydrolyzed by the enzyme preparations of mono and co-culture. Along with purified substrates, crude materials were also proved to be efficiently degraded by the cocktail of the CWDE. These results demonstrated that banana peels may be a potential substrate in solid-state fermentation for the production of plant cell wall degrading enzymes to be used for improving various biotechnological and industrial processes.

Production of plant cell wall degrading enzymes by monoculture and co-culture of Aspergillus niger and Aspergillus terreus under SSF of banana peels

PubMed Central

Rehman, Shazia; Aslam, Hina; Ahmad, Aqeel; Khan, Shakeel Ahmed; Sohail, Muhammad

2014-01-01

Filamentous fungi are considered to be the most important group of microorganisms for the production of plant cell wall degrading enzymes (CWDE), in solid state fermentations. In this study, two fungal strains Aspergillus niger MS23 and Aspergillus terreus MS105 were screened for plant CWDE such as amylase, pectinase, xylanase and cellulases (β-glucosidase, endoglucanase and filterpaperase) using a novel substrate, Banana Peels (BP) for SSF process. This is the first study, to the best of our knowledge, to use BP as SSF substrate for plant CWDE production by co-culture of fungal strains. The titers of pectinase were significantly improved in co-culture compared to mono-culture. Furthermore, the enzyme preparations obtained from monoculture and co-culture were used to study the hydrolysis of BP along with some crude and purified substrates. It was observed that the enzymatic hydrolysis of different crude and purified substrates accomplished after 26 h of incubation, where pectin was maximally hydrolyzed by the enzyme preparations of mono and co-culture. Along with purified substrates, crude materials were also proved to be efficiently degraded by the cocktail of the CWDE. These results demonstrated that banana peels may be a potential substrate in solid-state fermentation for the production of plant cell wall degrading enzymes to be used for improving various biotechnological and industrial processes. PMID:25763058

Production and Partial Characterization of α-Amylase Enzyme from Bacillus sp. BCC 01-50 and Potential Applications

PubMed Central

Qureshi, Abdul Sattar; Khushk, Imrana; Ali, Chaudhry Haider; Lashari, Safia; Bhutto, Muhammad Aqeel; Mangrio, Ghulam Sughra; Lu, Changrui

2017-01-01

Amylase is an industrially important enzyme and applied in many industrial processes such as saccharification of starchy materials, food, pharmaceutical, detergent, and textile industries. This research work deals with the optimization of fermentation conditions for α-amylase production from thermophilic bacterial strain Bacillus sp. BCC 01-50 and characterization of crude amylase. The time profile of bacterial growth and amylase production was investigated in synthetic medium and maximum enzyme titer was observed after 60 h. In addition, effects of different carbon sources were tested as a substrate for amylase production and molasses was found to be the best. Various organic and inorganic compounds, potassium nitrate, ammonium chloride, sodium nitrate, urea, yeast extract, tryptone, beef extract, and peptone, were used and beef extract was found to be the best among the nitrogen sources used. Temperature, pH, agitation speed, and size of inoculum were also optimized. Highest enzyme activity was obtained when the strain was cultured in molasses medium for 60 h in shaking incubator (150 rpm) at 50°C and pH 8. Crude amylase showed maximal activity at pH 9 and 65°C. Enzyme remained stable in alkaline pH range 9-10 and 60–70°C. Crude amylase showed great potential for its application in detergent industry and saccharification of starchy materials. PMID:28168200

Production and Partial Characterization of α-Amylase Enzyme from Bacillus sp. BCC 01-50 and Potential Applications.

PubMed

Simair, Altaf Ahmed; Qureshi, Abdul Sattar; Khushk, Imrana; Ali, Chaudhry Haider; Lashari, Safia; Bhutto, Muhammad Aqeel; Mangrio, Ghulam Sughra; Lu, Changrui

2017-01-01

Amylase is an industrially important enzyme and applied in many industrial processes such as saccharification of starchy materials, food, pharmaceutical, detergent, and textile industries. This research work deals with the optimization of fermentation conditions for α -amylase production from thermophilic bacterial strain Bacillus sp. BCC 01-50 and characterization of crude amylase. The time profile of bacterial growth and amylase production was investigated in synthetic medium and maximum enzyme titer was observed after 60 h. In addition, effects of different carbon sources were tested as a substrate for amylase production and molasses was found to be the best. Various organic and inorganic compounds, potassium nitrate, ammonium chloride, sodium nitrate, urea, yeast extract, tryptone, beef extract, and peptone, were used and beef extract was found to be the best among the nitrogen sources used. Temperature, pH, agitation speed, and size of inoculum were also optimized. Highest enzyme activity was obtained when the strain was cultured in molasses medium for 60 h in shaking incubator (150 rpm) at 50°C and pH 8. Crude amylase showed maximal activity at pH 9 and 65°C. Enzyme remained stable in alkaline pH range 9-10 and 60-70°C. Crude amylase showed great potential for its application in detergent industry and saccharification of starchy materials.

Xylanase and feruloyl esterase from actinomycetes cultures could enhance sugarcane bagasse hydrolysis in the production of fermentable sugars.

PubMed

Rahmani, Nanik; Kahar, Prihardi; Lisdiyanti, Puspita; Hermiati, Euis; Lee, Jaemin; Yopi; Prasetya, Bambang; Ogino, Chiaki; Kondo, Akihiko

2018-02-23

The addition of enzymes that are capable of degrading hemicellulose has a potential to reduce the need for commercial enzymes during biomass hydrolysis in the production of fermentable sugars. In this study, a high xylanase producing actinomycete strain (Kitasatospora sp. ID06-480) and the first ethyl ferulate producing actinomycete strain (Nonomuraea sp. ID06-094) were selected from 797 rare actinomycetes, respectively, which were isolated in Indonesia. The addition (30%, v/v) of a crude enzyme supernatant from the selected strains in sugarcane bagasse hydrolysis with low-level loading (1 FPU/g-biomass) of Cellic® CTec2 enhanced both the released amount of glucose and reducing sugars. When the reaction with Ctec2 was combined with crude enzymes containing either xylanase or feruloyl esterase, high conversion yield of glucose from cellulose at 60.5% could be achieved after 72 h-saccharification.

β-galactosidase from Aspergillus lacticoffeatus: A promising biocatalyst for the synthesis of novel prebiotics.

PubMed

Cardoso, Beatriz B; Silvério, Sara C; Abrunhosa, Luís; Teixeira, José A; Rodrigues, Lígia R

2017-09-18

β-galactosidase (EC 3.2.1.23) are interesting enzymes able to catalyze lactose hydrolysis and transfer reactions to produce lactose-based prebiotics with potential application in the pharmaceutical and food industry. In this work, Aspergillus lacticoffeatus is described, for the first time, as an effective β-galactosidase producer. The extracellular enzyme production was evaluated in synthetic and alternative media containing cheese whey and corn steep liquor. Although β-galactosidase production occurred in all media (expect for the one composed solely by cheese whey), the highest enzymatic activity values (460U/mL) were obtained for the synthetic medium. Ochratoxin A production in synthetic medium was also evaluated and 9days of fermentation was identified as a suitable fermentation time to obtain a crude extract enzyme with mycotoxin concentration below the legal comparable value established for wine and grape juices (2ng/mL). The optimal pH and temperature for the crude extract enzyme was found in the range of 3.5-4.5 and 50-60°C, respectively. The β-galactosidase activity was reduced in the presence of Ba 2+ , Fe 2+ , Li + , K + and galactose, while additives (except for ascorbic acid) and detergents exhibited a positive effect on enzymatic activity. This enzyme was able to catalyze the synthesis of prebiotics, namely lactulose (2.5g/L) and a galacto-oligosaccharide (trisaccharide, 6.3g/L), either when whole cells or crude enzyme was used as biocatalyst. The lactulose production using fungal whole cells is herein reported for the first time. Additionally, A. lacticoffeatus was also found to produce an enzyme with fructosyltransferase activity and other prebiotics, namely fructo-oligosaccharide 1-kestose (2.4g/L). Copyright © 2017 Elsevier B.V. All rights reserved.

Novel assay system for acidic Peptide:N-glycanase (aPNGase) activity in crude plant extract.

PubMed

Uemura, Ryota; Ogura, Mikako; Matsumaru, Chihiro; Akiyama, Tsuyoshi; Maeda, Megumi; Kimura, Yoshinobu

2018-04-15

Acidic peptide:N-glycanase (aPNGase) plays a pivotal role in plant glycoprotein turnover. For the construction of aPNGase-knockout or -overexpressing plants, a new method to detect the activity in crude plant extracts is required because endogenous peptidases present in the extract hamper enzyme assays using fluorescence-labeled N-glycopeptides as a substrate. In this study, we developed a new method for measuring aPNGase activity in crude extracts from plant materials.

Purification and characterization of polyphenol oxidase from rape flower.

PubMed

Sun, Han-Ju; Wang, Jing; Tao, Xue-Ming; Shi, Juan; Huang, Mei-Ying; Chen, Zhe

2012-01-25

The purification and partial enzymology characteristics of polyphenol oxidase (PPO) from rape flower were studied. After preliminary treatments, the crude enzyme solution was in turn purified with ammonium sulfate, dialysis, and Sephadex G-75 gel chromatography. The optimal conditions and stability of PPO were examined at different pH values and temperatures. Subsequently, PPO was also characterized by substrate (catechol) concentrations, inhibitors, kinetic parameters, and molecular weight. Results showed that the optimal pH for PPO activity was 5.5 in the presence of catechol and that PPO was relatively stable at pH 3.5-5.5. PPO was moderately stable at temperatures from 60 to 70 °C, whereas it was easily denatured at 80-90 °C. Ethylenediaminetetraacetic acid, sodium chloride, and calcium chloride had little inhibitive effects on PPO, whereas citric acid, sodium sulfite, and ascorbic acid had strongly inhibitive effects. The Michaelis-Menten constant (K(m)) and maximal reaction velocity (V(max)) of PPO were 0.767 mol/L and 0.519 Ab/min/mL of the crude PPO solution, respectively. PPO was finally purified to homogeneity with a purification factor of 4.41-fold and a recovery of 12.41%. Its molecular weight was 60.4 kDa, indicating that the PPO is a dimer. The data obtained in this research may help to prevent the enzymatic browning of rape flower during its storage and processing.

Studies on the production of alkaline α-amylase from Bacillus subtilis CB-18.

PubMed

Nwokoro, Ogbonnaya; Anthonia, Odiase

2015-01-01

Amylases are among the main enzymes used in food and other industries. They hydrolyse starch molecules into polymers composing glucose units. Amylases have potential applications in a number of industrial processes including foods and pharmaceutical industries. Alkaline α-amylase has the potential of hydrolysing starch under alkaline pH and is useful in the starch and textile industries and as an ingredient of detergents. Amylases are produced from plants, however, microbial production processes have dominated applications in the industries. Optimization of microbial production processes can result in improved enzyme yields. Amylase activity was assayed by incubating the enzyme solution (0.5 ml) with 1% soluble starch (0.5 ml) in 0.1 M Tris/HCl buffer (pH 8.5). After 30 minutes, the reaction was stopped by the addition of 4 mL of 3,5-dinitrosalicylic acid (DNS) reagent then heated for 10 min in boiling water bath and cooled in a refrigerator. Absorbance readings were used to estimate the units of enzyme activity from glucose standard curve. Hydrolysed native starches from cassava, rice, corn, coco yam, maize and potato and soluble starch were adjusted to pH 8.5 prior to incubation with crude enzyme solution. Reducing sugars produced were therefore determined. The effect of pH on enzyme activity of the alkaline α-amylase was determined by using buffer solutions of different pH (potassium phosphate buffer, 6.0-7.0; Tris-HCl buffer 7.5 to 9.0 and carbonate/bicarbonate buffer, pH 9.5-11) for enzyme assay. The pH stability profile of the enzyme was determined by incubating 0.5 ml of α-amylase enzyme in 0.1 M Tris/HCl buffer (pH 8.5) and 0.5 ml of 1% (w/v) soluble starch (Merck) in 0.1 M Tris/HCl buffer (pH 8.5) for 3 h in various buffers. The effect of temperature on enzyme activity was studied by incubating 0.5 mL of the enzyme solution contained in the test tube and 0.5 mL of 1% soluble starch (Merck) solution prepared in 0.1 M Tris/HCl buffer (pH 8.5) for 3 h at various temperatures (25, 30, 35, 40, 45, 50, 55 and 60°C) in a thermo static water bath. The reactions were stopped by adding DNS reagent. The enzyme activity was therefore determined. Thermal stability was studied by incubating 0.5 ml of enzyme solution in 0.1 M Tris/HCl buffer (pH 8.5) and 0.5 ml of 1% (w/v) soluble starch (Merck) in 0.1 M Tris/HCl buffer (pH 8.5) for 3 h at various temperatures (20, 30, 40, 50, 60 and 70°C) for 60 min. The enzyme displayed optimal activity at pH 8.0 at which it produced maximum specific activity of 34.3 units/mg protein. Maximum stability was at pH 8.0 to 9.0. Maximum activity was observed at temperature of 50°C while thermo stability of the enzyme was observed at 40-50°C. The enzyme displayed a wide range of activities on starch and caused the release of 5.86, 4.75, 5.98, 3.44, 3.96, 8.84 mg/mL reducing sugar from cassava, potato, cocoyam, corn, rice and soluble starch respectively. This investigation reports some biochemical characterization of alkaline α-amylase from Bacillus subtilis CB-18. The substrate specificities of this enzyme on various starches suggested that the alkaline α-amylase enzyme had combined activities on raw and soluble starch.

Developing Enzyme and Biomimetic Catalysts for Upgrading Heavy Crudes via Biological Hydrogenation and Hydrodesulfurization

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

Borole, A P

The recovery and conversion of heavy oils is limited due to the high viscosity of these crudes and their high heteroatom content. Conventional technology relies on thermochemical hydrogenation and hydrodesulfurization to address these problems and is energy intensive due to the high operating temperature and pressure. This project was initiated to explore biological catalysts for adding hydrogen to the heavy oil molecules. Biological enzymes are efficient at hydrogen splitting at very mild conditions such as room temperature and pressure, however, they are very specific in terms of the substrates they hydrogenate. The goal of the project was to investigate howmore » the specificity of these enzymes can be altered to develop catalysts for oil upgrading. Three approaches were used. First was to perform chemical modification of the enzyme surface to improve binding of other non-natural substrates. Second approach was to expose the deeply buried catalytic active site of the enzyme by removal of protein scaffolding to enable better interaction with other substrates. The third approach was based on molecular biology to develop genetically engineered systems for enabling targeted structural changes in the enzyme. The first approach was found to be limited in success due to the non-specificity of the chemical modification and inability to target the region near the active site or the site of substrate binding. The second approach produced a smaller catalyst capable of catalyzing hydrogen splitting, however, further experimentation is needed to address reproducibility and stability issues. The third approach which targeted cloning of hydrogenase in alternate hosts demonstrated progress, although further work is necessary to complete the cloning process. The complex nature of the hydrogenase enzyme structure-function relationship and role of various ligands in the protein require significant more research to better understand the enzyme and to enable success in strategies in developing catalysts with broader specificity as that required for crude upgrading.« less

Stability Test of Partially Purified Bromelain from Pineapple (Ananas comosus (L.) Merr) Core Extract in Artificial Stomach Fluid

NASA Astrophysics Data System (ADS)

Setiasih, S.; Adimas, A. Ch. D.; Dzikria, V.; Hudiyono, S.

2018-01-01

This study aimed to isolate and purify bromelain from pineapple core (Ananascomosus (L.) Merr) accompanied by a stability test of its enzyme activity in artificial gastric juice. Purification steps start with fractionation by a precipitation method were carried out stepwise using several concentration of ammonium sulfate salt, followed by dialysis prosess and ion exchange chromatography on DEAE-cellulose column. Each step of purification produced an increasing specific activity in enzyme fraction, starting with crude extract, respectively: 0.276 U/mg; 14.591 U/mg; and 16.05 U/mg. Bromelain fraction with the highest level of purity was obtained in 50-80% ammonium sulphate fraction after dialyzed in the amount of 58.15 times compared to the crude extract. Further purification of the enzyme by DEAE-cellulose column produced bromelain which had a purity level 160-fold compared to crude enzyme. The result of bromelain stability test in artificial stomach juice by milk clotting units assay bromelain fraction have proteolytic activity in clotting milk substrate. Exposing bromelain fraction in artificial stomach juice which gave the highest core bromelain proteolytic activity was achieved at estimated volume of 0.4-0.5 mL. Exposure in a period of reaction time to artificial stomach juice that contained pepsin showed relatively stable proteolytic activity in the first 4 hours.

Influence of media composition on the production of alkaline α-amylase from Bacillus subtilis CB-18.

PubMed

Ogbonnaya, Nwokoro; Odiase, Anthonia

2012-01-01

Starch, a homopolysaccharide is an important and an abundant food reserve and energy source. Starches are processed to yield different products which find many industrial applications. Alpha-amylases hydrolyze starch by cleaving α-1,4-glucosidic bonds and have been used in food, textile and pharmaceutical industries [Sun et al. 2010]. Enzymatic conversion of starch with amylase presents an economically superior alternative to the conventional method of starch gelatinization. Alkaline α-amylase has an important position in the global enzyme market as a constituent of detergent. In this paper, we screened soil bacteria and an isolate, alkalophilic Bacillus subtilis CB-18 was found to produce an alkaline α-amylase in different media. MATERIAL AND METHODS. Screening of the isolates for amylolytic activity was carried out by growing bacteria isolated from the soil in starch agar plates and subsequently staining the plates with iodine solution to reveal zones of hydrolysis of starch. The selected isolate, Bacillus subtlis CB-18 was grown in different media at alkaline pH to evaluate the influence of media composition on alkaline α-amylase production. Enzyme assay was carried out by growing the culture in a broth medium and obtaining cell - free culture supernatant after centrifugation at 2515 × g for 15 minutes Amylase activity was determined by incubating 0.5 ml of crude enzyme solution in 0.1M Tris/HCl buffer (pH 8.5) with 0.5 ml of 1% soluble starch solution. The reaction was terminated by the addition of DNS reagent and reducing sugar produced from the amylolytic reaction was determined. Bacillus subtilis CB-18 used for this work was selected because it produced 7 mm zone diameter on starch agar plate. This organism was cultured in different alkaline broth media containing 2% soluble starch as inducer carbohydrate for α-amylase production. Among the carbon sources used for enzyme production, sorbitol was the best to stimulate enzyme production with α-amylase activity of 758 U/mL after 48 h. Peptone was the best nitrogen source for enzyme production with α-amylase activity of 680 U/mL after 48 h. Metal ions including Ca (2+), Mn(2+) and Mg(2+) stimulated enzyme production while Hg(2+) and Ag(+) repressed enzyme production. The best enzyme yields were observed in basal media containing agro-based substrates. This work reports the production of alkaline α-amylase by Bacillus subtlis CB-18 in different media. Enzyme production was highest when agro-based media were used to formulate the media.

Comparison of a xylanase and a complex of non starch polysaccharide-degrading enzymes with regard to performance and bacterial metabolism in weaned piglets.

PubMed

Vahjen, Wilfried; Osswald, Tanja; Schäfer, Klaus; Simon, Ortwin

2007-04-01

Weaned piglets were fed a wheat based diet either non-supplemented or supplemented with a multi-enzyme preparation or a xylanase mono-enzyme preparation, respectively. Both enzyme preparations increased live weight gain nonsignificantly, but only animals of the xylanase group showed a trend (p = 0.076) for an improved feed conversion. Only precaecal digestibility of total amino acids was improved significantly when the mono-enzyme preparation was added. Improvements of digestibility of crude fat, crude protein and starch did not reach the significance level. Both enzyme preparations reduced jejunal viscosity, however viscosity in the colon was only reduced by the mono-enzyme preparation. Both enzymes significantly reduced Lactobacillus spp. cell numbers as well as bacterial metabolites in the stomach and showed similar nonsignificant modifications in jejunum contents except for acetate in the mono-enzyme group. Total jejunal bile acids were unchanged. Compared to the control, the ratio of the main conjugated to the main deconjugated bile acid was significantly higher in the mono-enzyme group. This study has shown that the mono- and multi-enzyme preparation can lead to improved performance in wheat based diets for piglets. Like in poultry, the main mode of action seems to be the reduction of small intestinal viscosity. However, the generation of fermentable carbohydrates by the multi-enzyme preparation may mask beneficial effects on performance due to the development of an active bile acid deconjugating microbiota in the small intestine.

Characterization of an anti-glucosyltransferase serum specific for insoluble glucan synthesis by Streptococcus mutans.

PubMed

Linzer, R; Slade, H D

1976-02-01

An anti-glucosyltransferase serum, which synthesized 96% insoluble glucans, was prepared against a purified enzyme preparation from Streptococcus mutans strain HS6 (serotype a). This serum was examined for its effects on glucan synthesis by crude enzyme preparations from eight strains (four serotypes) of S. mutans and for the ability of these preparations to promote adherence of S. mutans to a smooth surface. Glucosyltransferase activity was assayed by measuring the incorporation of glucose from [14C]glucose-labeled sucrose into water-insoluble and water-soluble (ethanol-insoluble) glucans. Anti-glucosyltransferase serum inhibited insoluble glucan synthesis by crude enzyme preparations from cells of the four serotypes of S. mutans. Enzymes from strains of types a, b, and d were inhibited between 70 to 90%; enzymes from type c strains were inhibited from 45 to 60%. The adherence to a glass surface of heat-killed cells from these four serotypes was likewise inhibited. Soluble glucan synthesis was not inhibited by the serum, and in some cases its synthesis increased as insoluble glucan synthesis decreased.

Inhibitory effect of soy saponins on the activity of β-lactamases, including New Delhi metallo-β-lactamase 1.

PubMed

Horie, Hitoshi; Chiba, Asuka; Wada, Shigeo

2018-05-01

β-Lactamase-producing bacteria encode enzymes that inactivate β-lactam antibiotics by catalyzing the hydrolysis of the β-lactam ring. Crude soy saponins were observed to have synergistic effects on the antimicrobial activity of β-lactam antibiotics against β-lactamase-producing Staphylococcus aureus strains. Furthermore, the activities of β-lactamases derived from Enterobacter cloacae , Escherichia coli , and S. aureus were decreased significantly in the presence of crude soy saponins. This inhibitory effect was also observed against the New Delhi metallo-β-lactamase 1 (NDM-1), an enzyme whose activity is not inhibited by the current β-lactamase inhibitors. The synergistic effect on the antimicrobial activity of β-lactam antibiotics by crude soy saponins was thought to result from the inhibition the β-lactamase activity. The components of crude soy saponins include several kinds of soyasaponins and soyasapogenols. It was revealed that soyasaponin V has the highest inhibitory activity against NDM-1. The combined use of soy saponins with β-lactam antibiotics is expected to serve as a new therapeutic modality, potentially enhancing the effectiveness of β-lactam antibiotics against infectious diseases caused by β-lactamase-producing bacteria, including those encoding NDM-1.

The effects of dietary protein levels on the population growth, performance, and physiology of honey bee workers during early spring.

PubMed

Zheng, Benle; Wu, Zaifu; Xu, Baohua

2014-01-01

This study was conducted to investigate the effects of dietary protein levels on honey bee colonies, specifically the population growth, physiology, and longevity of honey bee workers during early spring. Diets containing four different levels of crude protein (25.0, 29.5, 34.0, or 38.5%) and pure pollen (control) were evaluated. Twenty-five colonies of honey bees with sister queens were used in the study. We compared the effects of the different bee diets by measuring population growth, emergent worker weight, midgut proteolytic enzyme activity, hypopharyngeal gland development, and survival. After 48 d, the cumulative number of workers produced by the colonies ranged from 22,420 to 29,519, providing a significant fit to a quadratic equation that predicts the maximum population growth when the diet contains 31.7% crude protein. Significantly greater emergent worker weight, midgut proteolytic enzyme activity, hypopharyngeal gland acini, and survival were observed in the colonies that were fed diets containing 34.0% crude protein compared with the other crude protein levels. Although higher emergent worker weight and survival were observed in the colonies that were fed the control diet, there were no significant differences between the control colonies and the colonies that were fed 34.0% crude protein. Based on these results, we concluded that a dietary crude protein content of 29.5-34.0% is recommended to maximize the reproduction rate of honey bee colonies in early spring. © The Author 2014. Published by Oxford University Press on behalf of the Entomological Society of America.

Thrombus Degradation by Fibrinolytic Enzyme of Stenotrophomonas sp. Originated from Indonesian Soybean-Based Fermented Food on Wistar Rats

PubMed Central

Tjandrawinata, Raymond R.

2016-01-01

Objective. To evaluate thrombus degrading effect of a fibrinolytic enzyme from food origin Stenotrophomonas sp. of Indonesia. Methods. Prior to animal study, the enzyme safety was tested using cell culture. The effect on expression of tissue plasminogen activator was also analysed in the cell culture. For in vivo studies, 25 Wistar rats were used: normal control, negative control, treatment groups with crude and semipurified enzyme given orally at 25 mg/kg, and positive control group which received Lumbrokinase at 25 mg/kg. Blood clot in the tail was induced by kappa carrageenan injection at 1 mg/kg BW. Results. Experiment with cell culture confirmed the enzyme safety at the concentration used and increased expression of tPA. Decreasing of thrombus was observed in the positive group down to 70.35 ± 23.11% of the negative control animals (100%). The thrombus observed in the crude enzyme treatment was down to 56.99 ± 15.95% and 71.5 ± 15.7% for semipurified enzyme. Scanning electron microscopy showed clearly that bood clots were found in the animals injected with kappa carrageenan; however, in the treatment and positive groups, the clot was much reduced. Conclusions. Oral treatment of enzyme from Stenotrophomonas sp. of Indonesian fermented food was capable of degrading thrombus induced in Wistar rats. PMID:27635131

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.

Enzyme-linked immunosorbent assay using a recombinant baculovirus-expressed Bacillus anthracis protective antigen (PA): measurement of human anti-PA antibodies.

PubMed Central

Iacono-Connors, L C; Novak, J; Rossi, C; Mangiafico, J; Ksiazek, T

1994-01-01

We developed an antigen capture enzyme-linked immunosorbent assay (ELISA) which does not require purified protective antigen (PA) for detection of human antibodies to Bacillus anthracis PA. Lysates of Spodoptera frugiperda (Sf-9) cells infected with recombinant baculovirus containing the PA gene were used as the source of PA to develop the ELISA. Recombinant PA from crude Sf-9 cell lysates or PA purified from B. anthracis Sterne strain was captured by an anti-PA monoclonal antibody coated onto microtiter plates. We demonstrated that human serum antibody titers to PA were identical in the ELISA whether we used crude Sf-9 cell lysates containing recombinant baculovirus-expressed PA or purified Sterne PA. Finally, false-positive results observed in a direct ELISA were eliminated with this antigen capture ELISA. Thus, the antigen capture ELISA with crude preparations of baculovirus-expressed PA is reliable, safe, and inexpensive for determining anti-PA antibody levels in human sera. PMID:7496927

GH10 XynA is the main xylanase identified in the crude enzymatic extract of Paenibacillus sp. A59 when grown on xylan or lignocellulosic biomass.

PubMed

Ghio, Silvina; Insani, Ester M; Piccinni, Florencia E; Talia, Paola M; Grasso, Daniel H; Campos, Eleonora

2016-01-01

A novel bacterial isolate with polysaccharides degrading activity was identified as Paenibacillus sp., and named Paenibacillus sp. A59. Even though it is a strict mesophile, optimal xylanase activity of the crude enzymatic extract was achieved between 50°C and 70°C and more than 60% of the activity was retained after incubation for 48h at 50°C, indicating thermotolerance of the enzymes involved. The extract was also active on pre-treated sugarcane residue (SCR) and wheat straw, releasing xylobiose and xylose as the main products, therefore confirming its predominantly xylanolytic activity. By zymograms and mass spectrometry of crude enzymatic extracts of xylan or SCR cultures, a 32kDa GH10 beta- 1,4- endoxylanase with xylanase and no CMCase activity was identified. We named this enzyme XynA and it was the only xylanase identified under both conditions assayed, suggesting that it is a good candidate for recombinant expression and evaluation in hemicelluloses deconstruction applications. Also, a protein with two S-layer homology domains (SLH) and a large uncharacterized C-terminal domain as well as an ABC substrate binding protein were identified in crude extracts of SCR cultures. We propose that Paenibacillus sp. A59 uses a system similar to anaerobic and other Gram positive bacteria, with SLH-domain proteins anchoring polysaccharide-degrading enzymes close to the membrane and the substrate binding protein assisting translocation of simple sugars to the cell interior. Copyright © 2016 Elsevier GmbH. All rights reserved.

Two-stage statistical medium optimization for augmented cellulase production via solid-state fermentation by newly isolated Aspergillus niger HN-1 and application of crude cellulase consortium in hydrolysis of rice straw.

PubMed

Sandhu, Simranjeet Kaur; Oberoi, Harinder Singh; Babbar, Neha; Miglani, Kanupriya; Chadha, Bhupinder Singh; Nanda, Dhiraj Kumar

2013-12-26

Cellulolytic enzyme production by newly isolated Aspergillus niger HN-1 was statistically optimized using Plackett-Burman and central composite design (CCD). Optimum concentrations of 2, 0.40, 0.01, and 0.60 g L (-1) for KH2PO4, urea, trace elements solution, and CaCl2·2H2O, respectively, were suggested by Design-Expert software. The two-stage optimization process led to a 3- and 2-fold increases in the filter paper cellulase (FP) and β-glucosidase activities, respectively. FP, β-glucosidase, endoglucanase, exopolygalaturonase, cellobiohydrolase, xylanase, α-l-arabinofuranosidase, β-xylosidase, and xylan esterase activities of 36.7 ± 1.54 FPU gds(-1), 252.3 ± 7.4 IU gds(-1), 416.3 ± 22.8 IU gds(-1), 111.2 ± 5.4 IU gds(-1), 8.9 ± 0.50 IU gds(-1), 2593.5 ± 78.9 IU gds(-1), 79.4 ± 4.3 IU gds(-1), 180.8 ± 9.3 IU gds(-1), and 288.7 ± 11.8 IU gds(-1), respectively, were obtained through solid-state fermentation during the validation studies. Hydrolysis of alkali-treated rice straw with crude cellulases resulted in about 84% glucan to glucose, 89% xylan to xylose, and 91% arabinan to arabinose conversions, indicating potential for biomass hydrolysis by the crude cellulase consortium obtained in this study.

Chronic crude garlic-feeding modified adult male rat testicular markers: mechanisms of action

PubMed Central

Hammami, Imen; Amara, Souheila; Benahmed, Mohamed; El May, Michèle V; Mauduit, Claire

2009-01-01

Background Garlic or Allium sativum (As) shows therapeutic effects such as reduction of blood pressure or hypercholesterolemia but side-effects on reproductive functions remain poorly investigated. Because of garlic's chemical complexity, the processing methods and yield in preparations differ in efficacy and safety. In this context, we clarify the mechanisms of action of crushed crude garlic on testicular markers. Methods During one month of treatment, 24 male rats were fed 5%, 10% and 15% crude garlic. Results We showed that crude garlic-feeding induced apoptosis in testicular germ cells (spermatocytes and spermatids). This cell death process was characterized by increased levels of active CASP3 but not CASP6. Expression of the caspase inhibitors BIRC3 and BIRC2 was increased at all doses of As while expression of XIAP and BIRC5 was unchanged. Moreover, expression of the IAP inhibitor DIABLO was increased at doses 10% and 15% of As. The germ cell death process induced by As might be related to a decrease in testosterone production because of the reduced expression of steroidogenic enzymes (Star, Cyp11a, Hsd3b5 and Hsd17b). Evaluation of Sertoli markers showed that TUBB3 and GSTA2 expression was unchanged. In contrast, AMH, RHOX5 and CDKN1B expression was decreased while GATA4 expression was increased. Conclusion In summary, we showed that feeding with crude garlic inhibited Leydig steroidogenic enzyme expression and Sertoli cell markers. These alterations might induce apoptosis in testicular germ cells. PMID:19552815

Linamarase activities in Bacillus spp. responsible for thermophilic aerobic digestion of agricultural wastes for animal nutrition.

PubMed

Ugwuanyi, J Obeta; Harvey, L M; McNeil, B

2007-01-01

Thermophilic Bacillus spp. isolated from thermophilic aerobic digestion (TAD) of model agricultural slurry were screened for ability to secret linamarase activity and degrade linamarin, a cyanogenic glycoside toxin abundant in cassava. Screening was performed by both linamarin - picrate assay and by p-nitrophenyl beta-D-glucoside (PNPG) degradation, and results of both assays were related. Linamarase positive isolates were identified as Bacillus coagulans, Bacillus licheniformis and Bacillus stearothermophilus. Enzyme production was growth related and peak production was reached in 48 h in B. coagulans and 36 h in B. stearothermophilus. B. coagulans produced over 40 times greater activity than B. stearothermophilus. Enzyme productivity in shake flask was not strictly related to screening assay result. Crude enzyme of B. coagulans was optimally active at 75 degrees C while that of B. stearothermophilus was optimally active at 80 degrees C and both had optimum activity at pH 8.0. The thermophilic and neutrophilic- to marginally alkaline activity of the crude enzymes could be very useful in the detoxification and reprocessing of cyanogens containing cassava wastes by TAD for use in animal nutrition.

Keratinolytic activity of Aspergillus fumigatus fresenius.

PubMed

Santos RMDB; Firmino, A A; de Sá, C M; Felix, C R

1996-12-01

Aspergillus fumigatus can utilize chicken feather keratin as its sole carbon and nitrogen source. Because enzymatic conversion of native keratin into readily usable products is of economic interest, this fungus was studied for its capacity to produce and secrete keratin-hydrolyzing proteinases. Substantial keratin-azure hydrolyzing activity was present in the culture fluid of keratin-containing media. Considerably lower activity was present in cultures containing glucose and nitrate as the carbon and nitrogen sources, or keratin plus glucose and nitrate. Secretion of keratin-hydrolyzing activity in A. fumigatus was induced by keratin but repressed by low-molecular-weight carbon and nitrogen sources. The amount of keratinolytic enzyme present in the culture fluid was dependent on the initial pH of the culture medium. The crude enzyme also hydrolyzed native keratin and casein in vitro. Hydrolysis was optimal at pH 9 and 45 degrees C. The crude enzyme was remarkably thermostable. At 70 degrees C, it retained about 90% of its original activity for 1.5 h. The obtained results indicated that the A. fumigatus keratinolytic enzyme may be suitable for enzymatic improvement of feather meal.

Effects of analogues of substance P fragments on the MAO activity in rat brain.

PubMed

Turska, E; Lachowicz, L; Koziołkiewicz, W; Wasiak, T

1985-01-01

The influence in vitro of analogues of Sp5-11 and SP6-11 substance P fragments on the activity of monoamine oxidase (MAO) in homogenates and crude mitochondrial fractions of rat brain was examined. The rat brain was divided into: I--cerebral cortex, II--hippocampus, III--midbrain, IV--thalamus with hypothalamus, V--cerebellum and VI--medulla oblongata. The obtained results proved that the analogues of SP fragments inhibit selectively the activity of the enzyme in the homogenates of cerebral cortex, hippocampus, midbrain and cerebellum. In the crude mitochondrial fractions the applied analogues of SP fragments caused a slight increase of the enzyme activity. The most significant changes in the activity of MAO were observed in hippocampus homogenate fraction.

Sensitivity of salad greens (Lactuca sativa L. and Eruca sativa Mill.) exposed to crude extracts of toxic and non-toxic cyanobacteria.

PubMed

Bittencourt-Oliveira, M C; Hereman, T C; Macedo-Silva, I; Cordeiro-Araújo, M K; Sasaki, F F C; Dias, C T S

2015-05-01

We evaluated the effect of crude extracts of the microcystin-producing (MC+) cyanobacteria Microcystis aeruginosa on seed germination and initial development of lettuce and arugula, at concentrations between 0.5 μg.L(-1) and 100 μg.L(-1) of MC-LR equivalent, and compared it to crude extracts of the same species without the toxin (MC-). Crude extracts of the cyanobacteria with MC (+) and without MC (-) caused different effects on seed germination and initial development of the salad green seedlings, lettuce being more sensitive to both extracts when compared to arugula. Crude extracts of M. aeruginosa (MC+) caused more evident effects on seed germination and initial development of both species of salad greens than MC-. Concentrations of 75 μg.L(-1) and 100 μg.L(-1) of MC-LR equivalent induced a greater occurrence of abnormal seedlings in lettuce, due to necrosis of the radicle and shortening of this organ in normal seedlings, as well as the reduction in total chlorophyll content and increase in the activity of the antioxidant enzyme peroxidase (POD). The MC- extract caused no harmful effects to seed germination and initial development of seedlings of arugula. However, in lettuce, it caused elevation of POD enzyme activity, decrease in seed germination at concentrations of 75 μg.L(-1) (MC-75) and 100 μg.L(-1) (MC-100), and shortening of the radicle length, suggesting that other compounds present in the cyanobacteria extracts contributed to this result. Crude extracts of M. aeruginosa (MC-) may contain other compounds, besides the cyanotoxins, capable of causing inhibitory or stimulatory effects on seed germination and initial development of salad green seedlings. Arugula was more sensitive to the crude extracts of M. aeruginosa (MC+) and (MC-) and to other possible compounds produced by the cyanobacteria.

Ground water contamination by crude oil near Bemidji, Minnesota

USGS Publications Warehouse

Delin, G.N.; Essaid, H.I.; Cozzarelli, I.M.; Lahvis, M.H.; Bekins, B.A.

1998-01-01

Ground-water contamination by crude oil, and other petroleum-based liquids, is a widespread problem. An average of 83 crude-oil spills occurred per year during 1994-96 in the United States, each spilling about 50,000 barrels of crude oil (U.S. Office of Pipeline Safety, electronic commun., 1997). An understanding of the fate of organic contaminants (such as oil and gasoline) in the subsurface is needed to design innovative and cost-effective remedial solutions at contaminated sites.

Chloroplast-derived enzyme cocktails hydrolyse lignocellulosic biomass and release fermentable sugars

PubMed Central

Verma, Dheeraj; Kanagaraj, Anderson; Jin, Shuangxia; Singh, Nameirakpam D.; Kolattukudy, Pappachan E; Daniell, Henry

2009-01-01

Summary It is widely recognized that biofuel production from lignocellulosic materials is limited by inadequate technology to efficiently and economically release fermentable sugars from the complex multi-polymeric raw materials. Therefore, endoglucanases, exoglucanase, pectate lyases, cutinase, swollenin, xylanase, acetyl xylan esterase, beta glucosidase and lipase genes from bacteria or fungi were expressed in E. coli or tobacco chloroplasts. A PCR based method was used to clone genes without introns from Trichoderma reesei genomic DNA. Homoplasmic transplastomic lines showed normal phenotype and were fertile. Based on observed expression levels, up to 49, 64 and 10,751 million units of pectate lyases or endoglucanase can be produced annually, per acre of tobacco. Plant production cost of endoglucanase is 3,100-fold and pectate lyase is 1,057 or 1,480 fold lower than the same recombinant enzymes sold commercially, produced via fermentation. Chloroplast-derived enzymes had higher temperature stability and wider pH optima than enzymes expressed in E. coli. Plant crude-extracts showed higher enzyme activity than E. coli with increasing protein concentration, demonstrating their direct utility without purification. Addition of E. coli extracts to the chloroplast-derived enzymes significantly decreased their activity. Chloroplast-derived crude-extract enzyme cocktails yielded more (up to 3,625%) glucose from filter paper, pine wood or citrus peel than commercial cocktails. Furthermore, pectate lyase transplastomic plants showed enhanced resistance to Erwina soft rot. This is the first report of using plant-derived enzyme cocktails for production of fermentable sugars from lignocellulosic biomass. Limitations of higher cost and lower production capacity of fermentation systems are addressed by chloroplast-derived enzyme cocktails. PMID:20070870

Enhanced Inulin Saccharification by Self-Produced Inulinase from a Newly Isolated Penicillium sp. and its Application in D-Lactic Acid Production.

PubMed

Zheng, Zhaojuan; Xu, Qianqian; Liu, Peng; Zhou, Fan; Ouyang, Jia

2018-03-10

In order to find an alternative for commercial inulinase, a strain XL01 identified as Penicillium sp. was screened for inulinase production. The broth after cultivated was centrifuged, filtered, and used as crude enzyme for the following saccharification. At pH 5.0 and 50 °C, the crude enzyme released 84.9 g/L fructose and 20.7 g/L glucose from 120 g/L inulin in 72 h. In addition, simultaneous saccharification and fermentation of chicory flour for D-lactic acid production was carried out using the self-produced crude inulinase and Lactobacillus bulgaricus CGMCC 1.6970. A high D-lactic acid titer and productivity of 122.0 g/L and 1.69 g/(L h) was achieved from 120 g/L chicory flour in 72 h. The simplicity for inulinase production and the high efficiency for D-lactic acid fermentation provide a perspective and profitable industrial biotechnology for utilization of the inulin-rich biomass.

Optimization of Growth Conditions for Purification and Production of L-Asparaginase by Spirulina maxima

PubMed Central

El Baroty, Gamal S.

2016-01-01

L-asparaginase (L-AsnA) is widely distributed among microorganisms and has important applications in medicine and in food technology sectors. Therefore, the ability of the production, purification, and characterization of AsnA from Spirulina maxima (SM) were tested. SM cultures grown in Zarrouk medium containing different N2 (in NaNO3 form) concentrations (1.25, 2.50, and 5.0 g/L) for 18 days contained a significant various quantity of dry biomass yields and AsnA enzyme levels. MS L-AsnA activity was found to be directly proportional to the N2 concentration. The cultures of SM at large scales (300 L medium, 5 g/L N2) showed a high AsnA enzyme activity (898 IU), total protein (405 mg/g), specific enzyme activity (2.21 IU/mg protein), and enzyme yield (51.28 IU/L) compared with those in low N2 cultures. The partial purification of crude MS AsnA enzyme achieved by 80% ammonium sulfate AS precipitated and CM-Sephadex C-200 gel filtration led to increases in the purification of enzyme with 5.28 and 10.91 times as great as that in SM crude enzymes. Optimum pH and temperature of purified AsnA for the hydrolyzate were 8.5 and 37 ± 0.2°C, respectively. To the best of our knowledge, this is the first report on L-asparaginase production in S. maxima. PMID:27525017

Cholecystokinin-converting enzymes in brain.

PubMed Central

Malesci, A; Straus, E; Yalow, R S

1980-01-01

Crude extracts of porcine cerebral cortical tissue convert cholecystokinin (CCK) to its COOH-terminal fragments, the dodecapeptide (CCK-12) and the octapeptide (CCK-8). The Sephadex G-75 void volume eluate of the crude extract cleaves the arginine-isoleucine bond and effects conversion only to CCK-12; the Sephadex G-50 void volume eluate of the same extract cleaves the arginine-aspartate bond as well, so that both CCK-12 and CCK-8 are end products. Thus, there are at least two enzymes; the one involved in the conversion to CCK-12 is of larger molecular radius than the other. The Km for the cleavage of CCK at the arginine-isoleucine bond by the Sephadex G-75 void volume eluate enzyme is 1.1 X 10(-6) M; the Km for trypsin cleavage of the same bond is 4.7 x 10(-6) M. The lower Vmax for the brain enzyme (1.5 x 10(-11) mol/min per g of extract) compared with trypsin (66 x 10(-11) mol/min per g of trypsin) simply reflects the lesser degree of purify of the brain extract than of the highly purified trypsin. Images PMID:6987659

Enzymatic production of cellulose nanofibers from oil palm empty fruit bunch (EFB) with crude cellulase of Trichoderma sp.

NASA Astrophysics Data System (ADS)

Aditiawati, Pingkan; Dungani, Rudi; Amelia, Cindy

2018-03-01

Oil palm empty fruit bunch (EFB) biomass was used as a source for isolation of cellulose nanofibers (CNF) using enzymatic method. Non-cellulosic component were removed from biomass by delignification process using inoculum of Marasmius sp. Nanocellulose production began with cryocrushing pre-treatment, enzyme addition, and post-treatment with sonication. In enzyme addition, crushed EFB suspended in sodium-citrate buffer and various percentage of crude cellulase enzyme from Trichoderma sp. which is 50%, 100%, and 200% (v/w), followed by incubation in various period which is 2, 3, and 4 days. Particle size analyzer, Scanning electron microscopy and Fourier-transmmission infrared spectroscopy were used to determine the properties of CNF. Maximum CNF size distribution of 2, 3, and 4 days incubation period was 30.717 and 70 nm, respectively (50% (v/w)); 94.75 and 635 nm, respectively (100% (v/w)); 837.51 and 433 nm, respectively (200% (v/w)). Almost 100% yield achieved from variation of 50% (v/w) enzyme and 2 days incubation period. FTIR spectroscopy analysis showed that some impurities in nanocellulose. SEM analysis showed that fibril nanocellulose, with larger size than PSA, mainly because aggregation of nanocellulose.

Production and partial purification of proteases from Aspergillus oryzae grown in a medium based on whey protein as an exclusive nitrogen source.

PubMed

Kumura, H; Ishido, T; Shimazaki, K

2011-02-01

Several attempts have been made to incorporate whey proteins into curd to increase cheese yield. For some types of cheese, degradation of whey proteins that have been incorporated into the curd would be required to obtain acceptable flavor and texture. On the basis of the high potential for protease synthesis in Aspergillus oryzae, sodium nitrate as a nitrogen source in a minimal medium for fungi, known as Czapek-Dox medium, was replaced with whey protein isolate to induce the protease to hydrolyze whey protein using A. oryzae AHU7146. A solid-phase medium adjusted to pH 6 was suitable for this purpose when incubation was carried out at 25°C for 2 wk. The application of column chromatography enabled the resolution of 3 proteolytic components (1, 2, and 3). With respect to optimal temperature and zymographic analysis, component 1 was similar to component 3. In contrast, component 2 was less abundant than the other components and exhibited activity in the alkaline pH region. The degradation of β-lactoglobulin and α-lactalbumin in whey protein isolate solution by the crude enzyme was primarily attributed to the action of components 1 and 3, based on HPLC analysis and the N-terminal amino acid sequences; however, zymography demonstrated evident proteolysis due to component 2. Because heat-denatured whey protein aggregates were digestible by the crude enzyme, the proteolytic system from A. oryzae has the potential as an additive to stimulate the ripening of cheese enriched with whey protein. Copyright © 2011 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Yellow Mealworm Protein for Food Purposes - Extraction and Functional Properties

PubMed Central

Zhao, Xue; Vázquez-Gutiérrez, José Luis; Johansson, Daniel P.; Landberg, Rikard; Langton, Maud

2016-01-01

A protocol for extraction of yellow mealworm larvae proteins was established, conditions were evaluated and the resulting protein extract was characterised. The freeze-dried yellow mealworm larvae contained around 33% fat, 51% crude protein and 43% true protein on a dry matter basis. The true protein content of the protein extract was about 75%, with an extraction rate of 70% under optimised extraction conditions using 0.25 M NaOH, a NaOH solution:ethanol defatted worm ratio of 15:1 mL/g, 40°C for 1 h and extraction twice. The protein extract was a good source of essential amino acids. The lowest protein solubility in distilled water solution was found between pH 4 and 5, and increased with either increasing or decreasing pH. Lower solubility was observed in 0.5 M NaCl solution compared with distilled water. The rheological tests indicated that temperature, sample concentration, addition of salt and enzyme, incubation time and pH alterations influenced the elastic modulus of yellow mealworm protein extract (YMPE). These results demonstrate that the functional properties of YMPE can be modified for different food applications. PMID:26840533

Economic Methods of Ginger Protease'sextraction and Purification

NASA Astrophysics Data System (ADS)

Qiao, Yuanyuan; Tong, Junfeng; Wei, Siqing; Du, Xinyong; Tang, Xiaozhen

This article reports the ginger protease extraction and purification methods from fresh ginger rhizome. As to ginger protease extraction, we adapt the steps of organic solvent dissolving, ammonium sulfate depositing and freeze-drying, and this method can attain crude enzyme powder 0.6% weight of fresh ginger rhizome. The purification part in this study includes two steps: cellulose ion exchange (DEAE-52) and SP-Sephadex 50 chromatography, which can purify crude ginger protease through ion and molecular weight differences respectively.

Elimination of Isoxazolyl-Penicillins antibiotics in waters by the ligninolytic native Colombian strain Leptosphaerulina sp. considerations on biodegradation process and antimicrobial activity removal.

PubMed

Copete-Pertuz, Ledys S; Plácido, Jersson; Serna-Galvis, Efraím A; Torres-Palma, Ricardo A; Mora, Amanda

2018-07-15

In this work, Leptosphaerulina sp. (a Colombian native fungus) significantly removed three Isoxazolyl-Penicillin antibiotics (IP): oxacillin (OXA, 16000 μg L -1 ), cloxacillin (CLX, 17500 μg L -1 ) and dicloxacillin (DCX, 19000 μg L -1 ) from water. The biological treatment was performed at pH 5.6, 28 °C, and 160 rpm for 15 days. The biotransformation process and lack of toxicity of the final solutions (antibacterial activity (AA) and cytotoxicity) were tested. The role of enzymes in IP removal was analysed through in vitro studies with enzymatic extracts (crude and pre-purified) from Leptosphaerulina sp., commercial enzymes and enzymatic inhibitors. Furthermore, the applicability of mycoremediation process to a complex matrix (simulated hospital wastewater) was evaluated. IP were considerably abated by the fungus, OXA was the fastest degraded (day 6), followed by CLX (day 7) and DCX (day 8). Antibiotics biodegradation was associated to laccase and versatile peroxidase action. Assays using commercial enzymes (i.e. laccase from Trametes versicolor and horseradish peroxidase) and inhibitors (EDTA, NaCl, sodium acetate, manganese (II) ions) confirmed the significant role of enzymatic transformation. Whereas, biomass sorption was not an important process in the antibiotics elimination. Evaluation of AA against Staphylococcus aureus ATCC 6538 revealed that Leptosphaerulina sp. also eliminated the AA. In addition, the cytotoxicity assay (MTT) on the HepG2 cell line demonstrated that the IP final solutions were non-toxic. Finally, Leptosphaerulina sp. eliminated OXA and its AA from synthetic hospital wastewater at 6 days. All these results evidenced the potential of Leptosphaerulina sp. mycoremediation as a novel environmentally friendly process for the removal of IP from aqueous systems. Copyright © 2018 Elsevier B.V. All rights reserved.

Xylanases of marine fungi of potential use for biobleaching of paper pulp.

PubMed

Raghukumar, Chandralata; Muraleedharan, Usha; Gaud, V R; Mishra, R

2004-10-01

Microbial xylanases that are thermostable, active at alkaline pH and cellulase-free are generally preferred for biobleaching of paper pulp. We screened obligate and facultative marine fungi for xylanase activity with these desirable traits. Several fungal isolates obtained from marine habitats showed alkaline xylanase activity. The crude enzyme from NIOCC isolate 3 (Aspergillus niger), with high xylanase activity, cellulase-free and unique properties containing 580 U l(-1) xylanase, could bring about bleaching of sugarcane bagasse pulp by a 60 min treatment at 55 degrees C, resulting in a decrease of ten kappa numbers and a 30% reduction in consumption of chlorine during bleaching. The culture filtrate showed peaks of xylanase activity at pH 3.5 and pH 8.5. When assayed at pH 3.5, optimum activity was detected at 50 degrees C, with a second peak of activity at 90 degrees C. When assayed at pH 8.5, optimum activity was seen at 80 degrees C. The crude enzyme was thermostable at 55 degrees C for at least 4 h and retained about 60% activity. Gel filtration of the 50-80% ammonium sulphate-precipitated fraction of the crude culture filtrate separated into two peaks of xylanase with specific activities of 393 and 2,457 U (mg protein)(-1). The two peaks showing xylanase activity had molecular masses of 13 and 18 kDa. Zymogram analysis of xylanase of crude culture filtrate as well as the 50-80% ammonium sulphate-precipitated fraction showed two distinct xylanase activity bands on native PAGE. The crude culture filtrate also showed moderate activities of beta-xylosidase and alpha- l-arabinofuranosidase, which could act synergistically with xylanase in attacking xylan. This is the first report showing the potential application of crude culture filtrate of a marine fungal isolate possessing thermostable, cellulase-free alkaline xylanase activity in biobleaching of paper pulp.

Detection by denaturing gradient gel electrophoresis of ammonia-oxidizing bacteria in microcosms of crude oil-contaminated mangrove sediments.

PubMed

dos Santos, A C F; Marques, E L S; Gross, E; Souza, S S; Dias, J C T; Brendel, M; Rezende, R P

2012-01-27

Currently, the effect of crude oil on ammonia-oxidizing bacterium communities from mangrove sediments is little understood. We studied the diversity of ammonia-oxidizing bacteria in mangrove microcosm experiments using mangrove sediments contaminated with 0.1, 0.5, 1, 2, and 5% crude oil as well as non-contaminated control and landfarm soil from near an oil refinery in Camamu Bay in Bahia, Brazil. The evolution of CO(2) production in all crude oil-contaminated microcosms showed potential for mineralization. Cluster analysis of denaturing gradient gel electrophoresis-derived samples generated with primers for gene amoA, which encodes the functional enzyme ammonia monooxygenase, showed differences in the sample contaminated with 5% compared to the other samples. Principal component analysis showed divergence of the non-contaminated samples from the 5% crude oil-contaminated sediment. A Venn diagram generated from the banding pattern of PCR-denaturing gradient gel electrophoresis was used to look for operational taxonomic units (OTUs) in common. Eight OTUs were found in non-contaminated sediments and in samples contaminated with 0.5, 1, or 2% crude oil. A Jaccard similarity index of 50% was found for samples contaminated with 0.1, 0.5, 1, and 2% crude oil. This is the first study that focuses on the impact of crude oil on the ammonia-oxidizing bacterium community in mangrove sediments from Camamu Bay.

Contents of lecithin and choline in crude drugs.

PubMed

Yamasaki, K; Kikuoka, M; Nishi, H; Kokusenya, Y; Miyamoto, T; Matsuo, M; Sato, T

1994-01-01

The determination of lecithin and choline in crude drugs was established by a combination of high performance liquid chromatography (HPLC) with electrochemical detector (ECD) and enzyme reaction. Lecithin in crude drugs extracted with a mixture of chloroform-methanol (2:1) at room temperature was hydrolyzed by phospholipase D. The hydrolyzate was injected to HPLC, and choline was separated from impurities by reverse phase column. The choline was converted to betaine and hydrogen peroxide by passing through column packed with immobilized choline oxidase. This hydrogen peroxide was detected by ECD. The peak area of hydrogen peroxide derived from lecithin was proportional to the concentration of lecithin from 0.10 to 1.52 microgram/ml. Choline in crude drugs was extracted with ethanol under reflux and determined under the same HPLC conditions as lecithin. The peak area of hydrogen peroxide derived from choline was proportional to the concentration of choline from 0.01 to 0.45 microgram/ml. The contents of lecithin and choline in 31 kinds of crude drugs were determined by these established methods. The results showed that Cervi Parvum Cornu, Kokurozin, Foenigraeci Semen and Psoraleae Semen contained more lecithin than other crude drugs, while Angelicae Radix, Foenigraeci Semen, Psoraleae Semen, and especially Hippocampus were found to contain more choline than other crude drugs.

Characterization of Paenibacillus sp. MBT213 Isolated from Raw Milk and Its Ability to Convert Ginsenoside Rb1 into Ginsenoside Rd from Panax ginseng

PubMed Central

Cho, Soo Hyun; Park, Young W; Song, Gyu Yong

2017-01-01

This study was conducted to isolate and characterize Paenibacillus sp. MBT213 possessing β-glucosidase activity from raw milk, and examine the enzymatic capacity on the hydrolysis of a major ginsenoside (Rb1). Strain MBT213 was found to have a high hydrolytic ability on ginsenoside Rb1 by Esculin Iron Agar test. 16S rDNA analysis revealed that MBT213 was Paenibacillu sp. Crude enzyme of MBT213 strain exhibited high conversion capacity on ginsenoside Rb1 into ginsenoside Rd proven by TLC and HPLC analyses. The API ZYM kit confirmed that Paenibacillu sp. MBT213 exerted higher β-glucosidase and β-galactosidase activity than other strains. Optimum pH and temperature for crude enzyme were found at 7.0 and 35°C in hydrolysis of ginsenoside Rb1. After 10 d of optimal reaction conditions for the crude enzyme, ginsenoside Rb1 fully converted to ginsenoside Rd. Ginseng roots (20%) were fermented for 14 d, and analyzed by HPLC showed that amount of ginsenoside Rb1 significantly decreased, while that of ginsenoside Rd was significantly increased. The study confirmed that the β-glucosidase produced by Paenibacillus sp. MBT213 can hydrolyze the major ginsenoside Rb1 and convert to Rd during fermentation of the ginseng. The β-glucosidase activity of this novel Paenibacillus sp. MBT213 strain may be utilized in development of variety of health foods, dairy foods and pharmaceutical products. PMID:29147097

Antioxidant capacity of chewing stick miswak Salvadora persica.

PubMed

Mohamed, Saleh A; Khan, Jalaluddin A

2013-02-21

Chewing stick (miswak Salvadora persica L.) is an effective tool for oral hygiene. It possessed various biological properties including significant antibacterial and anti-fungal effects. In the present study, we evaluated the antioxidant compounds in miswak. Miswak root was extracted with 80% methanol. Methanol extract as antioxidant was evaluated by using DPPH, ABTS and phosphomolybdenum complex assays and analysis by GC-MS. Peroxidase, catalase and polyphenoloxidase assays were performed for crude extract of miswak root. The methanol extract of miswak contained the highest amount of crude extract among the various solvent extracts. The methanol extract showed a concentration dependent scavenging of DPPH and ABTS radicals with IC50 values 4.8 and 1.6 μg crude extract, respectively. The total antioxidant activities, based on the reduction of molybdenum (VI) to molybdenum (V), increased with increasing crude extract content. The correlation coefficients (R2) between total crude extract and DPPH, ABTS scavenging activities and the formation of phosphomolybdenum complex were 0.97, 0.99 and 0.95, respectively. The GC-MS analysis showed that the methanol extract doesn't contain phenolic and flavonoid compounds or under detected limit. After silylation of methanol extract, three compounds namely 2-furancarboxaldehyde-5-(hydroxymethyl), furan-2-carboxylic acid-3-methyl- trimethylsilyl ester and D-erythro-pentofuranose-2-deoxy-1,3,5-tris-O-(trimethylsilyl) were identified by GC-MS analysis. These furan derivatives as they contain hydroxyl groups could be possessed antioxidant activities. The antioxidant enzymes were also detected in the miswak extract with high level of peroxidase and low level of catalase and polyphenoloxidase. The synergistic actions of antioxidant compounds and antioxidant enzymes make miswak is a good chewing stick for oral hygiene and food purposes.

Three phase partitioning of zingibain, a milk-clotting enzyme from Zingiber officinale Roscoe rhizomes.

PubMed

Gagaoua, Mohammed; Hoggas, Naouel; Hafid, Kahina

2015-02-01

The present work describes for the first time an elegant non-chromatographic method, the three phase partitioning for the purification and recovery of zingibain, a milk-clotting enzyme, from Zingiber officinale rhizomes. Factors affecting partitioning efficiency such as (NH4)2SO4 saturation, crude extract to t-butanol ratio and pH on zingibain partitioning were investigated. Optimal purification parameters were 50% (NH4)2SO4 saturation with 1.0:1.0 ratio of crude extract:t-butanol at pH 7.0, which gave 14.91 purification fold with 215% recovery of zingibain. The enzyme was found to be exclusively partitioned in the aqueous phase. The enzyme showed a prominent single band on SDS-PAGE. It is a monomeric protein of 33.8 kDa and its isoelectric point is 4.38. The enzyme exhibited maximal proteolytic activity at a temperature of 60 °C and pH 7.0. It was found to be stable at 40-65 °C during 2 h. The enzyme was found to be highly stable against numerous metal ions and its activity was enhanced by Ca(2+), K(+) and Na(+). It was completely inhibited by heavy metal ions such as Cu(2+) and Hg(2+) and partially by Cd(+). Zingibain milk-clotting activity (MCA) was found to be highly stable when stored under freezing (-20 °C) for 30 days compared at 4 °C. Copyright © 2014 Elsevier B.V. All rights reserved.

Toxicity of crude oil chemically dispersed in a wave tank to embryos of Atlantic herring (Clupea harengus).

PubMed

Greer, Colleen D; Hodson, Peter V; Li, Zhengkai; King, Thomas; Lee, Kenneth

2012-06-01

Tests of crude oil toxicity to fish are often chronic, exposing embryos from fertilization to hatch to oil solutions prepared using standard mixing procedures. However, during oil spills, fish are not often exposed for long periods and the dynamic nature of the ocean is not easily replicated in the lab. Our objective was to determine if brief exposures of Atlantic herring (Clupea harengus) embryos to dispersed oil prepared by standard mixing procedures was as toxic as oil dispersed in a more realistic model system. Embryos were first exposed to chemically dispersed Alaska North Slope crude and Arabian light crude oil for 2.4 h to 14 d from fertilization to determine if exposure time affected toxicity. Toxicity increased with exposure time, but 2.4-h exposures at realistic concentrations of oil induced blue-sac disease and reduced the percentage of normal embryos at hatch; there was little difference in toxicity between the two oils. Secondly, oil was chemically dispersed in a wave tank to determine if the resultant oil solutions were as toxic to herring embryos as laboratory-derived dispersed oil using a single exposure period of 24 h. Samples taken 15 min postdispersion were more toxic than laboratory-prepared solutions, but samples taken at 5, 30, and 60 min postdispersion were less toxic. Overall, the laboratory- and wave tank-derived solutions of dispersed oil provided similar estimates of toxicity despite differences in the methods for preparing test solutions, suggesting that laboratory and wave tank data are a reliable basis for ecological risk assessments of spilled oil. Copyright © 2012 SETAC.

EVIDENCE FOR AN EXOCELLULAR SITE FOR THE ACID PHOSPHATASE OF SACCHAROMYCES MELLIS1

PubMed Central

Weimberg, Ralph; Orton, William L.

1964-01-01

Weimberg, Ralph (Northern Regional Research Laboratory, Peoria, Ill.), and William L. Orton. Evidence for an exocellular site for the acid phosphatase of Saccharomyces mellis. J. Bacteriol. 88:1743–1754. 1964.—Evidence is presented which demonstrates an exocellular location for acid phosphatase in Saccharomyces mellis. Derepressed intact cells exhibit acid phosphatase activity. The properties of the system are similar to those shown by the enzyme in cell-free extracts. There is no increase in total activity when cell-free extracts are prepared. Enzymatically active cell walls were prepared by leaching acetone-dried cells of this yeast in dilute acetate buffer (pH 6.5) plus β-mercaptoethanol. The insoluble residue, consisting mainly of cell-wall material and containing the phosphatase, was treated with a variety of hydrolytic enzymes and other chemicals. Only papain and crude snail gut extracts dissociated the enzyme from the particulate fraction in nearly quantitative amounts. The mechanism of release by these two enzymes probably differs. Of all enzymes tested, only the snail gut extract digested the cell walls. By dividing the procedure for making protoplasts of S. mellis into two steps, acid phosphatase may be dissociated from resting cells and recovered as an active soluble enzyme. The first step is to pretreat the cells with a thiol reagent. The second step is to digest the cell wall by enzymes present in crude snail gut extracts. Arsenite must be included in the second step to protect the phosphatase from inactivation. The phosphatase is quantitatively released before the cell becomes osmotically fragile. Images PMID:14240965

Cylindrospermopsin inhibits growth and modulates protease activity in the aquatic plants Lemna minor L. and Wolffia arrhiza (L.) Horkel.

PubMed

Jámbrik, Katalin; Máthé, C; Vasas, G; Bácsi, I; Surányi, G; Gonda, S; Borbély, G; M-Hamvas, Márta

2010-01-01

The toxic effects of cylindrospermopsin (cyanobacterial toxin) on animals have been examined extensively, but little research has focused on their effects on plants. In this study cylindrospermopsin (CYN) caused alterations of growth, soluble protein content and protease enzyme activity were studied on two aquatic plants Lemna minor and Wolffia arrhiza in short-term (5 days) experiments. For the treatments we used CYN containing crude extracts of Aphanizomenon ovalisporum (BGSD-423) and purified CYN as well. The maximal inhibitory effects on fresh weight of L. minor and W. arrhiza caused by crude extract were 60% and 54%, respectively, while the maximum inhibitory effects were 30% and 43% in the case of purified CYN at 20 μg ml(-1) CYN content of culture medium. In CYN-treated plants the concentration of soluble protein showed mild increases, especially in W. arrhiza. Protease isoenzyme activity gels showed significant alterations of enzyme activities under the influence of CYN. Several isoenzymes were far more active and new ones appeared in CYN-treated plants. Treatments with cyanobacterial crude extract caused stronger effects than the purified cyanobacterial toxins used in equivalent CYN concentrations.

Kinetic and thermodynamic parameters, and partial characterization of the crude extract of tannase produced by Saccharomyces cerevisiae CCMB 520.

PubMed

Lopes, Lúzia Morgana de Melo; Costa Batista, Larissa Hayannyelly; Gouveia, Marcos Juliano; Leite, Tonny Cley Campos; de Mello, Marcelo Rodrigues Figueira; de Assis, Sandra Aparecida; de Sena, Amanda Reges

2018-05-01

Tannase can be used in different industrial sectors such as in food (juices and wine) and pharmaceutical production (trimethoprim) because it catalyses the hydrolysis of hydrolysable tannins. The aim of the current study is to assess the tannase found in the crude extract of Saccharomyces cerevisiae CCMB 520, and to set its catalytic and thermodynamic properties. The enzyme was optimally active at pH 6.0 and temperature 30 °C. Tannase was activated by Na + , Ca 2+ , K + at 5 × 10 -3  mol/L. The half-life at 30 °C was 3465.7 min. The activation energy was 40.32 kJ/mol. The Gibbs free energy, enthalpy and entropy at 30 °C were 85.40, 48.10 and -0.12 kJ/mol K, respectively. Our results suggest that the tannase found in the crude extract of S. cerevisiae is an attractive enzyme for industrial applications, such as for beverage manufacturing and gallic acid production, due its catalytic and thermodynamic properties (heat-stable and resistant to metal ions).

High cell density fed-batch fermentations for lipase production: feeding strategies and oxygen transfer.

PubMed

Salehmin, M N I; Annuar, M S M; Chisti, Y

2013-11-01

This review is focused on the production of microbial lipases by high cell density fermentation. Lipases are among the most widely used of the enzyme catalysts. Although lipases are produced by animals and plants, industrial lipases are sourced almost exclusively from microorganisms. Many of the commercial lipases are produced using recombinant species. Microbial lipases are mostly produced by batch and fed-batch fermentation. Lipases are generally secreted by the cell into the extracellular environment. Thus, a crude preparation of lipases can be obtained by removing the microbial cells from the fermentation broth. This crude cell-free broth may be further concentrated and used as is, or lipases may be purified from it to various levels. For many large volume applications, lipases must be produced at extremely low cost. High cell density fermentation is a promising method for low-cost production: it allows a high concentration of the biomass and the enzyme to be attained rapidly and this eases the downstream recovery of the enzyme. High density fermentation enhances enzyme productivity compared with the traditional submerged culture batch fermentation. In production of enzymes, a high cell density is generally achieved through fed-batch operation, not through perfusion culture which is cumbersome. The feeding strategies used in fed-batch fermentations for producing lipases and the implications of these strategies are discussed. Most lipase-producing microbial fermentations require oxygen. Oxygen transfer in such fermentations is discussed.

Measurement of crude-cell-extract glycerol dehydratase activity in recombinant Escherichia coli using coupled-enzyme reactions.

PubMed

Sankaranarayanan, Mugesh; Seol, Eunhee; Kim, Yeonhee; Chauhan, Ashish Singh; Park, Sunghoon

2017-03-01

Glycerol dehydratase (GDHt), which converts glycerol to 3-hydroxypropionaldehyde, is essential to the production of 1,3-propanediol (1,3-PDO) or 3-hydroxypropionic acid (3-HP). A reliable GDHt activity assay in crude-cell extract was developed. In the assay, GDHt converted 1,2-propanediol (1,2-PDO) to propionaldehyde, which was further converted to 1-propionic acid by aldehyde dehydrogenase (KGSADH) or to 1-propanol by yeast-alcohol dehydrogenase (yADH), while the NADH concentration change was monitored spectrophotometrically. Cells should be disintegrated by Bead Beater/French Press, not by chemical methods (BugBuster ® /B-PER™), because the reagents significantly inactivated GDHt and coupling enzymes. Furthermore, in the assay mixture, a much higher activity of KGSADH (>200-fold) or yADH (>400-fold) than that of GDHt should have been maintained. Under optimal conditions, both KGSADH and yADH showed practically the same activity. The coupled-enzyme assay method established here should prove to be applicable to recombinant strains developed for the production of 3-HP and/or 1,3-PDO from glycerol.

Isolation of autochthonous non-white rot fungi with potential for enzymatic upgrading of Venezuelan extra-heavy crude oil

PubMed Central

Naranjo, Leopoldo; Urbina, Hector; De Sisto, Angela; Leon, Vladimir

2007-01-01

The increasing world demand for fuels makes it necessary to exploit the largest reserve of extra-heavy crude oil (EHCO) of the Orinoco Oil Belt from Venezuela. We propose the use of extracellular oxidative enzymes, in particular, lignin-degrading enzyme systems (LDS) of fungi, for enzymatic improvement of EHCO. Autochthonous non-white rot fungal strains able to use EHCO, and several polycyclic aromatic hydrocarbons (PAHs) as sole carbon source and energy, were isolated from EHCO-polluted soils and identified as belonging to the genera Fusarium, Penicillium , Trichoderma , Aspergillus , Neosartorya, Pseudallescheria, Cladosporium, Pestalotiopsis , Phoma and Paecillomyces. Phenotypic and biochemical assays revealed the ability of these filamentous fungi to synthesize extracellular oxidative enzymes, and suggested a relationship between the LDS and EHCO bioconversion. This work reports, for the first time, the use of o-phenylenediamine dihydrochloride (OPD) as substrate to measure extracellular ligninolytic peroxidases (ELP) in culture broths of filamentous fungi (Fusarium solani HP-1), and constitutes the first formal study of the fungal community associated with the EHCO of the Orinoco Oil Belt. PMID:18833334

Production of lipase and protease from an indigenous Pseudomonas aeruginosa strain and their evaluation as detergent additives: compatibility study with detergent ingredients and washing performance.

PubMed

Grbavčić, Sanja; Bezbradica, Dejan; Izrael-Živković, Lidija; Avramović, Nataša; Milosavić, Nenad; Karadžić, Ivanka; Knežević-Jugović, Zorica

2011-12-01

An indigenous Pseudomonas aeruginosa strain has been studied for lipase and protease activities for their potential application in detergents. Produced enzymes were investigated in order to assess their compatibility with several surfactants, oxidizing agents and commercial detergents. The crude lipase appeared to retain high activity and stability in the presence of several surfactants and oxidizing agents and it was insusceptible to proteolysis. Lutensol® XP80 and Triton® X-100 strongly activated the lipase for a long period (up to 40 and 30% against the control after 1h) while the protease activity was enhanced by the addition of Triton® WR1339 and Tween® 80. The washing performance of the investigated surfactants was significantly improved with the addition of the crude enzyme preparation. Studies were further undertaken to improve enzymes production. The optimization of fermentation conditions led to an 8-fold increase of lipase production, while the production of protease was enhanced by 60%. Copyright © 2011 Elsevier Ltd. All rights reserved.

Crude cellulase from oil palm empty fruit bunch by Trichoderma asperellum UPM1 and Aspergillus fumigatus UPM2 for fermentable sugars production.

PubMed

Ibrahim, M F; Razak, M N A; Phang, L Y; Hassan, M A; Abd-Aziz, S

2013-07-01

Cellulase is an enzyme that converts the polymer structure of polysaccharides into fermentable sugars. The high market demand for this enzyme together with the variety of applications in the industry has brought the research on cellulase into focus. In this study, crude cellulase was produced from oil palm empty fruit bunch (OPEFB) pretreated with 2% NaOH with autoclave, which was composed of 59.7% cellulose, 21.6% hemicellulose, and 12.3% lignin using Trichoderma asperellum UPM1 and Aspergillus fumigatus UPM2. Approximately 0.8 U/ml of FPase, 24.7 U/ml of CMCase and 5.0 U/ml of β-glucosidase were produced by T. asperellum UPM1 at a temperature of 35 °C and at an initial pH of 7.0. A 1.7 U/ml of FPase, 24.2 U/ml of CMCase, and 1.1 U/ml of β-glucosidase were produced by A. fumigatus UPM2 at a temperature of 45 °C and at initial pH of 6.0. The crude cellulase was best produced at 1% of substrate concentration for both T. asperellum UPM1 and A. fumigatus UPM2. The hydrolysis percentage of pretreated OPEFB using 5% of crude cellulase concentration from T. asperellum UPM1 and A. fumigatus UPM2 were 3.33% and 19.11%, with the reducing sugars concentration of 1.47 and 8.63 g/l, respectively.

A Novel Algorithm Combining Finite State Method and Genetic Algorithm for Solving Crude Oil Scheduling Problem

PubMed Central

Duan, Qian-Qian; Yang, Gen-Ke; Pan, Chang-Chun

2014-01-01

A hybrid optimization algorithm combining finite state method (FSM) and genetic algorithm (GA) is proposed to solve the crude oil scheduling problem. The FSM and GA are combined to take the advantage of each method and compensate deficiencies of individual methods. In the proposed algorithm, the finite state method makes up for the weakness of GA which is poor at local searching ability. The heuristic returned by the FSM can guide the GA algorithm towards good solutions. The idea behind this is that we can generate promising substructure or partial solution by using FSM. Furthermore, the FSM can guarantee that the entire solution space is uniformly covered. Therefore, the combination of the two algorithms has better global performance than the existing GA or FSM which is operated individually. Finally, a real-life crude oil scheduling problem from the literature is used for conducting simulation. The experimental results validate that the proposed method outperforms the state-of-art GA method. PMID:24772031

Crude oil contaminated soil washing in air sparging assisted stirred tank reactor using biosurfactants.

PubMed

Urum, Kingsley; Pekdemir, Turgay; Ross, David; Grigson, Steve

2005-07-01

This study investigated the removal of crude oil from soil using air sparging assisted stirred tank reactors. Two surfactants (rhamnolipid and sodium dodecyl sulfate, SDS) were tested and the effects of different parameters (i.e. temperature, surfactant concentrations, washing time, volume/mass ratio) were investigated under varying washing modes namely, stirring only, air sparging only and the combination of stirring and air sparging. The results showed that SDS removed more than 80% crude oil from non-weathered soil samples, whilst rhamnolipid showed similar oil removal at the third and fourth levels of the parameters tested. The oil removal ability of the seawater prepared solutions were better than those of the distilled water solutions at the first and second levels of temperature and concentration of surfactant solutions. This approach of soil washing was noted to be effective in reducing the amount of oil in soil. Therefore we suggested that a field scale test be conducted to assess the efficiency of these surfactants.

Phytoremediation of abandoned crude oil contaminated drill sites of Assam with the aid of a hydrocarbon-degrading bacterial formulation.

PubMed

Yenn, R; Borah, M; Boruah, H P Deka; Roy, A Sarma; Baruah, R; Saikia, N; Sahu, O P; Tamuli, A K

2014-01-01

Environmental deterioration due to crude oil contamination and abandoned drill sites is an ecological concern in Assam. To revive such contaminated sites, afield study was conducted to phytoremediate four crude oil abandoned drill sites of Assam (Gelakey, Amguri, Lakwa, and Borholla) with the aid of two hydrocarbon-degrading Pseudomonas strains designated N3 and N4. All the drill sites were contaminated with 15.1 to 32.8% crude oil, and the soil was alkaline in nature (pH8.0-8.7) with low moisture content, low soil conductivity and low activities of the soil enzymes phosphatase, dehydrogenase and urease. In addition, N, P, K, and C contents were below threshold limits, and the soil contained high levels of heavy metals. Bio-augmentation was achieved by applying Pseudomonas aeruginosa strains N3 and N4 followed by the introduction of screened plant species Tectona grandis, Gmelina arborea, Azadirachta indica, and Michelia champaca. The findings established the feasibility of the phytoremediation of abandoned crude oil-contaminated drill sites in Assam using microbes and native plants.

Screening of Organophosphorus Acid Anhydrases from different Sources by Western Blot Analysis

DTIC Science & Technology

1991-01-01

agents) have been found3 in both procaryotic and eukaryotic organisms. These enzymes, referred to as organophosphorous acid anhydrases (OPA anhydrases...of organisms, both procaryotic and eukaryotic. Previously, these enzymes were known as DFPases, somanases, paraoxonases or parathion hydrolases, based...cell (SLM Aminco) at 16,000 psi. Cellular debris was removed by centrifugation (46,000xg) for 30 minutes at 40C. The crude cell supernatant which

Characteristic features and biotechnological applications of cross-linked enzyme aggregates (CLEAs).

PubMed

Sheldon, Roger A

2011-11-01

Cross-linked enzyme aggregates (CLEAs) have many economic and environmental benefits in the context of industrial biocatalysis. They are easily prepared from crude enzyme extracts, and the costs of (often expensive) carriers are circumvented. They generally exhibit improved storage and operational stability towards denaturation by heat, organic solvents, and autoproteolysis and are stable towards leaching in aqueous media. Furthermore, they have high catalyst productivities (kilograms product per kilogram biocatalyst) and are easy to recover and recycle. Yet another advantage derives from the possibility to co-immobilize two or more enzymes to provide CLEAs that are capable of catalyzing multiple biotransformations, independently or in sequence as catalytic cascade processes.

Regulation of nitrate reductase by nitric oxide in Chinese cabbage pakchoi (Brassica chinensis L.).

PubMed

Du, Shaoting; Zhang, Yongsong; Lin, Xianyong; Wang, Yue; Tang, Caixian

2008-02-01

Nitrate reductase (NR), a committed enzyme in nitrate assimilation, involves generation of nitric oxide (NO) in plants. Here we show that the NR activity was significantly enhanced by the addition of NO donors sodium nitroprusside (SNP) and NONOate (diethylamine NONOate sodium) to the culturing solution, whereas it was decreased by NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide (cPTIO). Interestingly, both NO gas and SNP directly enhanced but cPTIO inhibited the NR activities of crude enzyme extracts and purified NR enzyme. The cPTIO terminated the interaction between NR-generated NO and the NR itself. Furthermore, the NR protein content was not affected by the SNP treatment. The investigation of the partial reactions catalysed by purified NR using various electron donors and acceptors indicated that the haem and molybdenum centres in NR were the two sites activated by NO. The results suggest that the activation of NR activity by NO is regulated at the post-translational level, probably via a direct interaction mechanism. Accordingly, the concentration of nitrate both in leaves and roots was decreased after 2 weeks of cultivation with SNP. The present study identifies a new mechanism of NR regulation and nitrate assimilation, which provides important new insights into the complex regulation of N-metabolism in plants.

Decolorization of synthetic brilliant green carpet industry dye through fungal co-culture technology.

PubMed

Kumari, Simpal; Naraian, Ram

2016-09-15

Aim of the present study was to evaluate the efficiency of fungal co-culture for the decolorization of synthetic brilliant green carpet industry dye. For this purpose two lignocellulolytic fungi Pleurotus florida (PF) and Rhizoctonia solani (RS) were employed. The study includes determination of enzyme profiles (laccase and peroxidase), dye decolorization efficiency of co-culture and crude enzyme extracts. Both fungi produced laccase and Mn peroxidase and successfully decolorized solutions of different concentrations (2.0, 4.0, 6.0, & 8.0(w/v) of dye. The co-culture resulted highest 98.54% dye decolorization at 2% (w/v) of dye as compared to monocultures (82.12% with PF and 68.89% with RS) during 12 days of submerged fermentation. The lower levels of dyes were rapidly decolorized, while higher levels in slow order as 87.67% decolorization of 8% dye. The promising achievement of the study was remarkable decolorizing efficiency of co-culture over monocultures. The direct treatment of the mono and co-culture enzyme extracts to dye also influenced remarkable. The highest enzymatic decolorization was through combined (PF and RS) extracts, while lesser by monoculture extracts. Based on the observations and potentiality of co-culture technology; further it can be exploited for the bioremediation of areas contaminated with hazardous environmental pollutants including textile and other industry effluents. Copyright © 2016 Elsevier Ltd. All rights reserved.

Enzymatic activities for lignin monomer intermediates highlight the biosynthetic pathway of syringyl monomers in Robinia pseudoacacia.

PubMed

Shigeto, Jun; Ueda, Yukie; Sasaki, Shinya; Fujita, Koki; Tsutsumi, Yuji

2017-01-01

Most of the known 4-coumarate:coenzyme A ligase (4CL) isoforms lack CoA-ligation activity for sinapic acid. Therefore, there is some doubt as to whether sinapic acid contributes to sinapyl alcohol biosynthesis. In this study, we characterized the enzyme activity of a protein mixture extracted from the developing xylem of Robinia pseudoacacia. The crude protein mixture contained at least two 4CLs with sinapic acid 4-CoA ligation activity. The crude enzyme preparation displayed negligible sinapaldehyde dehydrogenase activity, but showed ferulic acid 5-hydroxylation activity and 5-hydroxyferulic acid O-methyltransferase activity; these activities were retained in the presence of competitive substrates (coniferaldehyde and 5-hydroxyconiferaldehyde, respectively). 5-Hydroxyferulic acid and sinapic acid accumulated in the developing xylem of R. pseudoacacia, suggesting, in part at least, sinapic acid is a sinapyl alcohol precursor in this species.

Immobilization of halophilic Bacillus sp. EMB9 protease on functionalized silica nanoparticles and application in whey protein hydrolysis.

PubMed

Sinha, Rajeshwari; Khare, S K

2015-04-01

The present work targets the fabrication of an active, stable, reusable enzyme preparation using functionalized silica nanoparticles as an effective enzyme support for crude halophilic Bacillus sp. EMB9 protease. The immobilization efficiency under optimized conditions was 60%. Characterization of the immobilized preparation revealed marked increase in pH and thermal stability. It retained 80% of its original activity at 70 °C while t 1/2 at 50 °C showed a five-fold enhancement over that for the free protease. Kinetic constants K m and V max were indicative of a higher reaction velocity along with decreased affinity for substrate. The preparation could be efficiently reused up to 6 times and successfully hydrolysed whey proteins with high degree of hydrolysis. Immobilization of a crude halophilic protease on a nanobased scaffold makes the process cost effective and simple.

Simultaneous enzymatic hydrolysis and anaerobic biodegradation of lipid-rich wastewater from poultry industry

NASA Astrophysics Data System (ADS)

Dors, Gisanara; Mendes, Adriano A.; Pereira, Ernandes B.; de Castro, Heizir F.; Furigo, Agenor

2013-03-01

Simultaneous enzymatic hydrolysis and anaerobic biodegradation of lipid-rich wastewater from poultry industry with porcine pancreatic lipase at different concentrations (from 1.0 to 3.0 g L-1) were performed. The efficiency of the enzymatic pretreatment was measured by the Chemical Oxygen Demand (COD) removal and formation of methane. All samples pretreated with lipase showed a positive effect on the COD removal and formation of methane. After 30 days of anaerobic biodegradation the methane production varied from 569 ± 95 to 1,101 ± 10 mL for crude wastewater and pretreated at 3.0 g L-1 enzyme, respectively. COD removal of wastewater supplemented at different enzyme concentrations was found to be threefold higher than crude wastewater. The use of lipases seems to be a promising alternative for treating lipid-rich wastewaters such as those from the poultry industry.

Ground-water contamination by crude oil: Section B in U.S. Geological Survey Toxic Substances Hydrology Program: Proceedings of the technical meeting, Charleston, South Carolina, March 8-12, 1999: Volume 3 (Part C)

USGS Publications Warehouse

Delin, G.N.; Herkelrath, W.N.; Morganwalp, David W.; Buxton, Herbert T.

1999-01-01

Ground water contamination by crude oil, and other petroleum-based liquids, is a widespread problem. An average of 83 crude-oil spills occurred per year during 1994-96 in the United States, each spilling an average of about 50,000 barrels of crude oil (U.S. Office of Pipeline Safety, electronic commun., 1997). An understanding of the fate of organic contaminants (such as oil and gasoline) in the subsurface is needed to design innovative and cost-effective remedial solutions at contaminated sites.

Thermostable 𝜶-Amylase Activity from Thermophilic Bacteria Isolated from Bora Hot Spring, Central Sulawesi

NASA Astrophysics Data System (ADS)

Gazali, F. M.; Suwastika, I. N.

2018-03-01

α-Amylase is one of the most important enzyme in biotechnology field, especially in industrial application. Thermostability of α-Amylase produced by thermophilic bacteria improves industrial process of starch degradation in starch industry. The present study were concerned to the characterization of α-Amylase activity from indigenous thermophilic bacteria isolated from Bora hot spring, Central Sulawesi. There were 18 isolates which had successfully isolated from 90°C sediment samples of Bora hot spring and 13 of them showed amylolytic activity. The α-Amylase activity was measured qualitatively at starch agar and quantitatively based on DNS (3,5-Dinitrosalicylic acid) methods, using maltose as standard solution. Two isolates (out of 13 amylolytic bacteria), BR 002 and BR 015 showed amylolytic index of 0.8 mm and 0.5 mm respectively, after being incubated at 55°C in the 0.002% Starch Agar Medium. The α-Amylase activity was further characterized quantitatively which includes the optimum condition of pH and temperature of α-Amylase crude enzyme from each isolate. To our knowledge, this is the first report on isolation and characterization of a thermostable α-Amylase from thermophilic bacteria isolated from Central Sulawesi particularly from Bora hot spring.

Fumaric acid production using renewable resources from biodiesel and cane sugar production processes.

PubMed

Papadaki, Aikaterini; Papapostolou, Harris; Alexandri, Maria; Kopsahelis, Nikolaos; Papanikolaou, Seraphim; de Castro, Aline Machado; Freire, Denise M G; Koutinas, Apostolis A

2018-04-13

The microbial production of fumaric acid by Rhizopus arrhizus NRRL 2582 has been evaluated using soybean cake from biodiesel production processes and very high polarity (VHP) sugar from sugarcane mills. Soybean cake was converted into a nutrient-rich hydrolysate via a two-stage bioprocess involving crude enzyme production via solid state fermentations (SSF) of either Aspergillus oryzae or R. arrhizus cultivated on soybean cake followed by enzymatic hydrolysis of soybean cake. The soybean cake hydrolysate produced using crude enzymes derived via SSF of R. arrhizus was supplemented with VHP sugar and evaluated using different initial free amino nitrogen (FAN) concentrations (100, 200, and 400 mg/L) in fed-batch cultures for fumaric acid production. The highest fumaric acid concentration (27.3 g/L) and yield (0.7 g/g of total consumed sugars) were achieved when the initial FAN concentration was 200 mg/L. The combination of VHP sugar with soybean cake hydrolysate derived from crude enzymes produced by SSF of A. oryzae at 200 mg/L initial FAN concentration led to the production of 40 g/L fumaric acid with a yield of 0.86 g/g of total consumed sugars. The utilization of sugarcane molasses led to low fumaric acid production by R. arrhizus, probably due to the presence of various minerals and phenolic compounds. The promising results achieved through the valorization of VHP sugar and soybean cake suggest that a focused study on molasses pretreatment could lead to enhanced fumaric acid production.

Determination of Energy and Nutrient Utilization of Enzyme-treated Rump Round Meat and Lotus Root Designed for Senior People with Young and Age d Hens as an Animal Model

PubMed Central

Kim, Jong Woong; Kil, Dong Yong

2016-01-01

This study aimed to examine the nutrient utilization of rump round meat and lotus root using young (32 wk) and aged hens (108 wk) as an animal model. Rump round meat and lotus root were prepared with or without enzymatic treatment. For each age group of laying hens, a total of 24 Hy-Line Brown laying hens were randomly allotted to one of two dietary treatments with six replicates. For rump round meat, the true total tract retention rate (TTTR) of dry matter (DM) and nitrogen (N) were unaffected by either enzymatic treatment or hen age. However, aged hens had greater (p<0.01) TTTR of energy and crude fat than young hens. Enzymatic treatment did not influence the TTTR of energy or crude fat. In addition, we did not observe any significant interaction between the TTTR of DM, energy, N, or crude fat in rump round meat and hen age or enzymatic treatment. The TTTR of DM remained unchanged between controls and enzyme-treated lotus root for young hens. However, enzyme-treated lotus root exhibited greater (p<0.05) TTTR of DM than control lotus root for aged hens, resulting in a significant interaction (p<0.05). The TTTR of energy and N in lotus roots were greater (p<0.01) for aged hens than for young hens. In conclusion, enzymatic treatment exerted beneficial effects on energy and nutrient utilization in aged hens, suggesting the aged hen model is practical for simulation of metabolism of elderly individuals. PMID:27499671

Optimisation of synergistic biomass-degrading enzyme systems for efficient rice straw hydrolysis using an experimental mixture design.

PubMed

Suwannarangsee, Surisa; Bunterngsook, Benjarat; Arnthong, Jantima; Paemanee, Atchara; Thamchaipenet, Arinthip; Eurwilaichitr, Lily; Laosiripojana, Navadol; Champreda, Verawat

2012-09-01

Synergistic enzyme system for the hydrolysis of alkali-pretreated rice straw was optimised based on the synergy of crude fungal enzyme extracts with a commercial cellulase (Celluclast™). Among 13 enzyme extracts, the enzyme preparation from Aspergillus aculeatus BCC 199 exhibited the highest level of synergy with Celluclast™. This synergy was based on the complementary cellulolytic and hemicellulolytic activities of the BCC 199 enzyme extract. A mixture design was used to optimise the ternary enzyme complex based on the synergistic enzyme mixture with Bacillus subtilis expansin. Using the full cubic model, the optimal formulation of the enzyme mixture was predicted to the percentage of Celluclast™: BCC 199: expansin=41.4:37.0:21.6, which produced 769 mg reducing sugar/g biomass using 2.82 FPU/g enzymes. This work demonstrated the use of a systematic approach for the design and optimisation of a synergistic enzyme mixture of fungal enzymes and expansin for lignocellulosic degradation. Copyright © 2012 Elsevier Ltd. All rights reserved.

Early-branching Gut Fungi Possess A Large, And Comprehensive Array Of Biomass-Degrading Enzymes

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

Solomon, Kevin V.; Haitjema, Charles; Henske, John K.

The fungal kingdom is the source of almost all industrial enzymes in use for lignocellulose bioprocessing. Its more primitive members, however, remain relatively unexploited. We developed a systems-level approach that integrates RNA-Seq, proteomics, phenotype and biochemical studies of relatively unexplored early-branching free-living fungi. Anaerobic gut fungi isolated from herbivores produce a large array of biomass-degrading enzymes that synergistically degrade crude, unpretreated plant biomass, and are competitive with optimized commercial preparations from Aspergillus and Trichoderma. Compared to these model platforms, gut fungal enzymes are unbiased in substrate preference due to a wealth of xylan-degrading enzymes. These enzymes are universally catabolite repressed,more » and are further regulated by a rich landscape of noncoding regulatory RNAs. Furthermore, we identified several promising sequence divergent enzyme candidates for lignocellulosic bioprocessing.« less

21 CFR 864.9400 - Stabilized enzyme solution.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Stabilized enzyme solution. 864.9400 Section 864... and Blood Products § 864.9400 Stabilized enzyme solution. (a) Identification. A stabilized enzyme... enzyme solutions include papain, bromelin, ficin, and trypsin. (b) Classification. Class II (performance...

21 CFR 864.9400 - Stabilized enzyme solution.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Stabilized enzyme solution. 864.9400 Section 864... and Blood Products § 864.9400 Stabilized enzyme solution. (a) Identification. A stabilized enzyme... enzyme solutions include papain, bromelin, ficin, and trypsin. (b) Classification. Class II (performance...

Simultaneous hydrolysis and fermentation of unprocessed food waste into ethanol using thermophilic anaerobic bacteria.

PubMed

Dhiman, Saurabh Sudha; David, Aditi; Shrestha, Namita; Johnson, Glenn R; Benjamin, Kenneth M; Gadhamshetty, Venkataramana; Sani, Rajesh K

2017-11-01

The one-pot CRUDE (Conversion of Raw and Untreated Disposal into Ethanol) process was developed for simultaneous hydrolysis and fermentation of unprocessed food waste into ethanol using thermophilic (growing at 65°C) anaerobic bacteria. Unlike existing waste to energy technologies, the CRUDE process obviates the need for any pre-treatment or enzyme addition. A High-Temperature-High-Pressure (HTHP) distillation technique was also applied that facilitated efficient use of fermentation medium, inoculum recycling, and in-situ ethanol collection. For material balancing of the process, each characterized component was represented in terms of C-mol. Recovery of 94% carbon at the end confirmed the operational efficiency of CRUDE process. The overall energy retaining efficiency calculated from sugars to ethanol was 1262.7kJdryweightkg -1 of volatile solids using HTHP. These results suggest that the CRUDE process can be a starting point for the development of a commercial ethanol production process. Copyright © 2017 Elsevier Ltd. All rights reserved.

Highly-efficient enzymatic conversion of crude algal oils into biodiesel.

PubMed

Wang, Yao; Liu, Jin; Gerken, Henri; Zhang, Chengwu; Hu, Qiang; Li, Yantao

2014-11-01

Energy-intensive chemical conversion of crude algal oils into biodiesel is a major barrier for cost-effective algal biofuel production. To overcome this problem, we developed an enzyme-based platform for conversion of crude algal oils into fatty acid methyl esters. Crude algal oils were extracted from the oleaginous microalga Nannochloropsis oceanica IMET1 and converted by an immobilized lipase from Candida antarctica. The effects of different acyl acceptors, t-butanol as a co-solvent, oil to t-butanol ratio, oil to methanol ratio, temperature and reaction time on biodiesel conversion efficiency were studied. The conversion efficiency reached 99.1% when the conversion conditions were optimized, i.e., an oil to t-butanol weight ratio of 1:1, an oil to methanol molar ratio of 1:12, and a reaction time of 4h at 25°C. The enzymatic conversion process developed in this study may hold a promise for low energy consumption, low wastewater-discharge biochemical conversion of algal feedstocks into biofuels. Published by Elsevier Ltd.

Pseudomonas aeruginosa arylsulfatase: a purified enzyme for the mild hydrolysis of steroid sulfates.

PubMed

Stevenson, Bradley J; Waller, Christopher C; Ma, Paul; Li, Kunkun; Cawley, Adam T; Ollis, David L; McLeod, Malcolm D

2015-10-01

The hydrolysis of sulfate ester conjugates is frequently required prior to analysis for a range of analytical techniques including gas chromatography-mass spectrometry (GC-MS). Sulfate hydrolysis may be achieved with commercial crude arylsulfatase enzyme preparations such as that derived from Helix pomatia but these contain additional enzyme activities such as glucuronidase, oxidase, and reductase that make them unsuitable for many analytical applications. Strong acid can also be used to hydrolyze sulfate esters but this can lead to analyte degradation or increased matrix interference. In this work, the heterologously expressed and purified arylsulfatase from Pseudomonas aeruginosa is shown to promote the mild enzyme-catalyzed hydrolysis of a range of steroid sulfates. The substrate scope of this P. aeruginosa arylsulfatase hydrolysis is compared with commercial crude enzyme preparations such as that derived from H. pomatia. A detailed kinetic comparison is reported for selected examples. Hydrolysis in a urine matrix is demonstrated for dehydroepiandrosterone 3-sulfate and epiandrosterone 3-sulfate. The purified P. aeruginosa arylsulfatase contains only sulfatase activity allowing for the selective hydrolysis of sulfate esters in the presence of glucuronide conjugates as demonstrated in the short three-step chemoenzymatic synthesis of 5α-androstane-3β,17β-diol 17-glucuronide (ADG, 1) from epiandrosterone 3-sulfate. The P. aeruginosa arylsulfatase is readily expressed and purified (0.9 g per L of culture) and thus provides a new and selective method for the hydrolysis of steroid sulfate esters in analytical sample preparation. Copyright © 2015 John Wiley & Sons, Ltd.

Pyrimidine Biosynthesis in Lactobacillus leichmannii

PubMed Central

Hutson, Judith Y.; Downing, Mancourt

1968-01-01

Tracer studies of pyrimidine biosynthesis in Lactobacillus leichmannii (ATCC 7830) indicated that, while aspartate is utilized in the usual manner, the guanido carbon of arginine, rather than carbon dioxide, is utilized as a pyrimidine precursor. The guanido carbon of arginine also contributes, to some extent, to the carbon dioxide pool utilized for purine biosynthesis. The enzyme of the first reaction leading from arginine to pyrimidines, arginine deiminase, was investigated in crude bacterial extracts. It was inhibited by thymidylic acid and purine ribonucleotides, and to a lesser extent by purine deoxynucleotides and deoxycytidylic acid. Under the assay conditions employed, a number of nucleotides had no effect on the enzyme activity of the aspartate transcarbamylase of L. leichmannii. Growth of the cells in media containing uracil, compared to growth in media without uracil, resulted in a four- to fivefold decrease in the concentrations of aspartate transcar-bamylase and dihydroorotase and a twofold increase in the concentration of arginine deiminase, as estimated from specific enzyme activity in crude extracts of the cells. A small increase in specific enzyme activity of ornithine transcarbamylase and carbamate kinase was also observed in extracts obtained from cells grown on uracil. No appreciable change in concentration of any of the five enzymes studied was detected when the cells were grown in media containing thymidine or guanylic acid. A hypothetical scheme which suggests a relationship between the control of purine and pyrimidine biosynthesis in this bacterium and which is consistent with the experimental results obtained is presented. PMID:5686000

Phospholipase A2 from Bothrops alternatus (víbora de la cruz) venom. Purification and some characteristic properties.

PubMed

Nisenbom, H E; Seki, C; Vidal, J C

1986-01-01

One single protein species with phospholipase activity has been isolated from Bothrops alternatus venom by a procedure involving gel-filtration on Sephadex G-50 (Step 1), chromatography on SP-Sephadex C-50 (Step 2) and gel-filtration on Sephadex G-75 (Step 3). The purified sample behaved as a homogeneous, monodisperse protein with a molecular weight of 15,000 and isoelectric point of 5.04. The yield in enzyme activity was 48% of the starting material and the apparent purification was 51-fold. When assayed on 1,2-diheptanoyl- or 1,2-dimyristoyl-sn-glycero-3-phosphorylcholine, fatty acids and lysolecithins were the only reaction products, in accordance with the predicted stoichiometry. Studies on positional specificity suggested that the enzyme is a phospholipase A2. The enzyme requires Ca2+ ions for activity and exhibited stereochemical specificity, since the enantiomeric 2, 3-diheptanoyl-sn-glycero-1-phosphorylcholine was not hydrolyzed. Under the experimental conditions employed, reaction products representative of either phospholipase B or C activities could not be detected. After Step 1, the phospholipase activity recovered was higher than the total activity in the crude venom sample, which is explained by the separation of an inhibitor during enzyme purification. The inhibitor was responsible for the initial lag period that characterized the kinetics of the enzyme reaction with crude venom acting on aggregated substrates (lipoprotein, vesicles or micelles), while the rate of hydrolysis of monomeric lecithins was not affected.

Production and partial characterization of lipases from a newly isolated Penicillium sp. using experimental design.

PubMed

Wolski, E; Rigo, E; Di Luccio, M; Oliveira, J V; de Oliveira, D; Treichel, H

2009-07-01

The objective of this work was to investigate the lipase production by a newly isolated Penicillium sp., using experimental design technique, in submerged fermentation using a medium based on peptone, yeast extract, NaCl and olive oil, as well as to characterize the crude enzymatic extracts obtained. Lipase activity values of 9.5 U ml(-1) in 96 h of fermentation was obtained at the maximized operational conditions of peptone, yeast extract, NaCl and olive oil concentrations (g l(-1)) of 20.0, 5.0, 5.0 and of 10.0 respectively. The partial characterization of crude enzymatic extract obtained by submerged fermentation showed optimum activity at pH range from 4.9 to 5.5 and temperature from 37 degrees C to 42 degrees C. The crude extract maintained its initial activity at freezing temperatures up to 100 days. A newly isolated strain of Penicillium sp. used in this work yielded good lipase activities compared to the literature. The growing interest in lipase production is related to the potential biotechnological applications that these enzymes present. New lipase producers are relevant to finding enzymes with different catalytic properties of commercial interest could be obtained, without using genetically modified organisms (GMO).

Production of an acidic and thermostable lipase of the mesophilic fungus Penicillium simplicissimum by solid-state fermentation.

PubMed

Gutarra, Melissa L E; Godoy, Mateus G; Maugeri, Francisco; Rodrigues, Maria Isabel; Freire, Denise M G; Castilho, Leda R

2009-11-01

The production of a lipase by a wild-type Brazilian strain of Penicillium simplicissimum in solid-state fermentation of babassu cake, an abundant residue of the oil industry, was studied. The enzyme production reached about 90 U/g in 72 h, with a specific activity of 4.5 U/mg of total proteins. The crude lipase showed high activities at 35-60 degrees C and pH 4.0-6.0, with a maximum activity at 50 degrees C and pH 4.0-5.0. Enzyme stability was enhanced at pH 5.0 and 6.0, with a maximum half-life of 5.02 h at 50 degrees C and pH 5.0. Thus, this lipase shows a thermophilic and thermostable behavior, what is not common among lipases from mesophilic filamentous fungi. The crude enzyme catalysed the hydrolysis of triglycerides and p-nitrophenyl esters (C4:0-C18:0), preferably acting on substrates with medium-chain fatty acids. This non-purified lipase in addition to interesting properties showed a reduced production cost making feasible its applicability in many fields.

Lactosylamidine-based affinity purification for cellulolytic enzymes EG I and CBH I from Hypocrea jecorina and their properties.

PubMed

Ogata, Makoto; Kameshima, Yumiko; Hattori, Takeshi; Michishita, Kousuke; Suzuki, Tomohiro; Kawagishi, Hirokazu; Totani, Kazuhide; Hiratake, Jun; Usui, Taichi

2010-12-10

Selective adsorption and separation of β-glucosidase, endo-acting endo-β-(1→4)-glucanase I (EG I), and exo-acting cellobiohydrolase I (CBH I) were achieved by affinity chromatography with β-lactosylamidine as ligand. A crude cellulase preparation from Hypocrea jecorina served as the source of enzyme. When crude cellulase was applied to the lactosylamidine-based affinity column, β-glucosidase appeared in the unbound fraction. By contrast, EG I and CBH I were retained on the column and then separated from each other by appropriately adjusting the elution conditions. The relative affinities of the enzymes, based on their column elution conditions, were strongly dependent on the ligand. The highly purified EG I and CBH I, obtained by affinity chromatography, were further purified by Mono P and DEAE chromatography, respectively. EG I and CBH I cleave only at the phenolic bond in p-nitrophenyl glycosides with lactose and N-acetyllactosamine (LacNAc). By contrast, both scissile bonds in p-nitrophenyl glycosides with cellobiose were subject to hydrolysis although with important differences in their kinetic parameters. Copyright © 2010 Elsevier Ltd. All rights reserved.

Seahorse (Hippocampus reidi) as a bioindicator of crude oil exposure.

PubMed

Delunardo, Frederico Augusto Cariello; de Carvalho, Luciano Rodrigues; da Silva, Bruno Ferreira; Galão, Michel; Val, Adalberto Luís; Chippari-Gomes, Adriana R

2015-07-01

This study explored the suitability of the seahorse Hippocampus reidi (Ginsburg, 1933) for assessing biomarkers of genotoxic effects and its use as a sentinel organism to detect the effects of acute exposure to petroleum hydrocarbons. Fish were exposed to three concentrations of crude oil (10, 20 and 30 g/kg) for 96 h, and the activity of phase II biotransformation enzyme glutathione S-transferase (GST) was measured. In addition, we performed genotoxicity assays, such as comet assay, micronucleus (MN) test and nuclear abnormalities (NA) induction, on the erythrocytes of the fish species. Our results revealed that the inhibition of hepatic GST activity in H. reidi was dependent on increasing crude oil concentrations. In contrast, an increase in the damage index (DI) and MN frequency were observed with increased crude oil concentrations. These results indicate that the alkaline comet assay and micronucleus test were suitable and useful in the evaluation of the genotoxicity of crude oil, which could improve determinations of the impact of oil spills on fish populations. In addition, H. reidi is a promising "sentinel organism" to detect the genotoxic impact of petroleum hydrocarbons. Copyright © 2015 Elsevier Inc. All rights reserved.

A New Versatile Microarray-based Method for High Throughput Screening of Carbohydrate-active Enzymes*

PubMed Central

Vidal-Melgosa, Silvia; Pedersen, Henriette L.; Schückel, Julia; Arnal, Grégory; Dumon, Claire; Amby, Daniel B.; Monrad, Rune Nygaard; Westereng, Bjørge; Willats, William G. T.

2015-01-01

Carbohydrate-active enzymes have multiple biological roles and industrial applications. Advances in genome and transcriptome sequencing together with associated bioinformatics tools have identified vast numbers of putative carbohydrate-degrading and -modifying enzymes including glycoside hydrolases and lytic polysaccharide monooxygenases. However, there is a paucity of methods for rapidly screening the activities of these enzymes. By combining the multiplexing capacity of carbohydrate microarrays with the specificity of molecular probes, we have developed a sensitive, high throughput, and versatile semiquantitative enzyme screening technique that requires low amounts of enzyme and substrate. The method can be used to assess the activities of single enzymes, enzyme mixtures, and crude culture broths against single substrates, substrate mixtures, and biomass samples. Moreover, we show that the technique can be used to analyze both endo-acting and exo-acting glycoside hydrolases, polysaccharide lyases, carbohydrate esterases, and lytic polysaccharide monooxygenases. We demonstrate the potential of the technique by identifying the substrate specificities of purified uncharacterized enzymes and by screening enzyme activities from fungal culture broths. PMID:25657012

(Bio)degradation of RDX and HMX in Marine/Estuarine Water and Sediments

DTIC Science & Technology

2006-09-01

and capability to metabolize organic acids and sugar. Both strains HAW-EB2 and HAW-EB5T utilize malate , valerate, peptone and yeast extract as sole...MEDINA) confirming that the nitramines were metabolized by sediment indigenous microorganisms. Both nitramines were also removed in microcosms prepared...Thus far all enzymes or crude enzyme extract examined were found to metabolize RDX or HMX via a le transfer process leading to denitration although 2e

Purification and characterization of myrosinase from horseradish (Armoracia rusticana) roots.

PubMed

Li, Xian; Kushad, Mosbah M

2005-06-01

Myrosinase (beta-thioglucoside glucohydrolase; EC 3.2.3.147) from horseradish (Armoracia rusticana) roots was purified to homogeneity by ammonium sulfate fractionation, Q-sepharose, and concanavalin A sepharose affinity chromatography. The purified protein migrated as a single band with a mass of about 65 kDa on SDS-polyacrylamide gel electrophoresis. Using LC-MS/MS, this band was identified as myrosinase. Western blot analysis, using the anti-myrosinase monoclonal antibody 3D7, showed a single band of about 65 kDa for horseradish crude extract and for the purified myrosinase. The native molecular mass of the purified myrosinase was estimated, using gel filtration, to be about 130 kDa. Based on these data, it appeared that myrosinase from horseradish root consists of two subunits of similar molecular mass of about 65 kDa. The enzyme exhibited high activity at broad pH (pH 5.0-8.0) and temperature (37 and 45 degrees C). The purified enzyme remained stable at 4 degrees C for more than 1 year. Using sinigrin as a substrate, the Km and Vmax values for the purified enzyme were estimated to be 0.128 mM and 0.624 micromol min(-1), respectively. The enzyme was strongly activated by 0.5 mM ascorbic acid and was able to breakdown intact glucosinolates in a crude extract of broccoli.

The production and activity test of cellulases using bagasse substrate on Aspergillus niger isolated from Clove field, Kare, Madiun

NASA Astrophysics Data System (ADS)

Ardhi, Muh. Waskito; Sulistyarsi, Ani; Pujiati

2017-06-01

Aspergillus sp is a microorganism which has a high ability to produce cellulase enzymes. In producing Cellulase enzymes requires appropriate concentration and incubation time to obtain optimum enzyme activity. This study aimed to determine the effect of inoculum concentration and incubation time towards production and activity of cellulases from Aspergillus sp substrate bagasse. This research used experiments method; completely randomized design with 2 factorial repeated 2 times. The treatment study include differences inoculum (K) 5% (K1), 15% (K2) 25%, (K3) and incubation time (F) that is 3 days (F1), 6 days (F2), 9 days (F3), 12 days (F4). The data taken from the treatment are glucose reduction and protein levels of crude cellulase enzyme activity that use Nelson Somogyi and Biuret methods. Analysis of variance ANOVA data used two paths with significance level of 5% then continued with LSD test. The results showed that: Fhit>Ftab. Thus, there is effect of inoculum concentrations and incubation time toward activity of crude cellulases of Aspergillus sp. The highest glucose reduction of treatment is K3F4 (concentration of inoculum is 25% with 12 days incubation time) amount 12.834 g / ml and the highest protein content is K3F4 (concentration of inoculum is 25% with with 12 days incubation time) amount 0.740 g / ml.

Pelagia noctiluca (Scyphozoa) Crude Venom Injection Elicits Oxidative Stress and Inflammatory Response in Rats

PubMed Central

Bruschetta, Giuseppe; Impellizzeri, Daniela; Morabito, Rossana; Marino, Angela; Ahmad, Akbar; Spanò, Nunziacarla; La Spada, Giuseppa; Cuzzocrea, Salvatore; Esposito, Emanuela

2014-01-01

Cnidarian toxins represent a rich source of biologically active compounds. Since they may act via oxidative stress events, the aim of the present study was to verify whether crude venom, extracted from the jellyfish Pelagia noctiluca, elicits inflammation and oxidative stress processes, known to be mediated by Reactive Oxygen Species (ROS) production, in rats. In a first set of experiments, the animals were injected with crude venom (at three different doses 6, 30 and 60 µg/kg, suspended in saline solution, i.v.) to test the mortality and possible blood pressure changes. In a second set of experiments, to confirm that Pelagia noctiluca crude venom enhances ROS formation and may contribute to the pathophysiology of inflammation, crude venom-injected animals (30 µg/kg) were also treated with tempol, a powerful antioxidant (100 mg/kg i.p., 30 and 60 min after crude venom). Administration of tempol after crude venom challenge, caused a significant reduction of each parameter related to inflammation. The potential effect of Pelagia noctiluca crude venom in the systemic inflammation process has been here demonstrated, adding novel information about its biological activity. PMID:24727391

Generation of shrimp waste-based dispersant for oil spill response.

PubMed

Zhang, Kedong; Zhang, Baiyu; Song, Xing; Liu, Bo; Jing, Liang; Chen, Bing

2018-04-01

In this study, shrimp waste was enzymatically hydrolyzed to generate a green dispersant and the product was tested for crude oil dispersion in seawater. The hydrolysis process was first optimized based on the dispersant effectiveness (DE) of the product. The functional properties of the product were identified including stability, critical micelle concentration, and emulsification activity. Water was confirmed as a good solvent for dispersant generation when compared with three chemical solvents. The effects of salinity, mixing energy, and temperature on the dispersion of the Alaska North Slope (ANS) crude oil were examined. Microtox acute toxicity test was also conducted to evaluate the toxicity of the produced dispersant. In addition, DE of the product on three different types of crude oil, including ANS crude oil, Prudhoe Bay crude oil (PBC), and Arabian Light crude oil (ALC) was compared with that of the Corexit 9500, respectively. The research output could lead to a promising green solution to the oil spill problem and might result in many other environmental applications.

Characterization of truncated endo-β-1,4-glucanases from a compost metagenomic library and their saccharification potentials.

PubMed

Lee, Jae Pil; Lee, Hyun Woo; Na, Han Beur; Lee, Jun-Hee; Hong, Yeo-Jin; Jeon, Jeong-Min; Kwon, Eun Ju; Kim, Sung Kyum; Kim, Hoon

2018-04-23

A gene encoding an endo-β-1,4-glucanase (Cel6H-f481) was cloned from a compost metagenomic library. The gene, cel6H-f481, was composed of 1446 bp to encode a fused protein of 481 amino acid residues (50,429 Da), i.e., 445 residues (Cel6H-445) from the metagenome, and 36 residues from the pUC19 vector at N-terminus. Cel6H-445 belonged to glycosyl hydrolase (GH) family 6 and showed 71% identity with Actinotalea fermentans endoglucanase with low coverage. Several active bands of truncated forms were observed by activity staining of the crude extract. Major truncated enzymes of 35 (Cel6H-p35) and 23 kDa (Cel6H-p23) were separated by HiTrap Q chromatography. The two enzymes had the same optimum temperature (50 °C) and pH (5.5), but Cel6H-p35 was more thermostable than Cel6H-p23 and other GH6 endoglucanases reported. Both enzymes efficiently hydrolyzed carboxymethyl-cellulose (CMC) and barley β-glucan, but hardly hydrolyzed other substrates tested. The V max of Cel6H-p35 for CMC was 1.4 times greater than that of Cel6H-p23. The addition of the crude enzymes to a commercial enzyme set increased the saccharification of pretreated rice straw powder by up to 30.9%. These results suggest the N-terminal region of Cel6H-p35 contributes to thermostability and specific activity, and that the enzymes might be a useful additive for saccharification. Copyright © 2018 Elsevier B.V. All rights reserved.

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

SLACK, JEFFREY, M.

Wood is a potential source for biofuels such as ethanol if it can be digested into sugars and fermented by yeast. Biomass derived from wood is a challenging substrate for ethanol production since it is made of lignin and cellulose which cannot be broken down easily into fermentable sugars. Some insects, and termites in particular, are specialized at using enzymes in their guts to digest wood into sugars. If termite gut enzymes could be made abundantly by a recombinant protein expression vector system, they could be applied to an industrial process to make biofuels from wood. In this study, amore » large cDNA library of relevant termite genes was made using termites fed a normal diet, or a diet with added lignin. A subtracted library yielded genes that were overexpressed in the presence of lignin. Termite gut enzyme genes were identified and cloned into recombinant insect viruses called baculoviruses. Using our PERLXpress system for protein expression, these termite gene recombinant baculoviruses were prepared and used to infect insect larvae, which then expressed abundant recombinant termite enzymes. Many of these expressed enzymes were prepared to very high purity, and the activities were studied in conjunction with collaborators at Purdue University. Recombinant termite enzymes expressed in caterpillars were shown to be able to release sugars from wood. Mixing different combinations of these enzymes increased the amount of sugars released from a model woody biomass substrate. The most economical, fastest and energy conserving way to prepare termite enzymes expressed by recombinant baculoviruses in caterpillars was by making crude liquid homogenates. Making enzymes stable in homogenates therefore was a priority. During the course of these studies, improvements were made to the recombinant baculovirus expression platform so that caterpillar-derived homogenates containing expressed termite enzymes would be more stable. These improvements in the baculoviruses included significantly reducing proteases and preventing blackening immune reactions that occur when caterpillars are homogenized. Proteases may degrade enzymes and immune reaction blackening may inactivate enzymes thus compromising the ability of these crude recombinant expressed termite enzyme preparations to release sugars. Commercial preparations of fungal enzymes currently are used to digest wood for ethanol production. We demonstrated in this study that termite enzymes could improve the efficiency of fungal enzyme cocktails. Although the economic feasibility of using caterpillar expressed termite enzymes alone to treat wood was not proven, this work points to the potential to combine C-PERLXpressed insect enzymes with industrial enzyme cocktails to boost their efficiency at treating wood for biofuels.« less

Production, Characterization and Application of a Thermostable Tannase from Pestalotiopsis guepinii URM 7114

PubMed Central

Reges de Sena, Amanda; Claúdia de Barros dos Santos, Ana; Gouveia, Miquéas Jamesse; Figueira de Mello, Marcelo Rodrigues; Leite, Tonny Cley Campos; Moreira, Keila Aparecida

2014-01-01

Summary Tannase (EC 3.1.1.20) is an enzyme that hydrolyzes the ester and depside bonds of tannic acid to gallic acid and glucose. In the production of foods and beverages, it contributes to the removal of the undesirable effects of tannins. The aim of this study is to investigate the potential of endophytic fungi isolated from jamun (Syzygium cumini (L.) Skeels) leaves, and identified as Pestalotiopsis guepinii, in the production of tannase. Tannase was produced extracellularly by P. guepinii under submerged, slurry-state and solid-state fermentations. The submerged fermentation was found to be the most promising (98.6 U/mL). Response surface methodology was employed to evaluate the effect of variables (pH and temperature), and the results showed that the best conditions for tannase activity were pH=6.9 and 30 °C. Km was found to be 7.18·10–4 mol/L and vmax=250.00 U/mL. The tannase activity was the highest in the presence of Ca2+ at a concentration of 5·10–3 mol/L. Moreover, the enzyme was not inhibited by the tested chelators and detergents. The stability of the enzyme was also studied, and crude enzyme was evaluated in simulation of gastrointestinal digestion of monogastric animals. The crude enzyme was highly stable under simulated conditions; it retained 87.3% of its original activity after 6 h. The study contributes to the identification of microbial species that produce tannase, with potential application in biotechnology. PMID:27904319

Production, Characterization and Application of a Thermostable Tannase from Pestalotiopsis guepinii URM 7114.

PubMed

Reges de Sena, Amanda; Claúdia de Barros Dos Santos, Ana; Gouveia, Miquéas Jamesse; Figueira de Mello, Marcelo Rodrigues; Leite, Tonny Cley Campos; Moreira, Keila Aparecida; Aparecida de Assis, Sandra

2014-12-01

Tannase (EC 3.1.1.20) is an enzyme that hydrolyzes the ester and depside bonds of tannic acid to gallic acid and glucose. In the production of foods and beverages, it contributes to the removal of the undesirable effects of tannins. The aim of this study is to investigate the potential of endophytic fungi isolated from jamun ( Syzygium cumini (L.) Skeels) leaves, and identified as Pestalotiopsis guepinii , in the production of tannase. Tannase was produced extracellularly by P. guepinii under submerged, slurry-state and solid-state fermentations. The submerged fermentation was found to be the most promising (98.6 U/mL). Response surface methodology was employed to evaluate the effect of variables (pH and temperature), and the results showed that the best conditions for tannase activity were pH=6.9 and 30 °C. K m was found to be 7.18·10 -4 mol/L and v max =250.00 U/mL. The tannase activity was the highest in the presence of Ca 2+ at a concentration of 5·10 -3 mol/L. Moreover, the enzyme was not inhibited by the tested chelators and detergents. The stability of the enzyme was also studied, and crude enzyme was evaluated in simulation of gastrointestinal digestion of monogastric animals. The crude enzyme was highly stable under simulated conditions; it retained 87.3% of its original activity after 6 h. The study contributes to the identification of microbial species that produce tannase, with potential application in biotechnology.

Feasibility test of utilizing Saccharophagus degradans 2-40(T) as the source of crude enzyme for the saccharification of lignocellulose.

PubMed

Jung, Young Hoon; Kim, Hyun Kyung; Song, Du-Sup; Choi, In-Geol; Yang, Taek Ho; Lee, Hee Jong; Seung, Doyoung; Kim, Kyoung Heon

2014-04-01

In the conversion of lignocellulose into high-value products, including fuels and chemicals, the production of cellulase and the enzymatic hydrolysis for producing fermentable sugar are the largest contributors to the cost of production of the final products. The marine bacterium Saccharophagus degradans 2-40(T) can degrade more than ten different complex polysaccharides found in the ocean, including cellulose and xylan. Accordingly, S. degradans has been actively considered as a practical source of crude enzymes needed for the saccharification of lignocellulose to produce ethanol by others including a leading commercial company. However, the overall enzyme system of S. degradans for hydrolyzing cellulose and hemicellulose has not been quantitatively evaluated yet in comparison with commercial enzymes. In this study, the inductions and activities of cellulase and xylanase of cell-free lysate of S. degradans were investigated. The growth of S. degradans cells and the activities of cellulase and xylanase were promoted by adding 2 % of cellulose and xylan mixture (cellulose:xylan = 4:3 in mass ratio) to the aquarium salt medium supplemented with 0.2 % glucose. The specific cellulase activity of the cell-free lysate of S. degradans, as determined by the filter paper activity assay, was approximately 70 times lower than those of commercial cellulases, including Celluclast 1.5 L and Accellerase 1000. These results imply that significant improvement in the cellulase activity of S. degradans is needed for the industrial uses of S. degradans as the enzyme source.

Metabolism of Phloridzin by Erwinia herbicola: Nature of the Degradation Products, and the Purification and Properties of Phloretin Hydrolase

PubMed Central

Chatterjee, A. K.; Gibbins, L. N.

1969-01-01

Erwinia herbicola Y46 degrades phloridzin to yield phloretin, phloroglucinol, and phloretic acid, when grown on defined medium containing phloridzin as the sole source of carbon. The identities of the intermediates isolated from culture filtrates were established by co-chromatography and by ultraviolet absorption spectra. Only 3 of 11 strains of this species, and none of the 12 species of bacterial phytopathogens tested could effect this breakdown. Some of the latter organisms possessed β-glucosidase activity which liberated d-glucose from phloridzin. The enzyme phloretin hydrolase was purified from cells of E. herbicola Y46 grown on Yeast Beef Broth, by treatment of crude extracts with protamine sulfate, ammonium sulfate precipitation, elution from calcium phosphate gel, elution from diethylaminoethyl-cellulose, and concentration by ultrafiltration. The final preparation was free of β-glucosidase, had a specific activity of 213 units per mg of protein, and represented a 142-fold purification over the crude extract. The enzyme had a pH optimum of 6.7 to 6.8, and produced only phloroglucinol and phloretic acid as products of phloretin breakdown, there being an equimolar relationship between the cleavage of phloretin and the formation of the products. The Michaelis constant (Km) for the enzyme with phloretin as substrate was 3.8 × 10−5m, and the enzyme was sensitive to Hg2+ and Cu2+ ions. Phloroglucinol, phloretic acid, p-chloromercuribenzoate and iodoacetamide were without effect on the activity. The enzyme did not react with phloridzin, naringin, or naringenin. The physiological significance of the results is discussed. PMID:5354935

Capsule-Targeting Depolymerase, Derived from Klebsiella KP36 Phage, as a Tool for the Development of Anti-Virulent Strategy.

PubMed

Majkowska-Skrobek, Grażyna; Łątka, Agnieszka; Berisio, Rita; Maciejewska, Barbara; Squeglia, Flavia; Romano, Maria; Lavigne, Rob; Struve, Carsten; Drulis-Kawa, Zuzanna

2016-12-01

The rise of antibiotic-resistant Klebsiella pneumoniae , a leading nosocomial pathogen, prompts the need for alternative therapies. We have identified and characterized a novel depolymerase enzyme encoded by Klebsiella phage KP36 (depoKP36), from the Siphoviridae family. To gain insights into the catalytic and structural features of depoKP36, we have recombinantly produced this protein of 93.4 kDa and showed that it is able to hydrolyze a crude exopolysaccharide of a K. pneumoniae host. Using in vitro and in vivo assays, we found that depoKP36 was also effective against a native capsule of clinical K. pneumoniae strains, representing the K63 type, and significantly inhibited Klebsiella -induced mortality of Galleria mellonella larvae in a time-dependent manner. DepoKP36 did not affect the antibiotic susceptibility of Klebsiella strains. The activity of this enzyme was retained in a broad range of pH values (4.0-7.0) and temperatures (up to 45 °C). Consistently, the circular dichroism (CD) spectroscopy revealed a highly stability with melting transition temperature (T m ) = 65 °C. In contrast to other phage tailspike proteins, this enzyme was susceptible to sodium dodecyl sulfate (SDS) denaturation and proteolytic cleavage. The structural studies in solution showed a trimeric arrangement with a high β-sheet content. Our findings identify depoKP36 as a suitable candidate for the development of new treatments for K. pneumoniae infections.

Purification of α-glucosidase from mouse intestine by countercurrent chromatography coupled with a reverse micelle solvent system.

PubMed

He, Kai; Zou, Zongyao; Hu, Yinran; Yang, Yong; Xiao, Yubo; Gao, Pincao; Li, Xuegang; Ye, Xiaoli

2016-02-01

Countercurrent chromatography coupled with a reverse micelle solvent was applied to separate α-glucosidase, which is stable at pH 6.0-8.8, 15-50°C. The separation conditions are as follows: stationary phase: pH 4.0 Tris-HCl buffer phase containing 50 mM Tris-HCl and 50 mM KCl; mobile phase A: isooctane containing 50 mM anionic surfactant sodium di(2-ethylhexyl)sulfosuccinate; mobile phase B: 50 mM Tris-HCl buffer containing 500 mM KCl (pH 8.0); In total, 25 mL (23.9 mg) crude enzyme was injected through the injection valve, the enzymatic reaction and sodium dodecylsulfate polyacrylamide gel electrophoresis results imply that the activity of purified α-glucosidase is 6.63-fold higher than that of the crude enzyme. Therefore, countercurrent chromatography coupled with a reverse micelle solvent is capable for protein separation and enrichment. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Characterization of proteases from Planomicrobium sp. L-2 isolated from the gastrointestinal tract of Octopus variabilis (Sasaki)

NASA Astrophysics Data System (ADS)

Jin, Yulan; Wang, Yurong; Xiao, Lin; Lin, Xiukun

2016-05-01

A crude protease produced from Planomicrobium sp. L-2 is described, and its effectiveness as an additive in liquid detergent evaluated. We isolate the protease-producing Planomicrobium sp. L-2 from the gastrointestinal tract of Octopus variabilis. At least three caseinolytic protease clear bands were observed in zymogram analysis. The crude alkaline protease was highly tolerant of a pH range from 7.0 to 9.0, and temperatures to 50°C after incubation for 1 h. Proteolytic enzymes were stable towards three surfactants (5% Tween 80, 1% Triton X-100 and 0.05% SDS) and an oxidizing agent (1% hydrogen peroxide), in addition to being highly stable and compatible with popular commercial laundry powered detergent brands available in China. Our study demonstrates the potential these proteases have for development into novel classes of detergent additive. This study also suggests that the gastrointestinal tract of Octopus variabilis may be a rich source of commercially valuable strains of enzyme.

[Isolation and purification of alpha-glycerophosphate oxidase in a polyethylene glycol/(NH4 )2SO4 aqueous two-phase system].

PubMed

Meng, Yao; Jin, Jiagui; Liu, Shuangfeng; Yang, Min; Zhang, Qinglian; Wan, Li; Tang, Kun

2014-02-01

Alpha-glycerophosphate oxidase (alpha-GPO) from Enterococcus casseli flavus was successfully isolated and purified by using polyethylene glycol (PEG)/(NH4)2SO4 aqueous two-phase system (ATPS). The results showed that the chosen PEG/(NH4)2SO4 ATPS could be affected by PEG molecular weight, pH, concentration of PEG and (NH4)2SO4, and inorganic salt as well as additional amount of crude enzyme. After evaluating these influencing factors, the final optimum purification strategy was formed by 16.5% (m/m) PEG2000, 13.2% (m/m) (NH4)2SO4, pH 7.5 and 30% (m/m) additive crude enzyme, respectively. The NaCl was a negative influencing factor which would lead to lower purification fold and activity recovery. These conditions eventually resulted in the activity recovery of 89% (m/m), distribution coefficient of 1.2 and purification fold of 7.0.

The feasibility study of crude palm oil transesterification at 30 °C operation.

PubMed

Sim, Jia Huey; Kamaruddin, Azlina Harun; Bhatia, Subhash

2010-12-01

The objective of this research is to investigate the potential of transesterification of crude palm oil (CPO) to biodiesel at 30 degrees C. The mass transfer limitations problem crucial at 30 degrees C due to the viscosity of CPO has been addressed. The process parameters that are closely related to mass transfer effects like enzyme loading, agitation speed and reaction time were optimized. An optimum methanol to oil substrate molar ratio at 6.5:1 was observed and maintained throughout the experiments. The optimum operating condition for the transesterification process was found at 6.67 wt% of enzyme loading and at 150 rpm of agitation speed. The corresponding initial reaction and FAME yield obtained at 6 h were 89.29% FAME yield/hr and 85.01%, respectively. The 85% FAME yield obtained at 30 degrees C operation of CPO transesterification shows that the process is potentially feasible for the biodiesel synthesis. 2010 Elsevier Ltd. All rights reserved.

Accumulation of petroleum hydrocarbons in intracellular lipid bodies of the freshwater diatom Synedra acus subsp. radians.

PubMed

Shishlyannikov, Sergey M; Nikonova, Alyona A; Klimenkov, Igor V; Gorshkov, Alexander G

2017-01-01

The accumulation of hydrophobic compounds by phytoplankton plays a crucial role in the biogeochemical cycle of persistent organic pollutants (POPs) in aquatic environments. We studied the accumulation of polycyclic aromatic hydrocarbons (PAHs) in the freshwater diatom Synedra acus subsp. radians during its cultivation with crude oil hydrocarbons, using epifluorescent and laser confocal microscopy as well as gas chromatography-mass spectrometry (GC/MS) analysis. Our results revealed that in the presence of crude oil or an extract of a crude oil/n-hexane solution (light oil), S. acus subsp. radians accumulated PAHs in its lipid bodies. During cultivation in the presence of a crude oil/n-hexane solution, the cells selectively accumulated C12-C18 alkanes, with a preference for C15 and C16 homologues. The length of n-alkane hydrocarbon chains accumulated in cells was similar to the acyl chains of fatty acids of the diatom. We therefore suggest that the insertion of n-alkanes into the membrane lipid bilayer promotes the transmembrane transport of PAH in diatoms. Our results confirm the hypothesis that diatoms play a role in the elimination of hydrophobic hydrocarbons from aquatic systems.

Preliminary study : optimization of pH and salinity for biosurfactant production from Pseudomonas aeruginosa in diesel fuel and crude oil medium

NASA Astrophysics Data System (ADS)

Ikhwani, A. Z. N.; Nurlaila, H. S.; Ferdinand, F. D. K.; Fachria, R.; Hasan, A. E. Z.; Yani, M.; Setyawati, I.; Suryani

2017-03-01

Biosurfactant is secondary metabolite surface active compound produced by microorganisms which is nontoxic and eco-friendly. Microorganism producing biosurfactant that is quite potential to use in many applications is from Pseudomonas aeruginosa strains. Good quality of biosurfactant production from microbes is supported by the suitable nutrients and environmental factors. The aim of this research was to obtain preliminary o data upon the optimum pH and salinity for the production of biosurfactant from Pseudomonas aeruginosa ATCC 15442 in diesel fuel and crude oil medium. P. aeruginosa ATCC 15442 cultured in diesel fuel and crude oil as carbon source showed biosurfactant activity. P.aeruginosa-derived biosurfactant was capable to form stable emulsion for 24 hours (EI24) in hydrocarbons n-hexane solutions. The particular biosurfactant showed EI24 highest value at pH 7 (31.02%) and 1% NaCl (24.00%) when P. aeruginosa was grown in 10% diesel fuel medium in mineral salt solution. As for the media crude oil, the highest EI24 value was at pH 6 (52.16%) and 1% NaCl (33.30%).

Production of fibrinolytic protease from Streptomyces lusitanus isolated from marine sediments

NASA Astrophysics Data System (ADS)

SudeshWarma, S.; Merlyn keziah, S.; Subathra Devi, C.

2017-11-01

This study aim was to isolate, screen, characterize and optimize marine Streptomyces for fibrinolytic enzyme production. The potent actinomycete isolate was subjected to optimization. The parameters for optimization included pH, temperature, carbon, nitrogen sources. The crude supernatant produced was purified using size exclusion gel filtration chromatography. The optimized parameters for maximum productivity were found to be pH 7, 37°C, maltose and peptone respectively. The molecular weight of the purified enzyme was found to be 21kDa.

Isolation, purification and characterization of β-amylase from Dioscorea hispida Dennst

NASA Astrophysics Data System (ADS)

Oktiarni, Dwita; Lusiana, Simamora, Febri Yanti; Gaol, Jusni M. Lumban

2015-09-01

β-amylase (E.C 3.2.1.2) is an enzyme commonly found in plants and bacteria. The enzyme is an exo-acting carbohydrolase which hydrolyzes α-1.4-glucosidic linkages of starch, removing maltose units from the non-reducing end of the polysaccharide chain, producing β-maltose and β-limit dextrin as the final product. β-amylase is widely distributed in the higher plants such as sweet potato. Besides the use in starch hydrolysis, starch-converting enzymes are also used in a number of other industrial applications, such as laundry and porcelain detergents or as anti-stalling agents in baking. This enzyme was extracted from Dioscorea hispida Dennst in 0.05 M acetate buffer pH 4.8 and followed by ammonium sulfate fractionation at cold temperature (10°C). Ammonium sulfate fractionation was shared into fraction of 0-60%, 60-70%, 70-80% and 80-100%. The fraction containing high of specific activity (determined by Somogyi-Nelson and Lowry methods) was futher purified by dialysis. Fraction with high enzyme activity of β-amylase were fraction 60-70% and 70-80%, with specific activity of Dioscorea hispida Dennst were 1.32 and 1.55 mg sugar.mg protein-1.minute-1, whereas specific activity of crude extract enzyme was 0.21 mg sugar.mg protein-1.minute-1. After purified with dialysis, fraction with high enzyme activity of β-amylase were fraction of 60-70% and 70-80%, with specific activity of Dioscorea hispida Dennst was 2.72 and 4.24 mg sugar.mg protein-1.minute-1. The purified Dioscorea hispida Dennst β-amylase from dialysis showed increasing in spesific activity the crude enzyme as much as 24 folds. The characterization of enzyme showed that Dioscorea hispida Dennst derived enzyme had optimum pH of 5.5 and temperature of 70°C. The kinetic parameters of purified Dioscorea hispida Dennst β-amylase showed that the KMapp, Vmaxapp value and Hill constant were 0.0211 mg/ml, 9.63 mg sugar.minute-1 and 1.34, respectively.

Characterization of polyphenol oxidase from the Manzanilla cultivar (Olea europaea pomiformis) and prevention of browning reactions in bruised olive fruits.

PubMed

Segovia-Bravo, Kharla A; Jarén-Galan, Manuel; García-García, Pedro; Garrido-Fernandez, Antonio

2007-08-08

The crude extract of the polyphenol oxidase (PPO) enzyme from the Manzanilla cultivar (Olea europaea pomiformis) was obtained, and its properties were characterized. The browning reaction followed a zero-order kinetic model. Its maximum activity was at pH 6.0. This activity was completely inhibited at a pH below 3.0 regardless of temperature; however, in alkaline conditions, pH inhibition depended on temperature and was observed at values above 9.0 and 11.0 at 8 and 25 degrees C, respectively. The thermodynamic parameters of substrate oxidation depended on pH within the range in which activity was observed. The reaction occurred according to an isokinetic system because pH affected the enzymatic reaction rate but not the energy required to carry out the reaction. In the alkaline pH region, browning was due to a combination of enzymatic and nonenzymatic reactions that occurred in parallel. These results correlated well with the browning behavior observed in intentionally bruised fruits at different temperatures and in different storage solutions. The use of a low temperature ( approximately 8 degrees C) was very effective for preventing browning regardless of the cover solution used.

Dispersion of Louisiana crude oil in salt water environment by Corexit 9500A in the presence of natural coastal materials

NASA Astrophysics Data System (ADS)

Tansel, Berrin; Lee, Mengshan; Berbakov, Jillian; Tansel, Derya Z.; Koklonis, Urpiana

2014-04-01

Effectiveness of Corexit 9500A for dispersing Louisiana crude oil was evaluated in salt water solutions containing natural materials in relation to salinity and dispersant-to-oil ratio (DOR). Experimental results showed that both salinity and DOR had significant effects on dispersion of Louisiana crude oil in the presence of different natural materials. The natural materials added to the salt water solutions included sea sand (South Beach, Miami, Florida), red mangrove leaves (Rhizophora mangle), seaweed (Sargassum natans), and sea grass (Halodule wrightii). Dispersant effectiveness (amount of oil dispersed into the water) was reduced significantly with increasing salinity with the minimum effectiveness observed in the salinity range between 30 and 50 ppt in all aqueous samples containing natural materials. When significant amounts of floating oil were present, the partially submerged natural materials enhanced the transfer of oil into the water column, which improved the dispersion effectiveness. However, dispersant effectiveness was significantly reduced when the amount of floating oil was relatively small and could not be released back to the water column. Surface tension may not be an adequate parameter for monitoring the effectiveness of dispersants in salt water environment. When distilled water was used (i.e., zero salinity), surface tension was significantly reduced with increasing dispersant concentration. However, there was no clear trend in the surface tension of the salt water solutions (17-51 ppt) containing crude oil and natural materials with increasing dispersant concentration.

Enzyme stabilization by glass-derived silicates in glass-exposed aqueous solutions

USGS Publications Warehouse

Ives, J.A.; Moffett, J.R.; Arun, P.; Lam, D.; Todorov, T.I.; Brothers, A.B.; Anick, D.J.; Centeno, J.; Namboodiri, M.A.A.; Jonas, W.B.

2010-01-01

Objectives: To analyze the solutes leaching from glass containers into aqueous solutions, and to show that these solutes have enzyme activity stabilizing effects in very dilute solutions. Methods: Enzyme assays with acetylcholine esterase were used to analyze serially succussed and diluted (SSD) solutions prepared in glass and plastic containers. Aqueous SSD preparations starting with various solutes, or water alone, were prepared under several conditions, and tested for their solute content and their ability to affect enzyme stability in dilute solution. Results: We confirm that water acts to dissolve constituents from glass vials, and show that the solutes derived from the glass have effects on enzymes in the resultant solutions. Enzyme assays demonstrated that enzyme stability in purified and deionized water was enhanced in SSD solutions that were prepared in glass containers, but not those prepared in plastic. The increased enzyme stability could be mimicked in a dose-dependent manner by the addition of silicates to the purified, deionized water that enzymes were dissolved in. Elemental analyses of SSD water preparations made in glass vials showed that boron, silicon, and sodium were present at micromolar concentrations. Conclusions: These results show that silicates and other solutes are present at micromolar levels in all glass-exposed solutions, whether pharmaceutical or homeopathic in nature. Even though silicates are known to have biological activity at higher concentrations, the silicate concentrations we measured in homeopathic preparations were too low to account for any purported in vivo efficacy, but could potentially influence in vitro biological assays reporting homeopathic effects. ?? 2009 The Faculty of Homeopathy.

Dynamic Response of Mycobacterium vanbaalenii PYR-1 to BP Deepwater Horizon Crude Oil

PubMed Central

Kim, Seong-Jae; Kweon, Ohgew; Sutherland, John B.; Kim, Hyun-Lee; Jones, Richard C.; Burback, Brian L.; Graves, Steven W.; Psurny, Edward

2015-01-01

We investigated the response of the hydrocarbon-degrading Mycobacterium vanbaalenii PYR-1 to crude oil from the BP Deepwater Horizon (DWH) spill, using substrate depletion, genomic, and proteome analyses. M. vanbaalenii PYR-1 cultures were incubated with BP DWH crude oil, and proteomes and degradation of alkanes and polycyclic aromatic hydrocarbons (PAHs) were analyzed at four time points over 30 days. Gas chromatography-mass spectrometry (GC-MS) analysis showed a chain length-dependent pattern of alkane degradation, with C12 and C13 being degraded at the highest rate, although alkanes up to C28 were degraded. Whereas phenanthrene and pyrene were completely degraded, a significantly smaller amount of fluoranthene was degraded. Proteome analysis identified 3,948 proteins, with 876 and 1,859 proteins up- and downregulated, respectively. We observed dynamic changes in protein expression during BP crude oil incubation, including transcriptional factors and transporters potentially involved in adaptation to crude oil. The proteome also provided a molecular basis for the metabolism of the aliphatic and aromatic hydrocarbon components in the BP DWH crude oil, which included upregulation of AlkB alkane hydroxylase and an expression pattern of PAH-metabolizing enzymes different from those in previous proteome expression studies of strain PYR-1 incubated with pure or mixed PAHs, particularly the ring-hydroxylating oxygenase (RHO) responsible for the initial oxidation of aromatic hydrocarbons. Based on these results, a comprehensive cellular response of M. vanbaalenii PYR-1 to BP crude oil was proposed. This study increases our fundamental understanding of the impact of crude oil on the cellular response of bacteria and provides data needed for development of practical bioremediation applications. PMID:25888169

Production and purification of amylolytic enzymes for saccharification of microalgal biomass.

PubMed

Rodrigues, Éllen Francine; Ficanha, Aline Matuella Moreira; Dallago, Rogério Marcos; Treichel, Helen; Reinehr, Christian Oliveira; Machado, Tainara Paula; Nunes, Greice Borges; Colla, Luciane Maria

2017-02-01

The aim of this study was the production of amylolytic enzymes by solid state or submerged fermentations (SSF or SF, respectively), followed by purification using chemical process or microfiltration and immobilization of purified enzymes in a polyurethane support. The free and immobilized enzymes obtained were used to evaluate enzymatic hydrolysis of the polysaccharides of Spirulina. Microfiltration of the crude extracts resulted in an increase in their specific activity and thermal stability at 40°C and 50°C for 24h, as compared to extracts obtained by SSF and SF. Immobilization of polyurethane purified enzyme produced yields of 332% and 205% for the enzymes obtained by SF and SSF, respectively. Free or immobilized enzymes favor the generation of fermentable sugar, being the application of the purified and immobilized enzymes in the hydrolysis of microalgal polysaccharides considered a promising alternative towards development of the bioethanol production process from microalgal biomass. Copyright © 2016 Elsevier Ltd. All rights reserved.

Effects of the naturally-occurring contaminant microcystins on the Azolla filiculoides-Anabaena azollae symbiosis.

PubMed

Pereira, A L; Monteiro, B; Azevedo, J; Campos, A; Osório, H; Vasconcelos, V

2015-08-01

Harmful algal blooms (HABs) contaminate aquatic ecosystems and are responsible for animal poisoning worldwide. We conducted a toxicity test with the aquatic fern and the biofertilizer, Azolla filiculoides. The sporophytes were exposed to three concentrations (0.01, 0.1 and 1μgmL(-1)) of a microcystin (MC) cyanobacterial crude extract and purified MC-LR. The growth of A. filiculoides decreased only at 1μgmL(-1) crude extract concentration while with MC-LR it decreased at all the tested concentrations, indicating that the presence of other compounds in the crude extract altered toxicity and stimulated the fern growth at lower concentrations (0.01 and 0.1μgmL(-1)). Both phycoerythrocyanin and allophycocyanin levels decreased in all the concentrations of crude extract and MC-LR. The phycocyanin had a marked increase at 0.1μgmL(-1) crude extract concentration and a marked decrease at 1μgmL(-1) MC-LR concentration. These changes in the phycobiliprotein content indicate a shift in the antenna pigments of the cyanobionts of A. filiculoides. The changes in two oxidative stress enzymes, glutathione reductase for the crude extract assay and glutathione peroxidase for MC-LR assay, points towards the induction of stress defense responses. The low bioconcentration factor in both crude extract and MC-LR treatments can suggest the low uptake of microcystins, and indicates that the aquatic fern can be used as a biofertilizer and as animal feed but is not suitable for MC phytoremediation. Copyright © 2015 Elsevier Inc. All rights reserved.

Identification of a type-D feruloyl esterase from Neurospora crassa.

PubMed

Crepin, V F; Faulds, C B; Connerton, I F

2004-02-01

Feruloyl esterases constitute an interesting group of enzymes that have the potential for use over a broad range of applications in the agri-food industries. In order to expand the range of available enzymes, we have examined the presence of feruoyl esterase genes present in the genome sequence of the filamentous fungus Neurospora crassa. We have identified an orphan gene (contig 3.544), the translation of which shows sequence identity with known feruloyl esterases. This gene was cloned and the corresponding recombinant protein expressed in Pichia pastoris to confirm that the enzyme (NcFaeD-3.544) exhibits feruloyl esterase activity. Unusually the enzyme was capable of p-coumaric acid release from untreated crude plant cell wall materials. The substrate utilisation preferences of the recombinant enzyme place it in the recently recognised type-D sub-class of feruloyl esterase.

Proteolytic enzymes from Bromelia antiacantha as tools for controlled tissue hydrolysis in entomology.

PubMed

Macció, Laura; Vallés, Diego; Cantera, Ana Maria

2013-12-01

A crude extract with high proteolytic activity (78.1 EU/mL), prepared from ripe fruit of Bromelia antiacantha was used to hydrolyze and remove soft tissues from the epigyne of Apopyllus iheringi. This enzymatic extract presented four actives isoforms which have a broad substrate specificity action. Enzyme action on samples was optimized after evaluation under different conditions of pH, enzyme-substrate ratio and time (parameters selected based on previous studies) of treatment (pH 4.0, 6.0 and 8.0 at 42°C with different amount of enzyme). Scanning electron microscopy was used to evaluate conditions resulting in complete digestion of epigyne soft tissues. Optimal conditions for soft tissue removal were 15.6 total enzyme units, pH 6.0 for 18 h at 42°C.

Surfactant- and oxidant-stable alkaline proteases from Bacillus invictae: Characterization and potential applications in chitin extraction and as a detergent additive.

PubMed

Hammami, Amal; Hamdi, Marwa; Abdelhedi, Ola; Jridi, Mourad; Nasri, Moncef; Bayoudh, Ahmed

2017-03-01

A newly alkaline proteases producing strain was isolated from sea water. The strain was identified as Bacillus invictae on the basis of biochemical characteristics and 16S rRNA sequence analysis. The crude protease activity showed an optimal activity at approximately 60°C and in wide pH interval ranging from 9.0 to 11.0. At least six clear caseinolytic protease bands were observed in a zymogram. Phenylmethylsulfonyl fluoride (PMSF), a serine-protease inhibitor, was found to inhibit completely the protease activity. The crude alkaline proteases showed high stability toward solid and liquid detergents. Furthermore, wash performance analysis revealed that the crude enzyme could effectively remove blood stain when added to commercial detergent. In addition, the crude proteases were found to be effective in the deproteinization of shrimp shell waste. The percent of protein removal after 3h of hydrolysis at 50°C with an E/S ratio of 10U/mg of protein or after fermentation by the strain were about 76% and 82%, respectively. Thus, the results of the present study showed that the crude proteases of B. invectae could be effectively used in several industrial applications, as an eco-friendly agent. Copyright © 2016 Elsevier B.V. All rights reserved.

Energy Value of Cassava Products in Broiler Chicken Diets with or without Enzyme Supplementation

PubMed Central

Bhuiyan, M. M.; Iji, P. A.

2015-01-01

This study investigated the metabolizable energy (ME) intake, net energy of production (NEp), heat production (HP), efficiencies of ME use for energy, lipid and protein retention as well as the performance of broiler chickens fed diets based on cassava chips or pellets with or without supplementation with an enzyme product containing xylanase, amylase, protease and phytase. The two products, cassava chips and pellets, were analysed for nutrient composition prior to feed formulation. The cassava chips and pellets contained 2.2% and 2.1% crude protein; 1.2% and 1.5% crude fat; and 75.1% and 67.8% starch, respectively. Lysine and methionine were 0.077%, 0.075%, and 0.017%, 0.020% protein material, respectively, while calculated ME was 12.6 and 11.7 MJ/kg, respectively. Feed intake to day 21 was lower (p<0.01) on the diet containing cassava chips compared to diets with cassava pellets. Enzyme supplementation increased (p<0.01) feed intake on all diets. Live weight at day 21 was significantly (p<0.01) reduced on the diet based on cassava chips compared to pellets, but an improvement (p<0.01) was noticed with the enzyme supplementation. Metabolizable energy intake was reduced (p<0.01) by both cassava chips and pellets, but was increased (p<0.01) on all diets by enzyme supplementation. The NEp was higher (p<0.01) in the maize-based diets than the diets containing cassava. Enzyme supplementation improved (p<0.01) NEp in all the diets. Heat production was highest (p<0.01) on diets containing cassava pellets than on cassava chips. It is possible to use cassava pellets in diets for broiler chickens at a level close to 50% of the diet to reduce cost of production, and the nutritive value of such diets can be improved through supplementation of enzyme products containing carbohydrases, protease, and phytase. PMID:26194227

A new versatile microarray-based method for high throughput screening of carbohydrate-active enzymes.

PubMed

Vidal-Melgosa, Silvia; Pedersen, Henriette L; Schückel, Julia; Arnal, Grégory; Dumon, Claire; Amby, Daniel B; Monrad, Rune Nygaard; Westereng, Bjørge; Willats, William G T

2015-04-03

Carbohydrate-active enzymes have multiple biological roles and industrial applications. Advances in genome and transcriptome sequencing together with associated bioinformatics tools have identified vast numbers of putative carbohydrate-degrading and -modifying enzymes including glycoside hydrolases and lytic polysaccharide monooxygenases. However, there is a paucity of methods for rapidly screening the activities of these enzymes. By combining the multiplexing capacity of carbohydrate microarrays with the specificity of molecular probes, we have developed a sensitive, high throughput, and versatile semiquantitative enzyme screening technique that requires low amounts of enzyme and substrate. The method can be used to assess the activities of single enzymes, enzyme mixtures, and crude culture broths against single substrates, substrate mixtures, and biomass samples. Moreover, we show that the technique can be used to analyze both endo-acting and exo-acting glycoside hydrolases, polysaccharide lyases, carbohydrate esterases, and lytic polysaccharide monooxygenases. We demonstrate the potential of the technique by identifying the substrate specificities of purified uncharacterized enzymes and by screening enzyme activities from fungal culture broths. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Determination of Urease Biochemical Properties of Asparagus Bean (Vigna unguiculata ssp sesquipedalis L.)

NASA Astrophysics Data System (ADS)

Zusfahair; Ningsih, D. R.; Fatoni, A.; Pertiwi, D. S.

2018-04-01

Urease is enzyme that plays a role in nitrogen metabolism during plant germination. Plants that produce a lot of urease are grains. This study used asparagus bean as source of urease. The purpose of this research is to learn the effect of germination time on the activity of urease enzyme from asparagus bean and its biochemical properties. The research was started by germination of asparagus bean on day 2, 4, 6, 8, 10 and 12. Asparagus bean sprouts were extracted using acetone and separated by centrifugation to obtain the crude extract of urease. The biochemical properties of the crude extract of urease was further determined including: the effect of temperature, pH, substrate concentration, and metal addition to urease activity. The urease activity is determined by the Nessler method. The germination time of asparagus bean in yielding urease enzyme reached the optimum activity on the 8th day with activity value of 593.7 U/mL. The biochemical properties of urease from asparagus bean have optimum activity at 35 °C, pH 7.0 and substrate concentration 0.125% with activity value of 600 U/mL. Addition of CaCl2, SnCl2 and ZnCl2 metals decrease the activity of urease.

A novel aqueous micellar two-phase system composed of surfactant and sorbitol for purification of pectinase enzyme from Psidium guajava and recycling phase components.

PubMed

Amid, Mehrnoush; Murshid, Fara Syazana; Manap, Mohd Yazid; Hussin, Muhaini

2015-01-01

A novel aqueous two-phase system composed of a surfactant and sorbitol was employed for the first time to purify pectinase from Psidium guajava. The influences of different parameters, including the type and concentration of the surfactant and the concentration and composition of the surfactant/sorbitol ratio, on the partitioning behavior and recovery of pectinase were investigated. Moreover, the effects of system pH and the crude load on purification fold and the yield of purified pectinase were studied. The experimental results indicated that the pectinase was partitioned into surfactant-rich top phase, and the impurities were partitioned into the sorbitol-rich bottom phase with the novel method involving an ATPS composed of 26% (w/w) Triton X-100 and 23% (w/w) sorbitol at 54.2% of the TLL crude load of 20% (w/w) at pH 6.0. The enzyme was successfully recovered by this method with a high purification factor of 15.2 and a yield of 98.3%, whereas the phase components were also recovered and recycled at rates above 96%. This study demonstrated that this novel ATPS method can be used as an efficient and economical alternative to the traditional ATPS for the purification and recovery of the valuable enzyme.

Evaluation of biosurfactants grown in corn oil by Rhodococcus rhodochrous on removing of heavy metal ion from aqueous solution

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

Suryanti, Venty, E-mail: venty@mipa.uns.ac.id; Hastuti, Sri; Pujiastuti, Dwi

The potential application of biosurfactants to remove heavy metal ion from aqueous solution by batch technique was examined. The glycolipids type biosurfactants were grown in a media containing of 20% v/v corn oil with 7 days of fermentation by Rhodococcus rhodochrous. The biosurfactants reduced the surface tension of water of about 51% from 62 mN/m to 30 mN/m. The biosurfactant increased the E24 of water-palm oil emulsion of about 55% from 43% to 97% and could maintain this E24 value of above 50% for up to 9 days. Heavy metal ion removal, in this case cadmium ion, by crude andmore » patially purified biosurfactants has been investigated from aqueous solution at pH 6. Adsorption capacity of Cd(II) ion by crude biosurfactant with 5 and 10 minutes of contact times were 1.74 and 1.82 mg/g, respectively. Additionally, the adsorption capacity of Cd(II) ion by partially purified biosurfactant with 5 and 10 minutes of contact times were 0.79 and 1.34 mg/g, respectively. The results demonstrated that the adsorption capacity of Cd(II) ion by crude biosurfactant was higher than that of by partially purified biosurfactant. The results suggested that the biosurfactant could be used in the removal of heavy metal ions from aqueous solution.« less

Evaluation of biosurfactants grown in corn oil by Rhodococcus rhodochrous on removing of heavy metal ion from aqueous solution

NASA Astrophysics Data System (ADS)

Suryanti, Venty; Hastuti, Sri; Pujiastuti, Dwi

2016-02-01

The potential application of biosurfactants to remove heavy metal ion from aqueous solution by batch technique was examined. The glycolipids type biosurfactants were grown in a media containing of 20% v/v corn oil with 7 days of fermentation by Rhodococcus rhodochrous. The biosurfactants reduced the surface tension of water of about 51% from 62 mN/m to 30 mN/m. The biosurfactant increased the E24 of water-palm oil emulsion of about 55% from 43% to 97% and could maintain this E24 value of above 50% for up to 9 days. Heavy metal ion removal, in this case cadmium ion, by crude and patially purified biosurfactants has been investigated from aqueous solution at pH 6. Adsorption capacity of Cd(II) ion by crude biosurfactant with 5 and 10 minutes of contact times were 1.74 and 1.82 mg/g, respectively. Additionally, the adsorption capacity of Cd(II) ion by partially purified biosurfactant with 5 and 10 minutes of contact times were 0.79 and 1.34 mg/g, respectively. The results demonstrated that the adsorption capacity of Cd(II) ion by crude biosurfactant was higher than that of by partially purified biosurfactant. The results suggested that the biosurfactant could be used in the removal of heavy metal ions from aqueous solution.

Stability studies of saponins in Bacopa monnieri dried ethanolic extracts.

PubMed

Phrompittayarat, Watoo; Wittaya-areekul, Sakchai; Jetiyanon, Kanchalee; Putalun, Waraporn; Tanaka, Hiroyuki; Ingkaninan, Kornkanok

2008-11-01

Bacopa monnieri (L.) Wettst. (Brahmi) is currently used as a drug and food supplement for memory improvement. However, studies on the physical and chemical stability of the extract components, especially on the lead compound important for pre-formulation, have not yet been reported. In this study, the stabilities of the crude extract and the diluted crude extract were investigated at various temperatures using saponin glycosides, bacopaside I and bacoside A3 as markers for quantitative analysis. The stability testing of bacopaside I and bacoside A3 standard solution was performed at various temperatures and pH values. The quantity of both compounds under all conditions was analyzed using HPLC techniques. The moisture adsorption of the crude extract was determined at 5, 40, 60 and 80 degrees C at 75 % relative humidity using gravimetric methods. The results revealed that the crude extract quickly adsorbed moisture up to 54 % w/w at both 40 and 80 degrees C, while it only slowly adsorbed moisture at 5 degrees C. The amounts of intact bacopaside I and bacoside A3 in the crude extract decreased drastically at 80 degrees C, slowly at 40 and 60 degrees C, and remained unchanged at 5 degrees C during the period of investigation. Moreover, the amount of both compounds in the standard solution dropped sharply at a pH of 1.2 but slowly at pH 6.8 and 9.0, respectively. The pre-formulation data could be further used for improvement of the final product quality.

Guaiacol Peroxidase Zymography for the Undergraduate Laboratory

ERIC Educational Resources Information Center

Wilkesman, Jeff; Castro, Diana; Contreras, Lellys M.; Kurz, Liliana

2014-01-01

This laboratory exercise presents a novel way to introduce undergraduate students to the specific detection of enzymatic activity by electrophoresis. First, students prepare a crude peroxidase extract and then analyze the homogenate via electrophoresis. Zymography, that is, a SDS-PAGE method to detect enzyme activity, is used to specifically…

Effects of dietary inclusion of high concentrations of crude glycerin on meat quality and fatty acid profile of feedlot fed Nellore bulls

PubMed Central

D'Áurea, André P.; Fávaro, Vanessa R.; van Cleef, Flavia O. S.; Barducci, Robson S.; Almeida, Marco T. C.; Machado Neto, Otávio R.; Ezequiel, Jane M. B.

2017-01-01

Crude glycerin, the main by-product of biodiesel production, can replace dietary energy sources, such as corn. The objective of this study was to evaluate the inclusion of up to 30% of crude glycerin in dry matter (DM) of the total diets, and its effects on meat quality parameters of feedlot Nellore bulls. Thirty animals (227.7 ± 23.8 kg body weight; 18 months old) were housed in individual pens and fed 5 experimental diets, containing 0, 7.5, 15, 22.5 or 30% crude glycerin (DM basis). After 103 d (21 d adaptation) animals were slaughtered and the Longissimus muscle was collected. The characteristics assessed were chemical composition, fatty acid profile, cholesterol, shear force, pH, color, water-holding capacity, cooking loss and sensory properties. The increasing inclusion of crude glycerin in the diets did not affect the chemical composition of the Longissimus muscle (P > 0.10). A quadratic effect was observed when levels of crude glycerin were increased, on the concentration of pentadecanoic, palmitoleic and eicosenoic fatty acids in meat (P < 0.05), and on the activity of the delta-9 desaturase 16 and delta-9 desaturase 18 enzymes (P < 0.05). The addition of crude glycerin increased the gamma linolenic fatty acid concentration (P < 0.01), and altered the monounsaturated fatty acids in Longissimus muscle of animals (Pquad. < 0.05). Crude glycerin decreased cholesterol content in meat (P < 0.05), and promoted higher flavor score and greasy intensity perception of the meat (P < 0.01). The inclusion of up to 30% crude glycerin in Nellore cattle bulls`diets (DM basis) improves meat cholesterol and sensory attributes, such as flavor, without affecting significantly the physical traits, the main fatty acid concentrations and the chemical composition. PMID:28644883

High pressure processing of fresh seafoods.

PubMed

Simpson, B K

1998-01-01

Crude proteolytic enzyme extracts were prepared from the muscle tissues of two fish species, bluefish and sheephead, and subjected to high hydrostatic pressure treatments (from 1,000-3,000 atm), and monitored for residual activity for cathepsin C, collagenase, chymotrypsin-like and trypsin-like enzymes versus homologous enzymes from bovine. The fish enzymes were more sensitive to hydrostatic pressure than the mammalian enzymes. The extent of enzyme inactivation achieved depended on both the amount of pressure applied, the duration of pressurization, and on the source material. Pressure treatment of fresh fish flesh formed products whose color deteriorated (cooked appearance) with increasing pressure as well as holding time. Application of pressure also improved tissue firmness or strength of fresh fish up to 2,000 atm and a holding time of 10 min, beyond which texture generally deteriorated. The combined use of pressure in combination with the broad spectrum protease inhibitor, alpha 2-macroglobulin, enhanced the capacity of the hydrostatic pressure technology to achieve a more lasting inactivation of endogenous enzymes to form stable fish gels.

Characterization of PepB, a group B streptococcal oligopeptidase.

PubMed Central

Lin, B; Averett, W F; Novak, J; Chatham, W W; Hollingshead, S K; Coligan, J E; Egan, M L; Pritchard, D G

1996-01-01

Group B streptococci were recently reported to possess a cell-associated collagenase. Although the enzyme hydrolyzed the synthetic collagen-like substrate N-(3-[2-furyl]acryloyl)-Leu-Gly-Pro-Ala, we found that neither the highly purified enzyme nor crude group B streptococcal cell lysate solubilized a film of reconstituted rat tail collagen, an activity regarded as obligatory for a true collagenase. We cloned and sequenced the gene for the enzyme (pepB). The deduced amino acid sequence showed 66.4% identity to the PepF oligopeptidase from Lactococcus lactis, a member of the M3 or thimet family of zinc metallopeptidases. The group B streptococcal enzyme also showed oligopeptidase activity and degraded a variety of small bioactive peptides, including bradykinin, neurotensin, and peptide fragments of substance P and adrenocorticotropin. PMID:8757883

Characterization of chitinases of polycentric anaerobic rumen fungi.

PubMed

Novotná, Z; Fliegerová, K; Simůnek, J

2008-01-01

Chitinolytic systems of anaerobic polycentric rumen fungi of genera Orpinomyces and Anaeromyces were investigated in three crude enzyme fractions - extracellular, cytosolic and cell-wall. Endochitinase was found as a dominant enzyme with highest activity in the cytosolic fraction. Endochitinases of both genera were stable at pH 4.5-7.0 with optimum at 6.5. The Orpinomyces endochitinase was stable up to 50 degrees C with an optimum for enzyme activity at 50 degrees C; similarly, Anaeromyces endochitinase was stable up to 40 degrees C with optimum at 40 degrees C. The most suitable substrate for both endochitinases was fungal cell-wall chitin. Enzyme activities were inhibited by Hg(2+) and Mn(2+), and activated by Mg(2+) and Fe(3+). Both endochitinases were inhibited by 10 mmol/L SDS and activated by iodoacetamide.

Immobilization of indigenous holocellulase on iron oxide (Fe2O3) nanoparticles enhanced hydrolysis of alkali pretreated paddy straw.

PubMed

Kumar, Ajay; Singh, Surender; Tiwari, Rameshwar; Goel, Renu; Nain, Lata

2017-03-01

The holocellulase from Aspergillus niger SH3 was characterized and found to contain 125 proteins including cellulases (26), hemicellulases (21), chitinases (10), esterases (6), amylases (4) and hypothetical protein (32). The crude enzyme was immobilized on five different nanoparticles (NPs) via physical adsorption and covalent coupling methods. The enzyme-nanoparticle complexes (ENC) were screened for protein binding, enzymatic activities and immobilization efficiency. Magnetic enzyme-nanoparticle complexes (MENC) showed higher immobilization efficiency (60-80%) for most of the enzymes. MENC also showed better catalytic efficiencies in term of higher V max and lower K m than free enzyme. Saccharification yields from alkali treated paddy straw were higher (375.39mg/gds) for covalently immobilized MENC than free enzyme (339.99mg/gds). The immobilized enzyme was used for two cycles of saccharification with 55% enzyme recovery. Hence, this study for the first time demonstrated the immobilization of indigenous enzyme and its utilization for saccharification of paddy straw. Copyright © 2016 Elsevier B.V. All rights reserved.

Highly Stable l-Lysine 6-Dehydrogenase from the Thermophile Geobacillus stearothermophilus Isolated from a Japanese Hot Spring: Characterization, Gene Cloning and Sequencing, and Expression

PubMed Central

Heydari, Mojgan; Ohshima, Toshihisa; Nunoura-Kominato, Naoki; Sakuraba, Haruhiko

2004-01-01

l-Lysine dehydrogenase, which catalyzes the oxidative deamination of l-lysine in the presence of NAD, was found in the thermophilic bacterium Geobacillus stearothermophilus UTB 1103 and then purified about 3,040-fold from a crude extract of the organism by using four successive column chromatography steps. This is the first report showing the presence of a thermophilic NAD-dependent lysine dehydrogenase. The product of the enzyme catalytic activity was determined to be Δ1-piperideine-6-carboxylate, indicating that the enzyme is l-lysine 6-dehydrogenase (LysDH) (EC 1.4.1.18). The molecular mass of the purified protein was about 260 kDa, and the molecule was determined to be a homohexamer with subunit molecular mass of about 43 kDa. The optimum pH and temperature for the catalytic activity of the enzyme were about 10.1 and 70°C, respectively. No activity was lost at temperatures up to 65°C in the presence of 5 mM l-lysine. The enzyme was relatively selective for l-lysine as the electron donor, and either NAD or NADP could serve as the electron acceptor (NADP exhibited about 22% of the activity of NAD). The Km values for l-lysine, NAD, and NADP at 50°C and pH 10.0 were 0.73, 0.088, and 0.48 mM, respectively. When the gene encoding this LysDH was cloned and overexpressed in Escherichia coli, a crude extract of the recombinant cells had about 800-fold-higher enzyme activity than the extract of G. stearothermophilus. The nucleotide sequence of the LysDH gene encoded a peptide containing 385 amino acids with a calculated molecular mass of 42,239 Da. PMID:14766574

Highly stable L-lysine 6-dehydrogenase from the thermophile Geobacillus stearothermophilus isolated from a Japanese hot spring: characterization, gene cloning and sequencing, and expression.

PubMed

Heydari, Mojgan; Ohshima, Toshihisa; Nunoura-Kominato, Naoki; Sakuraba, Haruhiko

2004-02-01

L-Lysine dehydrogenase, which catalyzes the oxidative deamination of L-lysine in the presence of NAD, was found in the thermophilic bacterium Geobacillus stearothermophilus UTB 1103 and then purified about 3,040-fold from a crude extract of the organism by using four successive column chromatography steps. This is the first report showing the presence of a thermophilic NAD-dependent lysine dehydrogenase. The product of the enzyme catalytic activity was determined to be Delta1-piperideine-6-carboxylate, indicating that the enzyme is L-lysine 6-dehydrogenase (LysDH) (EC 1.4.1.18). The molecular mass of the purified protein was about 260 kDa, and the molecule was determined to be a homohexamer with subunit molecular mass of about 43 kDa. The optimum pH and temperature for the catalytic activity of the enzyme were about 10.1 and 70 degrees C, respectively. No activity was lost at temperatures up to 65 degrees C in the presence of 5 mM L-lysine. The enzyme was relatively selective for L-lysine as the electron donor, and either NAD or NADP could serve as the electron acceptor (NADP exhibited about 22% of the activity of NAD). The Km values for L-lysine, NAD, and NADP at 50 degrees C and pH 10.0 were 0.73, 0.088, and 0.48 mM, respectively. When the gene encoding this LysDH was cloned and overexpressed in Escherichia coli, a crude extract of the recombinant cells had about 800-fold-higher enzyme activity than the extract of G. stearothermophilus. The nucleotide sequence of the LysDH gene encoded a peptide containing 385 amino acids with a calculated molecular mass of 42,239 Da.

Subcellular distribution and activation by non-ionic detergents of guanylate cyclase in cerebral cortex of rat.

PubMed

Deguchi, T; Amano, E; Nakane, M

1976-11-01

Non-ionic detergents stimulated particulate guanylate cyclase activity in cerebral cortex of rat 8- to 12-fold while stimulation of soluble enzyme was 1.3- to 2.5-fold. Among various detergents, Lubrol PX was the most effective one. The subcellular distribution of guanylate cyclase activity was examined with or without 0.5% Lubrol PX. Without Lubrol PX two-thirds of the enzyme activity was detected in the soluble fraction. In the presence of Lubrol PX, however, two-thirds of guanylate cyclase activity was recovered in the crude mitochondrial fraction. Further fractionation revealed that most of the particulate guanylate cyclase activity was associated with synaptosomes. The sedimentation characteristic of the particulate guanylate cyclase activity was very close to those of choline acetyltransferase and acetylcholine esterase activities, two synaptosomal enzymes. When the crude mitochondrial fraction was subfractionated after osmotic shock, most of guanylate cyclase activity as assayed in the absence of Lubrol PX was released into the soluble fraction while the rest of the enzyme activity was tightly bound to synaptic membrane fractions. The total guanylate cyclase activity recovered in the synaptosomal soluble fraction was 6 to 7 times higher than that of the starting material. The specific enzyme activity reached more than 1000 pmol per min per mg protein, which was 35-fold higher than that of the starting material. The membrane bound guanylate cyclase activity was markedly stimulated by Lubrol PX. Guanylate cyclase activity in the synaptosomal soluble fraction, in contrast, was suppressed by the addition of Lubrol PX. The observation that most of guanylate cyclase activity was detected in synaptosomes, some of which was tightly bound to the synaptic membrane fraction upon hypoosmotic treatment, is consistent with the concept that cyclic GMP is involved in neural transmission.

Isolation, purification, and characterization of AgUCGalT1, a galactosyltransferase involved in anthocyanin galactosylation in purple celery (Apium graveolens L.).

PubMed

Feng, Kai; Xu, Zhi-Sheng; Liu, Jie-Xia; Li, Jing-Wen; Wang, Feng; Xiong, Ai-Sheng

2018-06-01

This study showed that a galactosyltransferase, AgUCGalT1, is involved in anthocyanin galactosylation in purple celery. Celery is a well-known vegetable because of its rich nutrients, low calories, and medicinal values. Its petioles and leaf blades are the main organs acting as nutrient sources. UDP-galactose: cyanidin 3-O-galactosyltransferase can transfer the galactosyl moiety from UDP-galactose to the 3-O-position of cyanidin through glycosylation. This process enhances the stability and water solubility of anthocyanins. In the present study, LC-MS data indicated that abundant cyanidin-based anthocyanins accumulated in the petioles of purple celery ('Nanxuan liuhe purple celery'). A gene encoding UDP-galactose: cyanidin 3-O-galactosyltransferase, namely AgUCGalT1, was isolated from purple celery and expressed in Escherichia coli BL21 (DE3). Sequence alignments revealed that the AgUCGalT1 protein contained a highly conserved putative secondary plant glycosyltransferase (PSPG) motif. The glycosylation product catalyzed by AgUCGalT1 was detected using UPLC equipment. The recombinant AgUCGalT1 had an optimal enzyme activity at 35 °C and pH 8.0, and showed highest enzyme activity toward cyanidin among the enzyme activities involving other substances, namely, peonidin, quercetin, and kaempferol. The expression levels of AgUCGalT1 were positively correlated with the total anthocyanin contents in purple and non-purple celery varieties. Crude enzymes extracted from purple celery exhibited glycosylation ability, whereas crude enzymes obtained from non-purple celery did not have this ability. This work provided evidence as a basis for investigations on the function of AgUCGalT1 in anthocyanin glycosylation in purple celery.

Breakthrough Adsorption Study of Crude Oil Removal Using Buffing Dust

NASA Astrophysics Data System (ADS)

Setyaningsih, L. W. N.; Yuliansyah, A. T.; Prasetyo, A.; Arimanintan, S. K.; Putri, D. R.

2018-05-01

The utilization of leather industry solid waste as adsorbent to separate oil from water emulsions of surfactant flooding process is a solution that is relatively inexpensive. This study was conducted aiming to obtain a mathematical model that is appropriate for the adsorption process of crude oil by buffing dust in emulsion phase with a continuous adsorption method. Variations in the column adsorption experiments were carried out, such as: flow rate of feed of water-crude oil-surfactant, the concentration of crude oil in the feed, and mass of adsorbent used. Data were evaluated using three models: Adams Bohart, Thomas and Yan. Best results are obtained on the following conditions, the feed flow rate of 60 mL/minute, the crude oil concentration in feed is 1.5% volume and the mass of adsorbent used was 10 g. The values of kinetic constant and adsorption capacity obtained from Yan Model was 21.7774 mL/mg/minute and 220.9581 mg/g with the relative error obtained is 5.4424%.

Perspective for using Nd-Fe-B magnets as a tool for the improvement of the production and transportation of Vietnamese crude oil with high paraffin content

NASA Astrophysics Data System (ADS)

Tung, N. P.; Vinh, N. Q.; Phong, N. T. P.; Long, B. Q. K.; Hung, P. V.

2003-04-01

Controlling the paraffin deposition is one of the most serious and costly problems for high-paraffin crude-oil production and transportation. This holds especially for offshore, deep-water wells and pipelines, like in Vietnam. For finding solutions for these problems, a series of experiments has been performed in order to study the effects of a magnetic field on the rheology of two often produced crude oil samples, using the high and variable magnetic-flux density of a Nd-Fe-B magnet. The resulting changes in the essential characteristics of the magnetically treated (MT) crude oils, like form and size of the paraffin clusters as well as the rheology parameters, have definitely proved the possibility to use a Nd-Fe-B magnetic tool as an environmentally friendly and effective method to control the paraffin deposition in crude oils of Vietnam that have a high paraffin content.

Studies on the enzymes of Sarcocystis suicanis: purification and characterization of an acid phosphatase.

PubMed

Farooqui, A A; Adams, D D; Hanson, W L; Prestwood, A K

1987-08-01

Percoll density gradient centrifugation was used for isolating large quantities of bradyzoites of Sarcocystis suicanis, which were used for enzymatic analysis. Crude extracts of bradyzoites contained activities suggestive of several acid hydrolases. Levels of acid and alkaline phosphatase were higher than those of beta-N-acetylhexosaminidase and beta-galactosidase. Acid phosphatase was purified 156-fold with an overall recovery of 54% using DEAE-Sepharose 4B and Sephadex G-200 chromatography. The partially purified enzyme was not a glycoprotein and had a molecular weight of approximately 170,000. The enzyme was markedly inhibited by Cu++, Hg++, and iodoacetamide, suggesting the presence of a sulfhydryl group. Sodium tartrate caused strong inhibition of the enzyme. The acid phosphatase of S. suicanis appears to be a unique enzyme that cannot be classified under high or low molecular weight acid phosphatases of widely diverse origin.

Metabolic Capabilities of the Members of the Order Halanaerobiales and Their Potential Biotechnological Applications

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

Roush, Daniel W; Elias, Dwayne A; Mormile, Dr. Melanie R.

The order Halanaerobiales contains a number of well-studied halophiles that possess great potential for biotechnological applications. The unique halophilic adaptations that these organisms utilize, such as salting-in mechanisms to increase their intercellular concentration of KCl, combined with their ability to ferment simple sugars, provides an excellent platform for biotechnological development over a wide range of salt levels and possible other extreme conditions, such as alkaline conditions. From fermented foods to oil reservoirs, members of Halanaerobiales are found in many environments. The environmental conditions many of these organisms grow are similar to industrially important processes, such as alkaline pre-treated biomass stocks,more » treatment of crude glycerol from biodiesel production, salty fermented foods, as well as bioremediation of contaminants under extreme conditions of salinity and in some cases, alkalinity. From salt stable enzymes to waste fermentations, bioremediation options, bioenergy, and microbially enhanced oil recovery (MEOR), Halanaerobiales can provide a wide spectrum of environmentally friendly solutions to current problems.« less

Poly(N-Isopropylacrylamide)-co-Acrylamide Hydrogels for the Controlled Release of Bromelain from Agroindustrial Residues of Ananas comosus.

PubMed

Croisfelt, Fernanda; Martins, Bianca C; Rescolino, Robson; Coelho, Diego F; Zanchetta, Beatriz; Mazzola, Priscila G; Goulart, Luis Ricardo; Pessoa, Adalberto; Tambourgi, Elias B; Silveira, Edgar

2015-12-01

This works reports the purification of bromelain extracted from Ananas comosus industrial residues by ethanol purification, its partial characterization from the crude extract as well as the ethanol purified enzyme, and its application onto poly(N-isopropylacrylamide)-co-acrylamide hydrogels. Bromelain was recovered within the 30-70 % ethanol fraction, which achieved a purification factor of 3.12-fold, and yielded more than 90 % of its initial activity. The resulting purified bromelain contained more than 360 U · mg(-1), with a maximum working temperature of 60 °C and pH of 8.0. Poly(N-isopropylacrylamide)-co-acrylamide hydrogels presented a swelling rate of 125 %, which was capable of loading 56 % of bromelain from the solution, and was able to release up to 91 % of the retained bromelain. Ethanol precipitation is suitable for bromelain recovery and application onto poly(N-isopropylacrylamide)-co-acrylamide hydrogels based on its processing time and the applied ethanol prices. Georg Thieme Verlag KG Stuttgart · New York.

An Ultra-Sensitive Monoclonal Antibody-Based Competitive Enzyme Immunoassay for Sterigmatocystin in Cereal and Oil Products

PubMed Central

Li, Min; Li, Peiwu; Wu, Hui; Zhang, Qi; Ma, Fei; Zhang, Zhaowei; Ding, Xiaoxia; Wang, Hengling

2014-01-01

Sterigmatocystin (STG), a biosynthesis precursor of aflatoxin B1, is well known for its toxic and carcinogenic effects in humans and animals. STG derivatives and protein conjugates are needed for generation of monoclonal antibodies (mAbs). This work describes a reliable and fast synthesis of novel STG derivatives, based on which novel STG bovine serum albumin conjugates were prepared. With the novel STG bovine serum albumin conjugates, three sensitive and specific mAbs against STG, named VerA 3, VerA 4, and VerA 6, were prepared by semi-solid hypoxanthine/aminopterin/thymidine (HAT) medium using a modified two-step screening procedure. They exhibited high affinity for STG and no cross-reactivity (CR) with aflatoxins B1, B2, G1, G2, and M1. Based on the most sensitive antibody VerA 3, an ultra-sensitive competitive enzyme-linked immunosorbent assay (ELISA) was developed for STG in wheat, maize, and peanuts. Assays were performed in the STG-GA-BSA-coated (0.5 µg·mL−1) ELISA format, in which the antibody was diluted to 1∶80,000. Several physicochemical factors influencing assay performance, such as pH, ionic strength, blocking solution, and diluting solution, were optimized. The final results showed that the assays had the detection limits of 0.08 ng·g−1 for wheat, 0.06 ng·g−1 for maize, and 0.1 ng·g−1 for peanuts, inter-assay and intra-assay variations of less than 10%, and recoveries ranging from 83% to 110%. These recoveries were in good agreement with those obtained by using HPLC-MS/MS method (90–104%), indicating the importance of the mAb VerA 3 in the study of STG in crude agricultural products. PMID:25184275

EXTRACTION AND PHYSICO-CHEMICAL STUDIES OF DIASTASE-LIKE ENZYME FROM PIPER BETLE PETIOLES: PART 1

PubMed Central

Ramasarma, G.V.S; Dutta, Sadhan Kumar

1995-01-01

Petioles of the plant piper betle-bengal variety have been subjected for extraction employing standard procedure and the crude extract obtained has been evaluated for its diastase like activity and other physico-chemical properties to investigate further its possible biological and pharmacological activities. PMID:22556729

Optimization of liquid fermentation of microbial consortium WSD-5 followed by saccharification and acidification of wheat straw.

PubMed

Wen, Boting; Yuan, Xufeng; Cao, Yanzhuan; Liu, Yan; Wang, Xiaofen; Cui, Zongjun

2012-08-01

The microbial consortium WSD-5 is composed of bacteria and fungi, and the cooperation and symbiosis of the contained microbes enhance the degradation ability of WSD-5. Experiment results showed that the highest cellulase and hemicellulase were obtained when ventilation volume was 4 L/min, stirring rate was 0 rpm, and substrate loading rate was 3%. After 6 days of cultivation, a 67.60% loss in wheat straw dry weight was observed. The crude enzyme secreted from WSD-5 after optimization was evaluated by experiments of saccharification and acidification. The maximum concentration of reducing sugars was 3254 mg/L after 48 h saccharification. The concentration of sCOD peaked on day 2 with a value of 4345 mg/L during acidification, and the biogas yield and methane yield were 22.3% and 32.3% higher than un-acidified samples. This study is the first attempt to explore both the saccharification and the acidification ability of crude enzymes secreted by microbial consortium. Copyright © 2012 Elsevier Ltd. All rights reserved.

Purification and characterization of a milk-clotting aspartic proteinase from globe artichoke (Cynara scolymus L.).

PubMed

Llorente, Berta E; Brutti, Cristina B; Caffini, Néstor O

2004-12-29

The study of proteinase expression in crude extracts from different organs of the globe artichoke (Cynara scolymus L.) disclosed that enzymes with proteolytic and milk-clotting activity are mainly located in mature flowers. Maximum proteolytic activity was recorded at pH 5.0, and inhibition studies showed that only pepstatin, specific for aspartic proteinases, presented a significant inhibitory effect. Such properties, in addition to easy enzyme inactivation by moderate heating, make this crude protease extract potentially useful for cheese production. Adsorption with activated carbon, together with anion exchange and affinity chromatography, led to the isolation of a heterodimeric milk-clotting proteinase consisting of 30- and 15-kDa subunits. MALDI-TOF MS of the 15-kDa chain determined a 15.358-Da mass, and the terminal amino sequence presented 96% homology with the smaller cardosin A subunit. The amino terminal sequence of the 30-kDa chain proved to be identical to the larger cardosin A subunit. Electrophoresis evidenced proteinase self-processing that was confirmed by immunoblots presenting 62-, 30-, and 15-kDa bands.

Purification of inulinases by changing the ionic strength of the medium and precipitation with alcohols.

PubMed

Golunski, Simone; Silva, Marceli F; Marques, Camila T; Rosseto, Vanusa; Kaizer, Rosilene R; Mossi, Altemir J; Rigo, Diane; Dallago, Rogério M; DI Luccio, Marco; Treichel, Helen

2017-01-01

The present study evaluated the purification of inulinase by changing the ionic strength of the medium by addition of NaCl and CaCl2 followed by precipitation with n-propyl alcohol or iso-propyl alcohol. The effects of the concentration of alcohols and the rate of addition of alcohols in the crude extract on the purification yield and purification factor were evaluated. Precipitation caused an activation of enzyme and allowed purification factors up to 2.4-fold for both alcohols. The purification factor was affected positively by the modification of the ionic strength of the medium to 0.5 mol.L-1 NaCl before precipitation with the alcohol (n-propyl or iso-propyl). A purification factor of 4.8-fold and an enzyme yield of 78.1 % could be achieved by the addition of 0.5 mol.L-1 of NaCl to the crude extract, followed by the precipitation with 50 % (v/v) of n-propyl alcohol, added at a flow rate of 19.9 mL/min.

Chronic effects of the Exxon Valdez oil spill on blood and enzyme chemistry of river otters

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

Duffy, L.K.; Bowyer, R.T.; Testa, J.W.

1994-04-01

River otters (Lutra canadensis) living in marine environments of Prince William Sound, Alaska, and exposed to crude oil from the Exxon Valdez spill in March 1989 showed elevated levels of blood haptoglobins, and interleukin-6 ir, as well as elevated activities of aspartate aminotransferase, alanine aminotransferase, and creatine kinase in summer 1991. Stepwise logistic regression, using a subset of these and other blood proteins and enzyme activities as potential independent variables, correctly classified 86.4% of 22 otters as inhabiting oiled or nonoiled areas. River otters abandoned latrine sites (an index to their abundance) over three times more often in oiled thanmore » in nonoiled areas, suggesting there may have been a delayed response in river otter populations to exposure to crude oil. This is the first clear model for the long-term effects of an oil spill on blood parameters of a free-ranging mammal using a nonlethal methodology. These effects occurred two years after the spill and following a major effort to clean oil from the shorelines of Prince William Sound.« less

Expression of a functional cold active β-galactosidase from Planococcus sp-L4 in Pichia pastoris.

PubMed

Mahdian, Seyed Mohammad Amin; Karimi, Ehsan; Tanipour, Mohammad Hossein; Parizadeh, Seyed Mohammad Reza; Ghayour-Mobarhan, Majid; Bazaz, Mojtaba Mousavi; Mashkani, Baratali

2016-09-01

Lactase deficiency problem discourages many adults from consuming milk as a major source of micro- and macronutrients. Enzymatic hydrolysis of lactose is an ideal solution for this problem but such processing adds significant costs. In this study, a cold active β-galactosidase from Planococcus sp-L4 (bgal) was optimized for expression of recombinant "BGalP" in Pichia pastoris. As a result of codon optimization, the codon adaptation index was improved from 0.58 to 0.85 after replacing rare codons. After transformation of two P. pastoris strains (KM71H and GS115), the activity of BGalP enzyme was measured in the culture supernatants using ortho-Nitrophenyl-β-galactoside (ONPG). Maximal activity was recorded as 3.7U/ml on day 11 in KM71H clone #2 which was 20% higher than the best GS115 clone. Activity measurements under different conditions indicated optimal activity at pH 6.5. It was active at temperatures ranging from 0 to 55°C with deactivation occurring at or above 60°C. Protein analysis of the crude ultra-filtrate showed the enzyme was ∼75kDa and was the major constituent (85%) of the sample. This enzyme have the potential to find utility for the breakdown of lactose in chilled milk and subsequently can be deactivated by pasteurization. The use of BGalP would minimize energy consumption thus decreasing cost and also help to preserve the nutritional elements of the milk. Copyright © 2015 Elsevier Inc. All rights reserved.

Effect of soaking in water and rumen digeta solutions on metabolizable energy content and chemical composition of barley seeds for use in poultry diet.

PubMed

Tabatabee, S N; Sadeghi, G H; Tabeidian, S A

2007-03-15

An experiment was carried out to evaluate the effect of soaking in water and different rumen digesta solutions on nutritional value of dry barley seeds. Treatments were included distilled water as control and rumen digesta that diluted with distilled water to obtain 20, 40 and 60% digesta solutions. Solutions have added to 10 kg of barley seed samples to achieve final 30% moisture content. After 21 days the chemical composition and energy content of barley seed were determined. Gross energy of barley seeds did not affected by different experimental treatments. Use of 20% rumen digesta solution resulted to a significant (p<0.01) increase in AME and AMEn content of barley seeds. Barley seed that treated with 40% of rumen digesta solution had highest TME and TMEn content and its different from seeds that treated with 60 and 100% rumen digesta solutions was significant (p<0.05). The chemical composition such as dry matter, crud protein, crude fat, crud fiber, ash and NFE were found to be similar and there was no significant difference. However, soaking in rumen digesta solutions increased crud protein, ether extract, crude fiber and ash content of barley seeds numerically.

[Characterization of haemolysis of the Vibrio parahaemolyticus no.93].

PubMed

Su, S C; Lee, C Y

1997-02-01

Vibrio parahaemolyticus is a causative bacterium of food poisoning, and the haemolysin produced by this organism has been considered as one of the important virulence factors. In order to understand the pathogenic mechanism of this bacterium, the characteristics of haemolysin from Vibrio parahaemolyticus isolated from Taiwan were studied. One of the clinical strains, V. parahaemolyticus No.93, presents a weak hemolytic zone on 7% NaCl-Wagatsuma medium. The DNA hybridization results show that V. parahemolyticus has neither tdh nor trh gene. V. parahaemolyticus No.93 shows obviously hemolytic zone on 3%-NaCl Wagatsuma medium (human blood). The crude extracellular protein of V. parahaemolyticus No. 93 was evaluated for its heat tolerance and enzyme activities by media assay. The results show that this crude extracellular protein is thermolabile. The crude extracellular protein of V. parahaemolyticus No.93 was analyzed on 10% SDS-PAGE and an apparent band of 64 kDa protein was observed. Furthermore, the crude extracellular protein was analyzed by running gelatin-SDS-PAGE and hemoglobin-SDS-PAGE, and three clear zones on 62 kDa, 52 kDa and 41 kDa were observed on both SDS-PAGEs. Thus we propose that the crude extracellular protein of the V. parahaemolyticus No.93 can degrade gelatin as well as hemoglobin. Whether these protease being the virulence factors of Vibrio parahaemolyticus No.93 needs to be further studied.

Production, isolation, and purification of L-asparaginase from Pseudomonas aeruginosa 50071 using solid-state fermentation.

PubMed

El-Bessoumy, Ashraf A; Sarhan, Mohamed; Mansour, Jehan

2004-07-31

The L-asparaginase (E. C. 3. 5. 1. 1) enzyme was purified to homogeneity from Pseudomonas aeruginosa 50071 cells that were grown on solid-state fermentation. Different purification steps (including ammonium sulfate fractionation followed by separation on Sephadex G-100 gel filtration and CM-Sephadex C50) were applied to the crude culture filtrate to obtain a pure enzyme preparation. The enzyme was purified 106-fold and showed a final specific activity of 1900 IU/mg with a 43% yield. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of the purified enzyme revealed it was one peptide chain with M(r) of 160 kDa. A Lineweaver-Burk analysis showed a K(m) value of 0.147 mM and V(max) of 35.7 IU. The enzyme showed maximum activity at pH 9 when incubated at 37 degrees C for 30 min. The amino acid composition of the purified enzyme was also determined.

Preliminary studies on immobilization of lipase using chicken eggshell

NASA Astrophysics Data System (ADS)

Salleh, S.; Serri, N. A.; Hena, S.; Tajarudin, H. A.

2016-06-01

A few advantages of enzyme immobilization are reusability of expensive enzyme, improvement of stability and activity compared to crude enzyme. Various organic components can be used as carrier for enzyme immobilization such as chicken eggshell. It can be used as a carrier for immobilization as its mineral component mostly contains of calcium carbonate. In the present study, Tributyrin method was used to test enzyme activity of Rhizomucour Miehei, Candida Antarctica and Candida Rugosa. Rhizomucour Miehei shows the highest enzyme activity (360.8 mol/min/mL lipase) and was used in further experiment. Experiment was continued to study incubation time for lipase immobilization on eggshell (1-4 hours) and reaction time of esterification of sugar ester (0-72 hours). Two hours incubation time for lipase immobilization was observed and gives the highest yield of sugar ester (78.13%). Fructose and stearic acid as substrate was used for the production of sugar ester. The highest percentage of sugar ester production was shown at 36 hours of reaction time.

Evaluation of a cleanser for petroleum-contaminated skin.

PubMed

Phieffer, Laura S; Banks, David M; Bosse, Michael J; Meyer, Martha H; Meyer, Ralph A; Smith, Kevin

2003-12-01

Extremity injuries contaminated with petroleum products pose clinical dilemmas. This project was designed to evaluate the efficacy of a dioctyl sulfosuccinate (DS) solution for cleansing petroleum-contaminated skin. One hundred Sprague-Dawley rats were subjected to a contamination protocol followed by a cleansing procedure. Four petroleum contaminants and five cleansing solutions were selected. The protocol consisted of shaving, initial punch biopsy, contamination, precleansing punch biopsy, standardized scrub protocol, and postcleansing punch biopsy. Biopsy samples were analyzed for petroleum residue using fluorometry. The 10% DS solution had the highest reduction of crude oil, grease, and tar: 99.6 +/- 0.4% (mean +/- SD) contaminant reduction for crude oil, 99.8 +/- 0.2% for grease, and 99.8 +/- 0.2% for tar. The other cleansers showed less efficacy (p < 0.05). Concentrated DS appears to be significantly more effective at cleaning petroleum products from skin than the commonly chosen surgical and commercial cleansers.

Solids precipitation in crude oils, gas-to-liquids and their blends

NASA Astrophysics Data System (ADS)

Ramanathan, Karthik

Gas-to-liquids (GTL) liquids are obtained from syngas by the Fischer-Tropsch synthesis. The blending of GTL liquids produced from natural gas/coal reserves and crude oils is a possibility in the near future for multiple reasons. Solids precipitation is a major problem in pipelines and refineries leading to significant additional operating costs. The effect of the addition of a paraffinic GTL liquid to crude oils on solids precipitation was investigated in this study. A Fourier transform infrared (FT-IR) spectroscopic technique was used to obtain solid-liquid equilibria (SLE) data for the various samples. The SLE of multiple systems of model oils composed of n-alkanes was investigated preliminarily. Blends of a model oil simulating a GTL liquid composition and a crude oil showed that the wax precipitation temperature (WPT) decreased upon blending. Three crude oils from different geographic regions (Alaskan North Slope, Colorado and Venezuela) and a laboratory-produced GTL liquid were used in the preparation of blends with five different concentrations of the GTL liquid. The wax precipitation temperatures of the blends were found to decrease with the increasing addition of the GTL liquid for all the oils. This effect was attributed to the solvent effect of the low molecular weight-paraffinic GTL liquid on the crude oils. The weight percent solid precipitated that was estimated as a function of temperature did not show a uniform trend for the set of crude oils. The asphaltene onset studies done on the blends with near-infrared spectroscopy indicated that the addition of GTL liquid could have a stabilizing effect on the asphaltenes in some oils. Analytical techniques such as distillation, solvent separation, HPLC, GC, and GPC were used to obtain detailed composition data on the samples. Two sets of compositional data with 49 and 86 pseudo-components were used to describe the three crude oils used in the blending work. The wax precipitation was calculated using a thermodynamic model based on a modified regular solution theory. A study was done to test the sensitivity of the thermodynamic model to varying levels of crude oil characterization input data for a fourth crude oil sample. The differentiation of the solute fraction (C25+) into the normal alkane, non-n-alkane and the aromatic fractions was found to be important for improving the predictive accuracy of the model. The n-alkane and non-n-alkane distribution used in the modeling of wax precipitation for the three crude oils blended with the GTL liquid gave the WPT's that agreed to within 5% of the experimental values. The precipitated solid amounts were overestimated using this method.

Static magnetic field effects on proteases with fibrinolytic activity produced by Mucor subtilissimus.

PubMed

Albuquerque, Wendell; Nascimento, Thiago; Brandão-Costa, Romero; Fernandes, Thiago; Porto, Ana

2017-02-01

The influence of a static magnetic field (SMF) on crude enzyme extracts with proteolytic activity is described and discussed. Proteolytic enzymes, which hydrolyze peptide bonds, and fibrinolytic enzymes, which dissolve fibrin clots, have industrial relevance, and applicability dependent on improvements of productivity and activity. We investigated whether a moderate SMF affects proteolysis in different in vitro tests: general proteolysis of azocasein substrate, and static and dynamic fibrinolytic processes (to compare fibrin gel configuration under exposure). Crude enzyme extracts, obtained from solid state fermentation of Mucor subtilissimus UCP (Universidade Católica de Pernambuco, Recife, Brazil) 1262, were used to carry out assays under slightly heterogeneous fields: a varied vertical SMF (for tests in Eppendorf tubes, from 0.100 to 0.170 T) and a varied horizontal SMF (for tests in Petri dishes, from 0.01 to 0.122 T), generated by two permanent magnets (NdFeB alloy). Results showed significant differences (P < 0.05) in static fibrinolysis assays after 24 h of exposure. The mean diameter of halos of fibrin degradation in the treated group increased by 21% compared to the control group; and the pixel number count of fibrin consumption (in a computational analysis of the area of each halo) enhanced by 30% with exposure. However, in dynamic fibrinolysis assays, no effects of SMF were observed. These results suggest a response of fibrin monomers to the SMF as a possible cause of the observed effects. Bioelectromagnetics. 38:109-120, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Cellulolytic and xylanolytic potential of high β-glucosidase-producing Trichoderma from decaying biomass.

PubMed

Okeke, Benedict C

2014-10-01

Availability, cost, and efficiency of microbial enzymes for lignocellulose bioconversion are central to sustainable biomass ethanol technology. Fungi enriched from decaying biomass and surface soil mixture displayed an array of strong cellulolytic and xylanolytic activities. Strains SG2 and SG4 produced a promising array of cellulolytic and xylanolytic enzymes including β-glucosidase, usually low in cultures of Trichoderma species. Nucleotide sequence analysis of internal transcribed spacer 2 (ITS2) region of rRNA gene revealed that strains SG2 and SG4 are closely related to Trichoderma inhamatum, Trichoderma piluliferum, and Trichoderma aureoviride. Trichoderma sp. SG2 crude culture supernatant correspondingly displayed as much as 9.84 ± 1.12, 48.02 ± 2.53, and 30.10 ± 1.11 units mL(-1) of cellulase, xylanase, and β-glucosidase in 30 min assay. Ten times dilution of culture supernatant of strain SG2 revealed that total activities were about 5.34, 8.45, and 2.05 orders of magnitude higher than observed in crude culture filtrate for cellulase, xylanase, and β-glucosidase, respectively, indicating that more enzymes are present to contact with substrates in biomass saccharification. In parallel experiments, Trichoderma species SG2 and SG4 produced more β-glucosidase than the industrial strain Trichoderma reesei RUT-C30. Results indicate that strains SG2 and SG4 have potential for low cost in-house production of primary lignocellulose-hydrolyzing enzymes for production of biomass saccharides and biofuel in the field.

Identification of the copper-zinc superoxide dismutase activity in Mycoplasma hyopneumoniae.

PubMed

Chen, J R; Weng, C N; Ho, T Y; Cheng, I C; Lai, S S

2000-05-11

Copper-zinc superoxide dismutase (Cu/ZnSOD), a key enzyme in defense against toxic oxygen-free radicals, is widespread in eukaryotes and several species of gram-negative bacteria. The presence of this enzyme in Mycoplasma hyopneumoniae (M. hyopneumoniae), the primary pathogen of mycoplasmal pneumonia in pigs, was examined since the polyclonal antibody against bovine Cu/ZnSOD was dominantly cross-reactive with the M. hyopneumoniae Cu/ZnSOD from whole cellular proteins. In situ activity staining on SDS-PAGE showed that the molecular mass of M. hyopneumoniae Cu/ZnSOD in reducing form was approximately 17kDa. The presence of Cu and Zn ions at the active site of the enzyme was confirmed on the basis of inhibition by KCN and by H(2)O(2). The activity of M. hyopneumoniae Cu/ZnSOD on both SDS- and native-polyacrylamide gels was completely inhibited by 2mM KCN and the gels showed no iron-containing SOD (FeSOD) or manganese-containing SOD (MnSOD) in the crude extracts. The activity of M. hyopneumoniae Cu/ZnSOD in crude extract was 70units/mg protein and was 55% inhibited by 5mM KCN and 56% inactivated by 40mM H(2)O(2). This enzyme was growth-stage dependent and evidenced markedly higher production during the early log phase. Different expression levels of Cu/ZnSOD activity in field isolates were also detected. Taken together, the presence of Cu/ZnSOD in M. hyopneumoniae was identified for the first time.

A novel liquid/liquid extraction process composed of surfactant and acetonitrile for purification of polygalacturonase enzyme from Durio zibethinus.

PubMed

Amid, Mehrnoush; Manap, Yazid; Azmira, Farhana; Hussin, Muhaini; Sarker, Zaidul Islam

2015-07-01

Polygalacturonase is one of the important enzymes used in various industries such as food, detergent, pharmaceutical, textile, pulp and paper. A novel liquid/liquid extraction process composed of surfactant and acetonitrile was employed for the first time to purify polygalacturonase from Durio zibethinus. The influences of different parameters such as type and concentration of surfactants, concentrations of acetonitrile and composition of surfactant/acetonitrile on partitioning behavior and recovery of polygalacturonase was investigated. Moreover, the effect of pH of system and crude load on purification fold and yield of purified polygalacturonase were studied. The results of the experiment indicated the polygalacturonase was partitioned into surfactant top rich phase with impurities being partitioned into acetonitrile bottom rich phase in the novel method of liquid/liquid process composed of 23% (w/w) Triton X-100 and 19% (w/w) acetonitrile, at 55.6% of TLL (tie line length) crude load of 25% (w/w) at pH 6.0. Recovery and recycling of components also was measured in each successive step of liquid/liquid extraction process. The enzyme was successfully recovered by the method with a high purification factor of 14.3 and yield of 97.3% while phase components were also recovered and recycled above 95%. This study demonstrated that the novel method of liquid/liquid extraction process can be used as an efficient and economical extraction method rather than the traditional methods of extraction for the purification and recovery of the valuable enzyme. Copyright © 2015 Elsevier B.V. All rights reserved.

Development of a solid-state fermentation process for production of an alpha amylase with potentially interesting properties.

PubMed

Hashemi, Maryam; Razavi, Seyed Hadi; Shojaosadati, Seyed Abbas; Mousavi, Seyyed Mohammad; Khajeh, Khosro; Safari, Mohammad

2010-09-01

Ca-independency with potential activity and stability at low pH are among the most interesting characteristics of alpha-amylase in starch industry. In this attempt the synergetic effect of low pH on activity of crude Ca-independent alpha-amylase isolated from a native Bacillus sp. KR-8104 in solid-state fermentation (SSF) was studied using wheat bran (WB) as a substrate. The effects of different parameters including moisturizing agents, solid substrate to moisture ratio, particle size, incubation temperature and period, inoculum (v/w) and supplementation with 1% (w/w) different carbon and nitrogen sources on enzyme production were investigated. Maximum enzyme production of 140U/g dry fermented substrate was obtained from wheat bran moistened with tap water at a ratio of 1:1.5 and supplemented with 1% (w/w) NH(4)NO(3) and 1% (w/w) lactose after 48h incubation at 37 degrees C. Even though the production of alpha-amylase was lower at 40 and 45 degrees C, the viable cell count was higher. In addition response surface methodology (RSM) was applied to find optimum conditions of temperature and pH on crude amylase activity. Using central composite design (CCD) a quadratic mathematical model equation was derived for the prediction of enzyme activity. The results showed that the model was in good agreement with experimental results, with R(2)=0.90 (p<0.0001) and the low pH has a synergetic effect on enzyme activity at higher temperature. Copyright 2010 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Catalytical Properties of Free and Immobilized Aspergillus niger Tannase.

PubMed

Flores-Maltos, Abril; Rodríguez-Durán, Luis V; Renovato, Jacqueline; Contreras, Juan C; Rodríguez, Raúl; Aguilar, Cristóbal N

2011-01-01

A fungal tannase was produced, recovered, and immobilized by entrapment in calcium alginate beads. Catalytical properties of the immobilized enzyme were compared with those of the free one. Tannase was produced intracellularly by the xerophilic fungus Aspergillus niger GH1 in a submerged fermentation system. Enzyme was recovered by cell disruption and the crude extract was partially purified. The catalytical properties of free and immobilized tannase were evaluated using tannic acid and methyl gallate as substrates. K(M) and V(max) values for free enzyme were very similar for both substrates. But, after immobilization, K(M) and V(max) values increased drastically using tannic acid as substrate. These results indicated that immobilized tannase is a better biocatalyst than free enzyme for applications on liquid systems with high tannin content, such as bioremediation of tannery or olive-mill wastewater.

Catalytical Properties of Free and Immobilized Aspergillus niger Tannase

PubMed Central

Flores-Maltos, Abril; Rodríguez-Durán, Luis V.; Renovato, Jacqueline; Contreras, Juan C.; Rodríguez, Raúl; Aguilar, Cristóbal N.

2011-01-01

A fungal tannase was produced, recovered, and immobilized by entrapment in calcium alginate beads. Catalytical properties of the immobilized enzyme were compared with those of the free one. Tannase was produced intracellularly by the xerophilic fungus Aspergillus niger GH1 in a submerged fermentation system. Enzyme was recovered by cell disruption and the crude extract was partially purified. The catalytical properties of free and immobilized tannase were evaluated using tannic acid and methyl gallate as substrates. K M and V max values for free enzyme were very similar for both substrates. But, after immobilization, K M and V max values increased drastically using tannic acid as substrate. These results indicated that immobilized tannase is a better biocatalyst than free enzyme for applications on liquid systems with high tannin content, such as bioremediation of tannery or olive-mill wastewater. PMID:21918717

Biological pretreatment of rice straw with Streptomyces griseorubens JSD-1 and its optimized production of cellulase and xylanase for improved enzymatic saccharification efficiency.

PubMed

Zhang, Dan; Luo, Yanqing; Chu, Shaohua; Zhi, Yuee; Wang, Bin; Zhou, Pei

2016-08-17

Biological pretreatment of rice straw and production of reducing sugars by hydrolysis of bio-pretreated material with Streptomyces griseorubens JSD-1 was investigated. After 10 days of incubation, various chemical compositions of inoculated rice straw were degraded and used for further enzymatic hydrolysis studies. The production of cellulolytic enzyme by S. griseorubens JSD-1 favored the conversion of cellulose to reducing sugars. The culture medium for cellulolytic enzyme production by using agro-industrial wastes was optimized through response surface methodology. According to the response surface analysis, the concentrations of 11.13, 20.34, 4.61, and 2.85 g L(-1) for rice straw, wheat bran, peptone, and CaCO3, respectively, were found to be optimum for cellulase and xylanase production. Then the hydrolyzed spent Streptomyces cells were used as a nitrogen source and the maximum filter paper cellulase, carboxymethylcellulase, and xylanase activities of 25.79, 78.91, and 269.53 U mL(-1) were achieved. The crude cellulase produced by S. griseorubens JSD-1 was subsequently used for the hydrolysis of bio-pretreated rice straw, and the optimum saccharification efficiency of 88.13% was obtained, indicating that the crude enzyme might be used instead of commercial cellulase during a saccharification process. These results give a basis for further study of bioethanol production from agricultural cellulosic waste.

A Novel Aqueous Micellar Two-Phase System Composed of Surfactant and Sorbitol for Purification of Pectinase Enzyme from Psidium guajava and Recycling Phase Components

PubMed Central

Murshid, Fara Syazana; Manap, Mohd Yazid; Hussin, Muhaini

2015-01-01

A novel aqueous two-phase system composed of a surfactant and sorbitol was employed for the first time to purify pectinase from Psidium guajava. The influences of different parameters, including the type and concentration of the surfactant and the concentration and composition of the surfactant/sorbitol ratio, on the partitioning behavior and recovery of pectinase were investigated. Moreover, the effects of system pH and the crude load on purification fold and the yield of purified pectinase were studied. The experimental results indicated that the pectinase was partitioned into surfactant-rich top phase, and the impurities were partitioned into the sorbitol-rich bottom phase with the novel method involving an ATPS composed of 26% (w/w) Triton X-100 and 23% (w/w) sorbitol at 54.2% of the TLL crude load of 20% (w/w) at pH 6.0. The enzyme was successfully recovered by this method with a high purification factor of 15.2 and a yield of 98.3%, whereas the phase components were also recovered and recycled at rates above 96%. This study demonstrated that this novel ATPS method can be used as an efficient and economical alternative to the traditional ATPS for the purification and recovery of the valuable enzyme. PMID:25756051

Real time monitoring of the minute dynamic variation at the crude oil-water interface.

PubMed

Duan, Ming; Ding, Ziling; Wang, Hu; Xiong, Yan

2018-02-21

Quantitative recording of the minute dynamic variation at an oil-water interface is always a great challenge. Dual polarization interferometry (DPI) presents an approach in monitoring the variations of mass, thickness, and density at the interface with high resolution. In this study, a planar crude oil-water interface was established by spin-coating on a DPI chip surface. Different concentrations of sodium dodecyl sulphate (SDS) and polyacrylamide (PAM) were injected into the interface. The absorption of free SDS molecules, in low concentrations, can be interpreted as a two-step absorption. With the existence of micelles in higher concentrations, the crude oil molecules tend to be taken away by the micelles. The absorptions of the polymers at the interface are different from SDS. The crude oil can hardly be taken away by the polymers. Instead, the hydrophobic segments of polymers insert into the oil film and hydrophilic groups stretch into the solution.

Subcellular Localization of Rice Leaf Aryl Acylamidase Activity 1

PubMed Central

Gaynor, John J.; Still, Cecil C.

1983-01-01

The intracellular localization of aryl acylamidase (aryl-acylamide amidohydrolase, EC 3.5.1.13) in rice (Oryza sativa L. var Starbonnet) leaves was investigated. The enzyme hydrolyzes and detoxifies the herbicide propanil (3,4-dichloropropionanilide) thereby accounting for immunity of the rice plant to herbicidal action. Fractionation of mesophyll protoplasts by differential centrifugation yielded the highest specific activity of amidase in the crude mitochondrial fraction. Further separation of density gradients of the silica sol Percoll also indicated that this enzyme was mitochondrial. By the use of biochemical markers, the purified mitochondrial fraction was shown to be substantially free of contamination from nuclei, chloroplasts, golgi, and plasma membranes. Subfractionation of the purified mitochondria suggests that this enzyme is located on the outer membrane. PMID:16662987

A newly high alkaline lipase: an ideal choice for application in detergent formulations

PubMed Central

2011-01-01

Background Bacterial lipases received much attention for their substrate specificity and their ability to function in extreme environments (pH, temperature...). Many staphylococci produced lipases which were released into the culture medium. Reports of thermostable lipases from Staphylococcus sp. and active in alkaline conditions are not previously described. Results A newly soil-isolated Staphylococcus sp. strain ESW secretes an induced lipase in the culture medium. The effects of temperature, pH and various components in a detergent on the activity and stability of Staphylococcus sp. lipase (SL1) were studied in a preliminary evaluation for use in detergent formulation solutions. The enzyme was highly active over a wide range of pH from 9.0 to 13.0, with an optimum at pH 12.0. The relative activity at pH 13.0 was about 60% of that obtained at pH 12.0. It exhibited maximal activity at 60°C. This novel lipase, showed extreme stability towards non-ionic and anionic surfactants after pre-incubation for 1 h at 40°C, and relative stability towards oxidizing agents. Additionally, the crude enzyme showed excellent stability and compatibility with various commercial solid and liquid detergents. Conclusions These properties added to the high activity in high alkaline pH make this novel lipase an ideal choice for application in detergent formulations. PMID:22123072

21 CFR 864.9400 - Stabilized enzyme solution.

Code of Federal Regulations, 2013 CFR

2013-04-01

...) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Products Used In Establishments That Manufacture Blood and Blood Products § 864.9400 Stabilized enzyme solution. (a) Identification. A stabilized enzyme solution is a reagent intended for medical purposes that is used to enhance the reactivity of red blood...

21 CFR 864.9400 - Stabilized enzyme solution.

Code of Federal Regulations, 2012 CFR

2012-04-01

...) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Products Used In Establishments That Manufacture Blood and Blood Products § 864.9400 Stabilized enzyme solution. (a) Identification. A stabilized enzyme solution is a reagent intended for medical purposes that is used to enhance the reactivity of red blood...

21 CFR 864.9400 - Stabilized enzyme solution.

Code of Federal Regulations, 2014 CFR

2014-04-01

...) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Products Used In Establishments That Manufacture Blood and Blood Products § 864.9400 Stabilized enzyme solution. (a) Identification. A stabilized enzyme solution is a reagent intended for medical purposes that is used to enhance the reactivity of red blood...

Biodegradation of crude oil using an efficient microbial consortium in a simulated marine environment.

PubMed

Bao, Mu-tai; Wang, Li-na; Sun, Pei-yan; Cao, Li-xin; Zou, Jie; Li, Yi-ming

2012-06-01

Ochrobactrum sp. N1, Brevibacillus parabrevis N2, B. parabrevis N3 and B. parabrevis N4 were selected when preparing a mixed bacterial consortium based on the efficiency of crude oil utilization. A crude oil degradation rate of the N-series microbial consortium reached upwards of 79% at a temperature of 25 °C in a 3.0% NaCl solution in the shake flask trial. In the mesocosm experiment, a specially designed device was used to simulate the marine environment. The internal tank size was 1.5 m (L)×0.8 m (W)×0.7 m (H). The microbial growth conditions, nutrient utilization and environmental factors were thoroughly investigated. Over 51.1% of the crude oil was effectively removed from the simulated water body. The escalation process (from flask trials to the mesocosm experiment), which sought to represent removal under conditions more similar to the field, proved the high efficiency of using N-series bacteria in crude oil degradation. Copyright © 2012 Elsevier Ltd. All rights reserved.

Energetic Metabolism and Biochemical Adaptation: A Bird Flight Muscle Model

ERIC Educational Resources Information Center

Rioux, Pierre; Blier, Pierre U.

2006-01-01

The main objective of this class experiment is to measure the activity of two metabolic enzymes in crude extract from bird pectoral muscle and to relate the differences to their mode of locomotion and ecology. The laboratory is adapted to stimulate the interest of wildlife management students to biochemistry. The enzymatic activities of cytochrome…

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.

Impact of a multicarbohydrase containing α-galactosidase and xylanase on ileal digestible energy, crude protein digestibility, and ileal amino acid digestibility in broiler chickens.

PubMed

Jasek, A; Latham, R E; Mañón, A; Llamas-Moya, S; Adhikari, R; Poureslami, R; Lee, J T

2018-06-08

Exogenous enzymatic supplementation of poultry feeds, including α-galactosidase and xylanase, has been shown to increase metabolically available energy, although little information has been published on the impact on amino acid digestibility. An experiment was conducted to investigate a multicarbohydrase containing α-galactosidase and xylanase on amino acid digestibility, ileal digestible energy (IDE), and CP in male broiler chicks. The experiment was a 2 × 2 (diet × enzyme) factorial arrangement with 15 replicates of 8 male broilers per replicate raised for 21 d in a battery setting. The 2 dietary treatments included a positive control (PC) and a negative control (NC) diet formulated to contain 2.5% less calculated AME and digestible amino acids. Each of these diets was fed with and without enzyme. Broilers were fed a starter diet from 0-14 d (crumble) and a grower from 14-21 d (pellet). Birds were sampled on day 21 to determine ileal amino acid digestibility, IDE, and CP digestibility. Titanium dioxide (TiO2) was used as an indigestible marker for the determination of digestibility coefficients. Total ileal amino acid digestibility was increased (P = 0.008) by 3.80% with the inclusion of enzyme. Methionine and lysine digestibility was improved (P < 0.05) with the inclusion of enzyme by 3.37% and 2.61%, respectively. Enzyme inclusion increased (P = 0.001) cysteine digestibility by 9.3%. Diet-influenced ileal amino acid digestibility with tryptophan, threonine, isoleucine, and valine digestibility being increased (P < 0.05) in the PC when compared to the NC. IDE was decreased (P = 0.037) in broilers fed the NC diet by 100 kcal/kg feed when compared to broilers fed the PC diet. Enzyme inclusion increased (P = 0.047) IDE value by 90 kcal/kg. Crude protein digestibility was not influenced by diet; however, similar improvements in CP digestibility with enzyme inclusion were observed as with energy. These data support the benefits of a multicarbohydrase containing α-galactosidase and xylanase inclusion to improve nutrient and ileal amino acid digestibility across multiple dietary nutrient profiles.

First report of neurotoxic effect of the cyanobacterium Cylindrospermopsis raciborskii on the motility of trematode metacercariae.

PubMed

Lopes, K C; Ferrão-Filho, A S; Santos, E G N; Santos, C P

2018-03-01

Cylindrospermopsis raciborskii (Woloszynska) is a photosynthetic cyanobacterium that can produce cytotoxic (cylindrospermopsin) and neurotoxic cyanotoxins (saxitoxins). In Brazil the strains of C. raciborskii are reported to produce only saxitoxins (STX) and their effect on fish parasites has not been tested to date. The fish Poecilia vivipara Bloch and Schneider is a common host for the trematode Pygidiopsis macrostomum Travassos off the coast of Rio de Janeiro, and this fish-parasite interaction is a model for behavioural and ecotoxicological studies. The aim of this work was to evaluate the motility of metacercariae of P. macrostomum from P. vivipara exposed to 40 mg l-1 and 400 mg l-1 of crude lyophilized extract of the cyanobacterium C. raciborskii (CYRF-01) for 48 h. The fish were separated into groups of ten individuals and, after exposure, five fish from each group were dissected for counting and checking the motility of metacercariae. The other five fish were dissected after 48 h in clean water. The detection and quantification of STX in the solutions of cyanobacteria, and the gills and guts of fish, were performed by an enzyme-linked immunosorbent assay. The crude extract of C. raciborskii caused temporary paralysis in metacercariae of P. macrostomum after exposure of fish to both concentrations, and the motility recovered after the fish were kept for 48 h in clean water. STX was detected in the guts and gills of all fish analysed, suggesting that this toxin is involved in the paralysis of metacercariae. This is the first report on the action of neurotoxins in metacercariae of fish.

Dextransucrase production using cashew apple juice as substrate: effect of phosphate and yeast extract addition.

PubMed

Chagas, Clarice M A; Honorato, Talita L; Pinto, Gustavo A S; Maia, Geraldo A; Rodrigues, Sueli

2007-05-01

Cashew apples are considered agriculture excess in the Brazilian Northeast because cashew trees are cultivated primarily with the aim of cashew nut production. In this work, the use of cashew apple juice as a substrate for Leuconostoc mesenteroides cultivation was investigated. The effect of yeast extract and phosphate addition was evaluated using factorial planning tools. Both phosphate and yeast extract addition were significant factors for biomass growth, but had no significant effect on maximum enzyme activity. The enzyme activities found in cashew apple juice assays were at least 3.5 times higher than the activity found in the synthetic medium. Assays with pH control (pH = 6.5) were also carried out. The pH-controlled fermentation enhanced biomass growth, but decreased the enzyme activity. Crude enzyme free of cells produced using cashew apple juice was stable for 16 h at 30 degrees C at a pH of 5.0.

Purification and characterization of an extracellular cellulase from Anoxybacillus gonensis O9 isolated from geothermal area in Turkey.

PubMed

Genc, Berna; Nadaroglu, Hayrunnisa; Adiguzel, Ahmet; Baltaci, Ozkan

2015-11-01

In the present study, cellulase was purified and characterized from Anoxybacillus gonensis (Gen bank Number: KM596794) which was isolated and characterized from Agri Diyadin Hot spring. It was found to synthesize cellulase which had a wide range of industrial applications. Twenty four-hour-cultured bacteria induced cellulase production and specific activities during the purification steps were 1.47, 81.06 and 109.4 EU mg(-1) protein at crude extract, ammonium sulphate precipitated and DEAE-Sephadex purification steps. The highest enzyme activity was observed at 50°C and the optimum range of pH was 3-10. Molecular weight of enzyme was determined approximately 40kDa. The kinetic parameters of cellulase against carboxymethylcellulose (CMC) were 153.4 pmol min(-1) mg for Vmax and 0.46mM for Km. Among effectors of the enzyme, Zn2+, Ca2+, Co2+ and EDTA decreased enzyme activity.

Abscisic Acid Metabolism by a Cell-free Preparation from Echinocystis lobata Liquid Endoserum 1

PubMed Central

Gillard, Douglas F.; Walton, Daniel C.

1976-01-01

A cell-free enzyme system capable of metabolizing abscisic acid has been obtained from Eastern Wild Cucumber (Echinocystis lobata Michx.) liquid endosperm. The reaction products were determined to be phaseic acid (PA) and dihydrophaseic acid (DPA) by co-chromatography on thin layer chromatograms as the free acids, methyl esters, and their respective oxidation or reduction products. The crude enzyme preparation was separated by centrifugation into a particulate abscisic acid (ABA)-hydroxylating activity and a soluble PA-reducing activity. The particulate ABA-hydroxylating enzyme showed a requirement for O2 and NADPH, inhibition by CO, and high substrate specificity for (+)-ABA. Acetylation of short term incubation mixtures gave evidence for the presence of 6′-hydroxymethyl-ABA as an intermediate in PA formation. Determinations of endogenous ABA and DPA concentrations suggest that the ABA-hydroxylating and PA-reducing enzymes are extensively metabolizing ABA in the intact E. lobata seed. PMID:16659768

Light-regulation of enzyme activity in anacystis nidulans (Richt.).

PubMed

Duggan, J X; Anderson, L E

1975-01-01

The effect of light on the levels of activity of six enzymes which are light-modulated in higher plants was examined in the photosynthetic procaryot Anacystis nidulans. Ribulose-5-phosphate kinase (EC 2.7.1.19) was found to be light-activated in vivo and dithiothreitol-activated in vitro while glucose-6-phosphate dehydrogenase (EC 1.1.1.49) was light-inactivated and dithiothreitol-inactivated. The enzymes fructose-1,6-diphosphate phosphatase (EC 3.1.3.11), sedoheptulose-1,7-diphosphate phosphatase, NAD- and NADP-linked glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12; EC 1.2.1.13) were not affected by light treatment of the intact algae, but sedoheptulose-diphosphate phosphatase and the glyceraldehyde-3-phosphate dehydrogenases were dithiothreitol-activated in crude extracts. Light apparently controls the activity of the reductive and oxidative pentose phosphate pathway in this photosynthetic procaryot as in higher plants, through a process which probably involves reductive modulation of enzyme activity.

Report: screening of selected medicinal plants for their enzyme inhibitory potential - a validation of their ethnopharmacological uses.

PubMed

Khuda, Fazli; Iqbal, Zafar; Khan, Ayub; Zakiullah; Shah, Yasar; Khan, Abad

2014-05-01

In present study four medicinal plants namely Valeriana wallichii, Xanthium strumarium, Achyranthes aspera and Duchesnea indica belonging to different families were collected in Khyber Pakhtunkhwa province and crude extract and subsequent fractions were analyzed for their inhibitory potential against acetylcholinesterase, butyrylcholinesterase and α-glucosidase enzymes. Valeriana wallichii, Xanthium strumarium and Achyranthes aspera were significantly active against cholinesterases. Chloroform and ethylacetate fractions of Valeriana wallichii exhibited significant activity against acetylcholinesterase (IC50: 61μg/ml) and butyrylcholinesterase enzymes (IC50: 58μg/ml), respectively. Similarly ethylacetate fraction of Achyranthes aspera showed significant activity against acetylcholinesterase (IC50: 61 μg/ml) and butyrylcholinesterase enzymes (IC50: 61 μg/ml), respectively. In case of α-glucosidase enzyme, the chloroform fraction of Xanthium strumarium exhibited significant inhibitory activity (IC50: 72 μg/ml) as compared to the standard compound acarbose (IC50: 483 μg/ml). Duchesnea indica showed no such activities.

Catalyzed enzyme electrodes

DOEpatents

Zawodzinski, Thomas A.; Wilson, Mahlon S.; Rishpon, Judith; Gottesfeld, Shimshon

1993-01-01

An enzyme electrode is prepared with a composite coating on an electrical conductor. The composite coating is formed from a casting solution of a perfluorosulfonic acid polymer, an enzyme, and a carbon supported catalyst. The solution may be cast directly on the conductor surface or may be formed as a membrane and applied to the surface. The perfluorosulfonic acid ionomer formed from the casting solution provides an insoluble biocompatible protective matrix for the enzyme and acts to retain the enzyme for long term availability in the electrode structure. The carbon supported catalyst provides catalytic sites throughout the layer for the oxidation of hydrogen peroxide from the enzyme reactions. The carbon support then provides a conductive path for establishing an electrical signal to the electrical conductor. In one embodiment, the electrical conductor is a carbon cloth that permits oxygen or other gas to be introduced to the perfluorosulfonic polymer to promote the enzyme reaction independent of oxygen in the solution being tested.

Using crude glycerol and thin stillage for the production of microbial lipids through the cultivation of Rhodotorula glutinis.

PubMed

Yen, Hong-Wei; Yang, Ya-Chun; Yu, Yi-Huan

2012-10-01

Single cell oils (SCO) produced from oleaginous microorganisms are a potential alternative oil feedstock for biodiesel production. The worldwide production of glycerol, a 10% (w/w) byproduct produced in the transesterfication process of oils converted to biodiesel, is increasing as more biodiesel is being produced. For the purposes of cost reduction, crude glycerol was regarded as a suitable carbon source for the cultivation of Rhodotorula glutinis. In addition to using renewable crude glycerol, waste solution collected from the brewing company (called thin stillage) was adopted as a substitute to replace a costly nitrogen source used in the medium. The results of using mixture of crude glycerol and thin stillage indicated about a 27% increase in total biomass as compared to that of using crude glycerol with a standard medium. Using glycerol instead of glucose as the carbon source could also alter the lipid profile, resulting in an increase in linolenic acid (C18:2) to comprise over 20% of the total lipid. Successfully using renewable crude glycerol and thin stillage for the cultivation of oleaginous microorganisms could greatly enhance the economic competition of biodiesel produced from SCO. Copyright © 2012 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Anti-inflammatory and enzyme inhibitory activities of a crude extract and a pterocarpan isolated from the aerial parts of Vitex agnus-castus.

PubMed

Ahmad, Bashir; Azam, Sadiq; Bashir, Shumaila; Khan, Ibrar; Adhikari, Achyut; Choudhary, Muhammad Iqbal

2010-11-01

A new compound, 6a,11a-dihydro-6H-[1] benzofuro [3,2-c][1,3]dioxolo[4,5-g]chromen-9-ol was isolated from the ethyl acetate fraction of Vitex agnus-castus. The structure of this compound was identified with the help of spectroscopic techniques ((13)C NMR, (1)H NMR, HMBC, HMQC, NOESY and COSY). The compound showed low urease- (32.0%) and chymotrypsin- (31.4%) inhibitory activity, and moderate (41.3%) anti-inflammatory activity. The crude extract and various fractions obtained from the aerial parts of the plant were also screened for possible in vitro hemagglutination, antibacterial and phytotoxic activities. No hemagglutination activity against human erythrocytes was observed in crude extracts and fractions of V. agnus-castus. The fractions and crude methanolic extract showed moderate and low antibacterial activity. Exceptions were the CHCl(3) fraction, which showed significant antibacterial activity against Klebsiella pneumonia (81% with MIC(50)=2.19 mg/mL), the n-hexane fraction, which exhibited no activity against Salmonella typhi, and the CHCl(3) and aqueous fractions, which showed no activity against Bacillus pumalis. Moderate phytotoxic activity (62.5%) was observed by n-hexane fraction of V. agnus-castus against Lemna minor L at 1000 μg/mL.

Response of the Cutworm Spodoptera litura to Sesame Leaves or Crude Extracts in Diet

PubMed Central

Ofosuhene Sintim, Henry; Tashiro, Toru; Motoyama, Naoki

2009-01-01

The effects of extracts of sesame, Sesamum indicum L. (Liamiales: Pedaliaceae), and whole leaves of some selected cultivars of sesame were tested using a natural host Spodoptera litura (F.) (Lepidoptera: Noctuidae). Indices taken using the immature stages include; diet utilization, growth and development and induction of detoxification enzymes. The results indicate that S. litura generally selects its food amongst cultivars within 6 hours after food presentation. Growth and development of the insect is controlled also by plant acceptability and quality. Although all the cultivars tested significantly limit insect growth and development the variety 56S-radiatum did not allow a complete life cycle as pupation from first instar stage was 0%. Generally the crucial period for immature S. litura was the larval period, especially the first two instars where the weight of an insect fed on an experimental diet was three times lower than that of a control diet. The larval developmental period was greater than 40 days as compared to 17 days for insects fed a control diet. S. litura also had lowered efficiency in utilizing ingested food, from a low of 13% in a sesame cultivar to 45% in the control diet. The key detoxification enzyme was a glutathione s-transferase that was confirmed by a 6-fold increase between S. litura fed a plant cultivar vs. a control diet towards the substrate 1,2-dichloro-4-nitrobenzene. First and second instars of S. litura have a relatively reduced detoxification of enzymes in response to plant cultivar diets leading to low survival. A 3% v/w crude extract of the cultivars increased enzyme induction towards all the tested substrates. PMID:20050772

Development of a highly efficient oil degumming process using a novel phosphatidylinositol-specific phospholipase C enzyme.

PubMed

Cerminati, Sebastián; Eberhardt, Florencia; Elena, Claudia E; Peirú, Salvador; Castelli, María E; Menzella, Hugo G

2017-06-01

Enzymatic degumming using phospholipase C (PLC) enzymes may be used in environmentally friendly processes with improved oil recovery yields. In this work, phosphatidylinositol-specific phospholipase C (PIPLC) candidates obtained from an in silico analysis were evaluated for oil degumming. A PIPLC from Lysinibacillus sphaericus was shown to efficiently remove phosphatidylinositol from crude oil, and when combined with a second phosphatidylcholine and phosphatidylethanolamine-specific phospholipase C, the three major phospholipids were completely hydrolyzed, providing an extra yield of oil greater than 2.1%, compared to standard methods. A remarkably efficient fed-batch Escherichia coli fermentation process producing ∼14 g/L of the recombinant PIPLC enzyme was developed, which may facilitate the adoption of this cost-effective oil-refining process.

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 Characterization of the Crown Gall-specific Enzyme, Octopine Synthase 1

PubMed Central

Hack, Ethan; Kemp, John D.

1980-01-01

A single enzyme catalyzes the synthesis of all four N2-(1-carboxyethyl)-amino acid derivatives found in a crown gall tumor tissue induced by Agrobacterium tumefaciens (E. F. Sm. and Town.) Conn strain B6 on sunflower (Helianthus annuus L.). This enzyme, octopine synthase, has been purified by ammonium sulfate fractionation and chromatography on diethylaminoethylcellulose, blue agarose, and hydroxylapatite. The purified enzyme has all the N2-(1-carboxyethyl)-amino acid synthesizing activities found in crude preparations, and the relative activities with six amino acids remain nearly constant during purification. Although the maximum velocities (V) and Michaelis constants (Km) differ, the ratio V/Km is the same for all amino acid substrates. Thus an equimolar mixture of amino acids will give rise to an equimolar mixture of products. The kinetic properties of the enzyme are consistent with a partially ordered mechanism with arginine (NADPH, then arginine or pyruvate). Octopine synthase is a monomeric enzyme with a molecular weight of 39,000 by gel filtration and 38,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Images PMID:16661312

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

Tao Zhu; Jack A. Walker; J. Liang

Due to increasing oil demand, oil companies are moving into arctic environments and deep-water areas for oil production. In these regions of lower temperatures, wax deposits begin to form when the temperature in the wellbore falls below wax appearance temperature (WAT). This condition leads to reduced production rates and larger pressure drops. Wax problems in production wells are very costly due to production down time for removal of wax. Therefore, it is necessary to develop a solution to wax deposition. In order to develop a solution to wax deposition, it is essential to characterize the crude oil and study phasemore » behavior properties. The main objective of this project was to characterize Alaskan North Slope crude oil and study the phase behavior, which was further used to develop a dynamic wax deposition model. This report summarizes the results of the various experimental studies. The subtasks completed during this study include measurement of density, molecular weight, viscosity, pour point, wax appearance temperature, wax content, rate of wax deposition using cold finger, compositional characterization of crude oil and wax obtained from wax content, gas-oil ratio, and phase behavior experiments including constant composition expansion and differential liberation. Also, included in this report is the development of a thermodynamic model to predict wax precipitation. From the experimental study of wax appearance temperature, it was found that wax can start to precipitate at temperatures as high as 40.6 C. The WAT obtained from cross-polar microscopy and viscometry was compared, and it was discovered that WAT from viscometry is overestimated. From the pour point experiment it was found that crude oil can cease to flow at a temperature of 12 C. From the experimental results of wax content, it is evident that the wax content in Alaskan North Slope crude oil can be as high as 28.57%. The highest gas-oil ratio for a live oil sample was observed to be 619.26 SCF/STB. The bubblepoint pressure for live oil samples varied between 1600 psi and 2100 psi. Wax precipitation is one of the most important phenomena in wax deposition and, hence, needs to be modeled. There are various models present in the literature. Won's model, which considers the wax phase as a non-ideal solution, and Pedersen's model, which considers the wax phase as an ideal solution, were compared. Comparison indicated that Pedersen's model gives better results, but the assumption of wax phase as an ideal solution is not realistic. Hence, Won's model was modified to consider different precipitation characteristics of the various constituents in the hydrocarbon fraction. The results obtained from the modified Won's model were compared with existing models, and it was found that predictions from the modified model are encouraging.« less

Enhanced Phytoremediation of Crude Oil-Polluted Soil by Four Plant Species: Effect of Inorganic and Organic Bioaugumentation.

PubMed

Nwaichi, Eucharia Oluchi; Frac, Magdalena; Nwoha, Paul Aleruchi; Eragbor, Progress

2015-01-01

A field experiment investigating the removal and/or uptake of Polycyclic Aromatic Hydrocarbons (PAHs) and specific metals (As, Cd, Cr) from a crude oil polluted agricultural soil was performed during the 2013 wet season using four plant species: Fimbristylis littoralis, Hevea brasilensis (Rubber plants), Cymbopogom citratus (Lemon grass), and Vigna subterranea (Bambara nuts). Soil functional diversity and soil-enzyme interactions were also investigated. The diagnostic ratios and the correlation analysis identified mixed petrogenic and pyrogenic sources as the main contributors of PAHs at the study site. A total of 16 PAHs were identified, 6 of which were carcinogenic. Up to 42.4 mg kg(-1) total PAHs was recorded prior to the experiments. At 90 d, up to 92% total PAH reduction and 96% As removal were achieved using F. littoralis, the best performing species. The organic soil amendment (poultry dung) rendered most of the studied contaminants unavailable for uptake. However, the organic amendment accounted for over 70% of the increased dehydrogenase, phosphatase, and proteolytic enzymes activities in the study. Overall, the combined use of soil amendments and phytoremediation significantly improved the microbial community activity, thus promoting the restoration of the ecosystem.

Bioconversion of Cyanidin-3-Rutinoside to Cyanidin-3-Glucoside in Black Raspberry by Crude α-L-Rhamnosidase from Aspergillus Species.

PubMed

Lim, Taehwan; Jung, Hana; Hwang, Keum Taek

2015-11-01

Cyanidin-3-glucoside (C3G) has been known to be more bioavailable than cyanidin-3- rutinoside (C3R), the most abundant anthocyanin in black raspberry (Rubus occidentalis). The aim of this study was to enhance the bioavailability of anthocyanins in black raspberry by cleaving L-rhamnose in C3R using crude enzyme extracts (CEEs) from Aspergillus usamii KCTC 6956, A. awamori KCTC 60380, A. niger KCCM 11724, A. oryzae KCCM 12698, and A. kawachii KCCM 32819. The enzyme activities of the CEEs were determined by a spectrophotometric method using rho-nitrophenyl-rhamnopyranoside and rho-nitrophenyl-glucopyranoside. The CEE from A. usamii had the highest α-L-rhamnosidase activity with 2.73 U/ml at 60°C, followed by those from A. awamori and A. niger. When bioconversion of C3R to C3G in black raspberry was analyzed by HPLC-DAD, the CEEs from A. usamii and A. awamori hydrolyzed 95.7% and 95.6% of C3R to C3G, respectively, after 2 h incubation. The CEEs from A. kawachii and A. oryzae did not convert C3R to C3G in black raspberry.

Physico-chemical pretreatment and enzymatic hydrolysis of cotton stalk for ethanol production by Saccharomyces cerevisiae.

PubMed

Singh, Anita; Bajar, Somvir; Bishnoi, Narsi R

2017-11-01

The aim of this work was to study the physico-chemical pretreatment and enzymatic hydrolysis of cotton stalk for ethanol production by Saccharomyces cerevisiae. Firstly, factors affecting pretreatment were screened out by Plackett-Burman design (PBD) and most significant factors were further optimized by Box-Behnken design (BBD). As shown by experimental study, most significant factors were FeCl 3 concentration (FC), irradiation time (IT) and substrate concentration (SC) affecting pretreatment of cotton stalk among all studied factors. Under optimum conditions of pretreatment FC 0.15mol/l, IT 20min and SC 55g/l, the release of reducing sugar was 6.6g/l. Hydrolysis of pretreated cotton stalk was done by crude on-site produced enzymes and hydrolysate was concentrated. Ethanol production by Saccharomyces cerevisiae using concentrated cotton stalk hydrolysate was 9.8g p /l, with ethanol yield 0.37g p /g s on consumed sugars. The data indicated that microwave FeCl 3 pretreated cotton stalk hydrolyses by crude unprocessed enzyme cocktail was good, and ethanol can be produced by fermentation of hydrolysate. Copyright © 2017 Elsevier Ltd. All rights reserved.

Purification, immobilization and characterization of tannase from Penicillium variable.

PubMed

Sharma, Shashi; Agarwal, Lata; Saxena, Rajendra Kumar

2008-05-01

Tannase from Penicillium variable IARI 2031 was purified by a two-step purification strategy comprising of ultra-filtration using 100 kDa molecular weight cutoff and gel-filtration using Sephadex G-200. A purification fold of 135 with 91% yield of tannase was obtained. The enzyme has temperature and pH optima of 50 degrees C and 5 degrees C, respectively. However, the functional temperature range is from 25 to 80 degrees C and functional pH range is from 3.0 to 8.0. This tannase could successfully be immobilized on Amberlite IR where it retains about 85% of the initial catalytic activity even after ninth cycle of its use. Based on the Michaelis-Menten constant (Km) of tannase, tannic acid is the best substrate with Km of 32 mM and Vmax of 1.11 micromol ml(-1)min(-1). Tannase is inhibited by phenyl methyl sulphonyl fluoride (PMSF) and N-ethylmaleimide retaining only 28.1% and 19% residual activity indicating that this enzyme belongs to the class of serine hydrolases. Tannase in both crude and crude lyophilized forms is stable for one year retaining more than 60% residual activity.

Enzymatic production of pectic oligosaccharides from onion skins.

PubMed

Babbar, Neha; Baldassarre, Stefania; Maesen, Miranda; Prandi, Barbara; Dejonghe, Winnie; Sforza, Stefano; Elst, Kathy

2016-08-01

Onion skins are evaluated as a new raw material for the enzymatic production of pectic oligosaccharides (POS) with a targeted degree of polymerization (DP). The process is based on a two-stage process consisting of a chelator-based crude pectin extraction followed by a controlled enzymatic hydrolysis. Treatment of the extracted crude onion skin's pectin with various enzymes (EPG-M2, Viscozyme and Pectinase) shows that EPG-M2 is the most appropriate enzyme for tailored POS production. The experiments reveal that the highest amount of DP2 and DP3 is obtained at a time scale of 75-90min with an EPG-M2 concentration of 26IU/mL. At these conditions the production amounts 2.5-3.0% (w/w) d.m for DP2 and 5.5-5.6% (w/w) d.m for DP3 respectively. In contrast, maximum DP4 production of 5.2-5.5% (w/w) d.m. is obtained with 5.2IU/mL at a time scale of 15-30min. Detailed LC-MS analysis reveals the presence of more methylated oligomers compared to acetylated forms in the digests. Copyright © 2016 Elsevier Ltd. All rights reserved.

In Vivo antiplatelet activity aggregation assay of bromelain fractionate by ethanol from extract pineapple core (Ananas comosus [l.] merr.)

NASA Astrophysics Data System (ADS)

Musfiroh, F. F.; Setiasih, S.; Handayani, S.; Hudiyono, S.; Ilyas, N. M.

2018-01-01

Processed fruit from pineapple is one of largest commodities tropical fruit production in Indonesia and will bring the waste from the skin and core. This study aims to isolate bromelain from the pineapple core (Ananas comusus) are purified by fractionation using ethanol and continued by activity test as an antiplatelets agent by in vivo method using white mice male ddy type with acetosal as positive control. Fractionation of crude enzyme bromelain with ethanol produces highest specific activity on ethanol 30-60% fraction (fraction 2) 3.107 Unit/mg and the protein content 61.25 mg with the degree of purity of 155 times compared to crude enzyme. Antiplatelet aggregation tests from in vivo method shows that faction of bromelain with doses 70 μg/KgBW, 140 μg/KgBW, and 210 μg/KgBW can increase a meaningful bleeding time. The highest percentage of increase shown by the isolate at a dose of 210 μg/KgBW in the amount of 515.10 ± 182.23%, when compared to aspirin (231.20 ± 140.66), the value is relatively higher.

An oxidant- and solvent-stable protease produced by Bacillus cereus SV1: application in the deproteinization of shrimp wastes and as a laundry detergent additive.

PubMed

Manni, Laila; Jellouli, Kemel; Ghorbel-Bellaaj, Olfa; Agrebi, Rym; Haddar, Anissa; Sellami-Kamoun, Alya; Nasri, Moncef

2010-04-01

The current increase in amount of shrimp wastes produced by the shrimp industry has led to the need in finding new methods for shrimp wastes disposal. In this study, an extracellular organic solvent- and oxidant-stable metalloprotease was produced by Bacillus cereus SV1. Maximum protease activity (5,900 U/mL) was obtained when the strain was grown in medium containing 40 g/L shrimp wastes powder as a sole carbon source. The optimum pH, optimum temperature, pH stability, and thermal stability of the crude enzyme preparation were pH 8.0, 60 degrees C, pH 6-9.5, and <55 degrees C, respectively. The crude protease was extremely stable toward several organic solvents. No loss of activity was observed even after 60 days of incubation at 30 degrees C in the presence of 50% (v/v) dimethyl sulfoxide and ethyl ether; the enzyme retained more than 70% of its original activity in the presence of ethanol and N,N-dimethylformamide. The protease showed high stability toward anionic (SDS) and non-ionic (Tween 80, Tween 20, and Triton X-100) surfactants. Interestingly, the activity of the enzyme was significantly enhanced by oxidizing agents. In addition, the enzyme showed excellent compatibility with some commercial liquid detergents. The protease of B. cereus SV1, produced under the optimal culture conditions, was tested for shrimp waste deproteinization in the preparation of chitin. The protein removal with a ratio E/S of 20 was about 88%. The novelties of the SV1 protease include its high stability to organic solvents and surfactants. These unique properties make it an ideal choice for application in detergent formulations and enzymatic peptide synthesis. In addition, the enzyme may find potential applications in the deproteinization of shrimp wastes to produce chitin.

Rational design to improve thermostability and specific activity of the truncated Fibrobacter succinogenes 1,3-1,4-β-D-glucanase.

PubMed

Huang, Jian-Wen; Cheng, Ya-Shan; Ko, Tzu-Ping; Lin, Cheng-Yen; Lai, Hui-Lin; Chen, Chun-Chi; Ma, Yanhe; Zheng, Yingying; Huang, Chun-Hsiang; Zou, Peijian; Liu, Je-Ruei; Guo, Rey-Ting

2012-04-01

1,3-1,4-β-D-Glucanase has been widely used as a feed additive to help non-ruminant animals digest plant fibers, with potential in increasing nutrition turnover rate and reducing sanitary problems. Engineering of enzymes for better thermostability is of great importance because it not only can broaden their industrial applications, but also facilitate exploring the mechanism of enzyme stability from structural point of view. To obtain enzyme with higher thermostability and specific activity, structure-based rational design was carried out in this study. Eleven mutants of Fibrobacter succinogenes 1,3-1,4-β-D-glucanase were constructed in attempt to improve the enzyme properties. In particular, the crude proteins expressed in Pichia pastoris were examined firstly to ensure that the protein productions meet the need for industrial fermentation. The crude protein of V18Y mutant showed a 2 °C increment of Tm and W203Y showed ∼30% increment of the specific activity. To further investigate the structure-function relationship, some mutants were expressed and purified from P. pastoris and Escherichia coli. Notably, the specific activity of purified W203Y which was expressed in E. coli was 63% higher than the wild-type protein. The double mutant V18Y/W203Y showed the same increments of Tm and specific activity as the single mutants did. When expressed and purified from E. coli, V18Y/W203Y showed similar pattern of thermostability increment and 75% higher specific activity. Furthermore, the apo-form and substrate complex structures of V18Y/W203Y were solved by X-ray crystallography. Analyzing protein structure of V18Y/W203Y helps elucidate how the mutations could enhance the protein stability and enzyme activity.

Escherichia coli NemA is an efficient chromate reductase that can be biologically immobilized to provide a cell free system for remediation of hexavalent chromium.

PubMed

Robins, Katherine J; Hooks, David O; Rehm, Bernd H A; Ackerley, David F

2013-01-01

Hexavalent chromium is a serious and widespread environmental pollutant. Although many bacteria have been identified that can transform highly water-soluble and toxic Cr(VI) to insoluble and relatively non-toxic Cr(III), bacterial bioremediation of Cr(VI) pollution is limited by a number of issues, in particular chromium toxicity to the remediating cells. To address this we sought to develop an immobilized enzymatic system for Cr(VI) remediation. To identify novel Cr(VI) reductase enzymes we first screened cell extracts from an Escherichia coli library of soluble oxidoreductases derived from a range of bacteria, but found that a number of these enzymes can reduce Cr(VI) indirectly, via redox intermediates present in the crude extracts. Instead, activity assays for 15 candidate enzymes purified as His6-tagged proteins identified E. coli NemA as a highly efficient Cr(VI) reductase (k(cat)/K(M)= 1.1×10(5) M(-1) s(-1) with NADH as cofactor). Fusion of nemA to the polyhydroxyalkanoate synthase gene phaC from Ralstonia eutropha enabled high-level biosynthesis of functionalized polyhydroxyalkanoate granules displaying stable and active NemA on their surface. When these granules were combined with either Bacillus subtilis glucose dehydrogenase or Candida boidinii formate dehydrogenase as a cofactor regenerating partner, high levels of chromate transformation were observed with only low initial concentrations of expensive NADH cofactor being required, the overall reaction being powered by consumption of the cheap sacrificial substrates glucose or formic acid, respectively. This system therefore offers promise as an economic solution for ex situ Cr(VI) remediation.

Escherichia coli NemA Is an Efficient Chromate Reductase That Can Be Biologically Immobilized to Provide a Cell Free System for Remediation of Hexavalent Chromium

PubMed Central

Robins, Katherine J.; Hooks, David O.; Rehm, Bernd H. A.; Ackerley, David F.

2013-01-01

Hexavalent chromium is a serious and widespread environmental pollutant. Although many bacteria have been identified that can transform highly water-soluble and toxic Cr(VI) to insoluble and relatively non-toxic Cr(III), bacterial bioremediation of Cr(VI) pollution is limited by a number of issues, in particular chromium toxicity to the remediating cells. To address this we sought to develop an immobilized enzymatic system for Cr(VI) remediation. To identify novel Cr(VI) reductase enzymes we first screened cell extracts from an Escherichia coli library of soluble oxidoreductases derived from a range of bacteria, but found that a number of these enzymes can reduce Cr(VI) indirectly, via redox intermediates present in the crude extracts. Instead, activity assays for 15 candidate enzymes purified as His6-tagged proteins identified E. coli NemA as a highly efficient Cr(VI) reductase (kcat/KM  = 1.1×105 M−1s−1 with NADH as cofactor). Fusion of nemA to the polyhydroxyalkanoate synthase gene phaC from Ralstonia eutropha enabled high-level biosynthesis of functionalized polyhydroxyalkanoate granules displaying stable and active NemA on their surface. When these granules were combined with either Bacillus subtilis glucose dehydrogenase or Candida boidinii formate dehydrogenase as a cofactor regenerating partner, high levels of chromate transformation were observed with only low initial concentrations of expensive NADH cofactor being required, the overall reaction being powered by consumption of the cheap sacrificial substrates glucose or formic acid, respectively. This system therefore offers promise as an economic solution for ex situ Cr(VI) remediation. PMID:23527133

Simultaneously saccharification and fermentation approach as a tool for enhanced fossil fuels biodesulfurization.

PubMed

Paixão, Susana M; Arez, Bruno F; Roseiro, José C; Alves, Luís

2016-11-01

Biodesulfurization can be a complementary technology to the hydrodesulfurization, the commonly physical-chemical process used for sulfur removal from crude oil. The desulfurizing bacterium Gordonia alkanivorans strain 1B as a fructophilic microorganism requires fructose as C-source. In this context, the main goal of this work was the optimization of a simultaneous saccharification and fermentation (SSF) approach using the Zygosaccharomyces bailii strain Talf1 crude enzymes with invertase activity and sucrose as a cheaper fructose-rich commercial C-source (50% fructose) towards dibenzothiophene (DBT) desulfurization by strain 1B. The determination of optimal conditions, for both sucrose hydrolysis and DBT desulfurization was carried out through two sequential experimental uniform designs according to the Doehlert distribution for two factors: pH (5.5-7.5) and temperature (28-38 °C), with the enzyme load of 1.16 U/g/L; and enzyme load (0-4 U/g/L) and temperature (28-38 °C), with pH at 7.5. Based on 2-hydroxybiphenyl production, the analysis of the response surfaces obtained pointed out for pH 7.5, 32 °C and 1.8 U/g/L as optimal conditions. Further optimized SSF of sucrose during the DBT desulfurization process permitted to attain a 4-fold enhanced biodesulfurization. This study opens a new focus of research through the exploitation of sustainable low cost sucrose-rich feedstocks towards a more economical viable bioprocess scale-up. Copyright © 2016 Elsevier Ltd. All rights reserved.

Effects of traditional Chinese medicine formula on ruminal fermentation, enzyme activities and nutrient digestibility of beef cattle.

PubMed

Zhu, Zhi; Song, Zhen-Hui; Cao, Li-Ting; Wang, Yong; Zhou, Wen-Zhang; Zhou, Pei; Zuo, Fu-Yuan

2018-04-01

This study was conducted to evaluate effects of traditional Chinese medicine formula (TCMF) combined with several herbs on ruminal fermentation, enzyme activities and nutrient digestibility. Twenty finishing bulls were assigned to control or different TCMFs (Yufeisan-1, -2, -3; 2.5% dry matter (DM) in concentrate). Results showed that DM intake was higher (P < 0.05) in the Yufeisan-3 group than others. Compared to control, apparent digestibility of crude protein and neutral detergent fiber were increased (P < 0.05) by Yufeisan-3. No changes were observed in ruminal pH, concentrations of ammonia-N, microbial crude protein and total volatile fatty acid, whereas ratio of acetate to propionate was lower (P < 0.05) and propionate proportion tended to be higher (P < 0.1) in three TCMFs than control. Ruminal xylanase (P = 0.061) and carboxymethylcellulase (P < 0.05) activities were higher in Yufeisan-3 than control. No changes were observed in abundance of total bacteria, fungi and protozoa, whereas Fibrobacter succinogenes (P = 0.062) and Ruminococcus flavefaciens (P < 0.05) were increased and total methanogens was reduced (P = 0.069) by Yufeisan-3 compared to control. Yufeisan-3 improved nutrient digestibility and ruminal enzyme activity, and modified fermentation and microbial community, maybe due to the presence of Herba agastaches, Cortex phellodendri and Gypsum fibrosum. © 2018 Japanese Society of Animal Science.

A novel beta-glucosidase from the cell wall of maize (Zea mays L.): rapid purification and partial characterization

NASA Technical Reports Server (NTRS)

Nematollahi, W. P.; Roux, S. J.

1999-01-01

Plants have a variety of glycosidic conjugates of hormones, defense compounds, and other molecules that are hydrolyzed by beta-glucosidases (beta-D-glucoside glucohydrolases, E.C. 3.2.1.21). Workers have reported several beta-glucosidases from maize (Zea mays L.; Poaceae), but have localized them mostly by indirect means. We have purified and partly characterized a 58-Ku beta-glucosidase from maize, which we conclude from a partial sequence analysis, from kinetic data, and from its localization is not identical to any of those already reported. A monoclonal antibody, mWP 19, binds this enzyme, and localizes it in the cell walls of maize coleoptiles. An earlier report showed that mWP19 inhibits peroxidase activity in crude cell wall extracts and can immunoprecipitate peroxidase activity from these extracts, yet purified preparations of the 58 Ku protein had little or no peroxidase activity. The level of sequence similarity between beta-glucosidases and peroxidases makes it unlikely that these enzymes share epitopes in common. Contrary to a previous conclusion, these results suggest that the enzyme recognized by mWP19 is not a peroxidase, but there is a wall peroxidase closely associated with the 58 Ku beta-glucosidase in crude preparations. Other workers also have co-purified distinct proteins with beta-glucosidases. We found no significant charge in the level of immunodetectable beta-glucosidase in mesocotyls or coleoptiles that precedes the red light-induced changes in the growth rate of these tissues.

Preparation of Multiwalled Carbon Nanotubes/Hydroxyl-Terminated Silicone Oil Fiber and Its Application to Analysis of Crude Oils

PubMed Central

Tong, Ting; Zhang, Wanfeng; Dai, Wei; He, Sheng; Chang, Zhenyang; Gao, Xuanbo

2014-01-01

A simple and efficient method to analyze the volatile and semivolatile organic compounds in crude oils has been developed based on direct immersion solid-phase microextraction coupled to comprehensive two-dimensional gas chromatography/time-of-flight mass spectrometry (DI-SPME-GC × GC/TOFMS). A novel fiber, multiwalled carbon nanotubes/hydroxyl-terminated silicone oil (MWNTs-TSO-OH), was prepared by sol-gel technology. Using standard solutions, the extraction conditions were optimized such as extraction mode, extraction temperature, extraction time, and salts effect. With the optimized conditions, a real crude oil sample was extracted and then analyzed in detail. It shows that the proposed method is very effective in simultaneously analyzing the normal and branched alkanes, cycloalkanes, aromatic hydrocarbons, and biomarkers of crude oil such as steranes and terpanes. Furthermore, the method showed good linearity (r > 0.999), precision (RSD < 8%), and detection limits ranging from 0.2 to 1.6 ng/L. PMID:24578659

Effects of sour crude oil on fatigue properties of steel plates for shipbuilding

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

Ouchi, H.; Kobayashi, J.; Ishikawa, T.

1994-12-31

The concentration of diffusible hydrogen introduced into steel was measured, and fatigue crack growth tests and fatigue life tests were carried out in sour crude oil containing a high concentration of hydrogen sulfide and under electrolytic hydrogen-charging conditions in neutral solution, using a high strength steel produced by the thermo-mechanical control process (TMCP) and a mild steel which are steels for hull plates. Comparison of the results demonstrated that a very small amount of hydrogen such as that introduced into steel from sour crude oil under atmospheric pressure accelerated the fatigue crack growth in the high {Delta}K regime and shortenedmore » the fatigue life in the high stress range region, but did not shorten the fatigue life in the low stress region. The electrolytic hydrogen-charging condition appeared to be appropriate as a fatigue-crack-growth test environment to simulate sour crude oil. The deterioration of fatigue characteristics of the TMCP high strength steel was similar with that of the mild steel.« less

Isolation and characterization of coagulant extracted from Moringa oleifera seed by salt solution.

PubMed

Okuda, T; Baes, A U; Nishijima, W; Okada, M

2001-02-01

It is known that M. oleifera contains a natural coagulant in the seeds. In our previous research, the method using salt water to extract the active coagulation component from M. oleifera seeds was developed and compared with the conventional method using water. In this research, the active coagulation component was purified from a NaCl solution crude extract of Moringa oleifera seeds. The active component was isolated and purified from the crude extract through a sequence of steps that included salting-out by dialysis, removal of lipids and carbohydrates by homogenization with acetone, and anion exchange. Specific coagulation activity of the active material increased up to 34 times more than the crude extract after the ion exchange. The active component was not the same as that of water extract. The molecular weight was about 3000 Da. The Lowry method and the phenol-sulfuric acid method indicated that the active component was neither protein nor polysaccharide. The optimum pH of the purified active component for coagulation of turbidity was pH 8 and above. Different from the conventional water extracts, the active component can be used for waters with low turbidity without increase in the dissolved organic carbon concentration.

Interfacial activity in alkaline flooding enhanced oil recovery

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

Chan, M.K.

1981-01-01

The ionization of long-chained organic acids in the crude oil to form soaps was shown to be primarily responsible for the lowering of oil-water interfacial tension at alkaline pH. These active acids can be concentrated by silica gel chromatography into a minor polar fraction. An equilibrium chemical model was proposed based on 2 competing reactions: the ionization of acids to form active anions, and the formation of undissociated soap between acid anions and sodium ions. It correlates the interfacial activity with the interfacial concentration of active acid anions which is expressed in terms of the concentrations of the chemical speciesmore » in the system. The model successfully predicts the observed oil-alkaline solution interfacial phenomenon, including its dependence on pH, alkali and salt concentrations, type of acid present and type of soap formed. Flooding at different alkali concentrations to activate different acid species present in the crude was shown to give better recovery than flooding at a single high alkali concentration. Treating the crude oil with a dilute solution of mineral acids liberates additional free active acids and yields better interfacial activity during subsequent alkali contact.« less

Inhibition of Phenylamide Hydrolysis by Bacillus sphaericus with Methylcarbamate and Organophosphorus Insecticides

PubMed Central

Engelhardt, G.; Wallnöfer, P. R.

1975-01-01

The degradation of the phenylamide herbicides monolinuron, linuron, and solan by cultures of Bacillus sphaericus ATCC 12123 was inhibited by the methylcarbamate insecticides metmercapturon, aldicarb, propoxur, and carbaryl and by the organophosphorus insecticides fenthion and parathion. The extent of inhibition was largest with metmercapturon and smallest with parathion. Inhibition of hydrolysis of the two phenylurea herbicides was greater than of the acylanilide compound. Tests with crude enzyme preparations of aryl acylamidase derived from B. sphaericus showed that the inhibition of the hydrolysis of linuron with methylcarbamates is a competitive one. The insecticides tested did not induce the enzyme, nor could they serve as its substrate. PMID:1155931

Enzyme-linked immunosorbent assay for total sennosides using anti-sennside A and anti-sennoside B monoclonal antibodies.

PubMed

Morinaga, Osamu; Uto, Takuhiro; Sakamoto, Seiichi; Tanaka, Hiroyuki; Shoyama, Yukihiro

2009-01-01

Total sennosides concentration is a very important factor when rhubarb and senna will be used as crude drugs. However, one-step analytical technique for total sennosides has not been reported except HPLC. An enzyme-linked immunosorbent assay (ELISA) for total sennosides concentration by using the combination of anti-sennoside A (SA) and anti-sennoside B (SB) monoclonal antibodies (MAbs) in a single assay has been investigated. Total sennosides concentration in rhubarb and senna samples determined by newly developed assay system showed good agreement with those analyzed by ELISA using anti-SA MAb and anti-SB MAb, respectively.

Protoplast formation and yeast cell-wall structure. The action of the enzymes of the snail, Helix pomatia

PubMed Central

Anderson, F. B.; Millbank, J. W.

1966-01-01

1. The digestive juice of the snail Helix pomatia was used in the study of the degradation of isolated cell-wall preparations from a strain of Saccharomyces carlsbergensis. 2. The crude enzyme system was fractionated by gel filtration and the activities of the more specific fractions thus obtained were examined. 3. Results are discussed with respect to (a) the nature of various factors that are essential for protoplast formation and cell-wall dissolution and (b) structures envisaged in yeast cell walls that are responsible for the observed variations in susceptibility to attack by snail juice. PMID:5964965

Identification of the catalytic triad of the lipase/acyltransferase from Aeromonas hydrophila.

PubMed Central

Brumlik, M J; Buckley, J T

1996-01-01

Aeromonas hydrophila secretes a lipolytic enzyme that has several properties in common with the mammalian enzyme lecithin-cholesterol acyltransferase. We have recently shown that it is a member of a newly described group of proteins that contain five similar blocks of sequence arranged in the same order in their primary structures (C. Upton and J. T. Buckley, Trends Biochem. Sci. 233:178-179, 1995). Assuming that, like other lipases, these enzymes have a Ser-Asp-His catalytic triad, we used these blocks to predict which aspartic acid and histidine would be at the active site of the Aeromonas enzyme. Targeted residues were replaced with other amino acids by site-directed mutagenesis, and the effects on secretion and activity were assessed. Changing His-291 to asparagine completely abolished enzyme activity, although secretion by the bacteria was not affected. Only very small amounts of the D116N mutant appeared in the culture supernatant, likely because it is sensitive to periplasmic proteases it encounters en route. Assays of crude preparations containing this variant showed no detectable enzyme activity. We conclude that, together with Ser-16, which we have identified previously, Asp-116 and His-291 compose the catalytic triad of the enzyme. PMID:8606184

A multi-step chromatographic strategy to purify three fungal endo-β-glucanases.

PubMed

McCarthy, Tracey; Tuohy, Maria G

2011-01-01

Fungi and fungal enzymes have traditionally occupied a central role in biotechnology. Understanding the biochemical properties of the variety of enzymes produced by these eukaryotes has been an area of research interest for decades and again more recently due to global interest in greener bio-production technologies. Purification of an individual enzyme allows its unique biochemical and functional properties to be determined, can provide key information as to the role of individual biocatalysts within a complex enzyme system, and can inform both protein engineering and enzyme production strategies in the development of novel green technologies based on fungal biocatalysts. Many enzymes of current biotechnological interest are secreted by fungi into the extracellular culture medium. These crude enzyme mixtures are typically complex, multi-component, and generally also contain other non-enzymatic proteins and secondary metabolites. In this chapter, we describe a multi-step chromatographic strategy required to isolate three new endo-β-glucanases (denoted EG V, EG VI, and EG VII) with activity against cereal mixed-linkage β-glucans from the thermophilic fungus Talaromyces emersonii. This work also illustrates the challenges frequently involved in isolating individual extracellular fungal proteins in general.

[Scientific Evaluation of Crude Drugs and Kampo Medicines Using the Eastern Blotting Method and Its Application to Biological Metabolic Studies].

PubMed

Morinaga, Osamu

2018-01-01

　The scientific evaluation of crude drugs and kampo medicines (KMs) was demonstrated using the eastern blotting method with monoclonal antibodies (MAbs) against bioactive natural compounds. Scutellariae radix is one of the most important crude drugs used in KMs. Part of its pharmaceutical properties is due to the flavone glycoside baicalin (BI). A quantitative analysis method based on eastern blotting was developed for BI using an anti-BI MAb. A rapid, simple, sensitive, specific analytical system was subsequently established for BI with the eastern blotting technique using dot-blot and chemiluminescent methods. This system was useful as a high-throughput analytical method for the determination of BI in KMs as well as HPLC and enzyme-linked immunosorbent assay systems. Furthermore, an eastern blotting method was applied to the biological metabolic study of glycyrrhizic acid (GL), the major active constituent of licorice, for investigation of metabolites of GL such as 3-monoglucuronyl-glycyrrhetinic acid (3MGA) because licorice causes pseudoaldosteronism as a side effect. This approach may make it possible to determine the pathogenic agents of licorice-induced pseudoaldosteronism.

Enzymatic degradation of oligosaccharides in pinto bean flour.

PubMed

Song, Danfeng; Chang, Sam K C

2006-02-22

The use of dry edible beans is limited due to the presence of flatulence factors, the raffinose oligosaccharides. Our objective was to investigate the process for the removal of oligosaccharides from pinto bean using enzymatic treatment and to compare it to removal by soaking and cooking methods. Crude enzyme preparation was produced by six fungal species on wheat bran- and okara-based substrates with soy tofu whey. The loss of raffinose oligosaccharides after soaking pinto beans for 16 h at the room temperature was 10%, after cooking for 90 min was 52%, and after autoclaving for 30 min was 58%. On the other hand, the treatment using crude alpha-galactosidase (60 U mL(-1)) produced by Aspergillus awamori NRRL 4869 from wheat bran-based substrate with soy tofu whey on pinto bean flour for 2 h completely hydrolyzed raffinose oligosaccharides. These results supported that the enzymatic treatment was the most effective among various processing methods tested for removing the raffinose oligosaccharides, and hence, crude alpha-galactosidases from fungi have potential use in the food industry.

Effects of Different Pretreatments to Fresh Fruit on Chemical and Thermal Characteristics of Crude Palm Oil.

PubMed

Tang, Minmin; Xia, Qiuyu; Holland, Brendan J; Wang, Hui; Zhang, Yufeng; Li, Rui; Cao, Hongxing

2017-12-01

This study selected 5 methods, including boiling, hot air drying, high-pressurized steam, freezing, and microwave radiation to pretreat fresh oil palm fruit before solvent extraction of the oil. Using fresh fruit as a control, the pretreatment methods were compared for the effects on the activity of the 2 main enzymes in the fruit and some physicochemical properties of the crude palm oil. The results indicated, although all the 5 pretreatments could inactivate lipase and peroxidase in the treated flesh significantly (P < 0.05), the high-pressurized steam was the most effective. There were also differences in the unsaturated fatty acid contents of the 6 oils. The crude oil from frozen fruit contained significantly more vitamin E (37829.33 ppm) than previously reported. Microwave radiation was shown to significantly decrease the free fatty acid content and the peroxide value, while increasing the oxidative stability index. Thermal behaviors of the oils were significantly different to each other with the exception a few parameters (P < 0.05). © 2017 Institute of Food Technologists®.

Pilot-scale bioconversion of rice and sunflower agro-residues into medicinal mushrooms and laccase enzymes through solid-state fermentation with Ganoderma lucidum.

PubMed

Postemsky, P D; Bidegain, M A; González-Matute, R; Figlas, N D; Cubitto, M A

2017-05-01

Solid-state fermentation was evaluated at the pilot-scale for the bioconversion and valorization of rice husks and straw (RSH), or sunflower seed hulls (SSH), into medicinal mushrooms and crude extracts, with laccase activity. The average mushroom yield was 56kg dry weight per ton of agro-residues. Laccase activity in crude aqueous extracts showed its maximum value of 10,927Ukg -1 in RSH (day 10, Exudate phase) and 16,442Ukg -1 in SSH (day 5, Full colonization phase), the activity at the Residual substrate phase being 511Ukg -1 in RSH and 803Ukg -1 in SSH, respectively. Crude extracts obtained with various protocols revealed differences in the extraction yields. Lyophilization followed by storage at 4°C allowed the preservation of laccase activity for more than one month. It is proposed that standard mushroom farms could increase their profits by obtaining laccase as a byproduct during the gaps in mycelium running. Copyright © 2017 Elsevier Ltd. All rights reserved.

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.

Hydrometallurgical methods of recovery of scandium from the wastes of various technologies

NASA Astrophysics Data System (ADS)

Molchanova, T. V.; Akimova, I. D.; Smirnov, K. M.; Krylova, O. K.; Zharova, E. V.

2017-03-01

The recovery of scandium from the wastes of the production of uranium, titanium, iron-vanadium, and alumina is studied. The applied acid schemes of scandium transfer to a solution followed by ion-exchange recovery and extraction concentration of scandium ensure the precipitation of crude scandium oxides containing up to 5% Sc2O3. Scandium oxides of 99.96-99.99% purity are formed after additional refining of these crude oxides according to an extraction technology using a mixture 15% multiradical phosphine oxide or Cyanex-925 + 15% tributyl phosphate in kerosene.

On Boiling of Crude Oil under Elevated Pressure

NASA Astrophysics Data System (ADS)

Pimenova, Anastasiya V.; Goldobin, Denis S.

2016-02-01

We construct a thermodynamic model for theoretical calculation of the boiling process of multicomponent mixtures of hydrocarbons (e.g., crude oil). The model governs kinetics of the mixture composition in the course of the distillation process along with the boiling temperature increase. The model heavily relies on the theory of dilute solutions of gases in liquids. Importantly, our results are applicable for modelling the process under elevated pressure (while the empiric models for oil cracking are not scalable to the case of extreme pressure), such as in an oil field heated by lava intrusions.

Enzymatic synthesis of dithiolopyrrolone antibiotics using cell-free extract of Saccharothrix algeriensis NRRL B-24137 and biochemical characterization of two pyrrothine N-acyltransferases in this extract.

PubMed

Saker, Safwan; Almousa Almaksour, Ziade; Chorin, Anne-Claire; Lebrihi, Ahmed; Mathieu, Florence

2014-01-01

Saccharothrix algeriensis NRRL B-24137 produces naturally different dithiolopyrrolone derivatives. The enzymatic activity of pyrrothine N-acyltransferase was determined to be responsible for the transfer of an acyl group from acyl-CoA to pyrrothine core. This activity was also reported to be responsible for the diversity of the dithiolopyrrolone derivatives. Based on this fact, nine dithiolopyrrolone derivatives were produced in vitro via the crude extract of Sa. algeriensis. Three of them have never been obtained before by natural fermentation: acetoacetyl-pyrrothine, hydroxybutyryl-pyrrothine, and dimethyl thiolutin (holomycin). Two acyltransferase activities, acetyltransferase and benzoyltransferase catalyzing the incorporation of linear and cyclic acyl groups to the pyrrothine core, respectively, were biochemically characterized in this crude extract. The first one is responsible for formation of acetyl-pyrrothine and the second for benzoyl-pyrrothine. Both enzymes were sensitive to temperature changes: For example, the loss of acetyltransferase and benzoyltransferase activity was 53% and 80% respectively after pre-incubation of crude extract for 60 min at 20°C. The two enzymes were more active in neutral and basal media (pH 7-10) than in the acidic one (pH 3-6). The optimum temperature and pH of acetyltransferase were 40°C and 7, with a Km value of 7.9 μM and a Vmax of 0.63 μM/min when acetyl-CoA was used as limited substrate. Benzoyltransferase had a temperature and a pH optimum at 55°C and 9, a Km value of 14.7 μM, and a Vmax of 0.67 μM/min when benzoyl- CoA was used as limited substrate.

Characterization of thermostable cellulase produced by Bacillus strains isolated from solid waste of carrageenan

NASA Astrophysics Data System (ADS)

Listyaningrum, N. P.; Sutrisno, A.; Wardani, A. K.

2018-03-01

Cellulase-producing bacteria was isolated from solid waste of carrageenan and identified as Bacillus licheniformis C55 by 16S rRNA sequencing. The optimum condition for cellulase production was obtained at pH and temperature of 8.0 and 50°C, respectively in a medium containing glucose as carbon source and 1.0% carboxymethyl cellulose (CMC) to stimulate the cellulase production. Most remarkably, the enzyme retained its relative activity over 50% after incubation at 50°C for 90 minutes. Substrate specificity suggested that the enzyme is an endoglucanase. The molecular mass of Bacillus licheniformis C55 crude cellulase was found about 18 kDa by SDS-PAGE analysis. This thermostable enzyme would facilitate development of more efficient and cost-effective forms of the process to convert lignocellulosic biomass into high-value products.

Ion-Exchange Chromatography: Basic Principles and Application.

PubMed

Cummins, Philip M; Rochfort, Keith D; O'Connor, Brendan F

2017-01-01

Ion-Exchange Chromatography (IEC) allows for the separation of ionizable molecules on the basis of differences in charge properties. Its large sample-handling capacity, broad applicability (particularly to proteins and enzymes), moderate cost, powerful resolving ability, and ease of scale-up and automation have led to it becoming one of the most versatile and widely used of all liquid chromatography (LC) techniques. In this chapter, we review the basic principles of IEC, as well as the broader criteria for selecting IEC conditions. By way of further illustration, we outline basic laboratory protocols to partially purify a soluble serine peptidase from bovine whole brain tissue, covering crude tissue extract preparation through to partial purification of the target enzyme using anion-exchange chromatography. Protocols for assaying total protein and enzyme activity in both pre- and post-IEC fractions are also described.

Characterization of arylalkylamine N-acetyltransferase (AANAT) activities and action spectrum for suppression in the band-legged cricket, Dianemobius nigrofasciatus (Orthoptera: Gryllidae).

PubMed

Izawa, Norimitsu; Suzuki, Takeshi; Watanabe, Masakatsu; Takeda, Makio

2009-04-01

Arylalkylamine N-acetyltransferase (AANAT), constituting a large family of enzymes, catalyzes the transacetylation from acetyl-CoA to monoamine substrates, although homology among species is not very high. AANAT in vertebrates is photosensitive and mediates circadian regulation. Here, we analyzed AANAT of the cricket, Dianemobius nigrofasciatus. The central nervous system contained AANAT activity. The optimum pHs were 6.0 (a minor peak) and 10.5 (a major peak) with crude enzyme solution. We analyzed the kinetics at pH 10.5 using the sample containing collective AANAT activities, which we term AANAT. Lineweaver-Burk plot and secondary plot yielded a K(m) for tryptamine as substrate of 0.42 microM, and a V(max) of 9.39 nmol/mg protein/min. The apparent K(m) for acetyl-CoA was 59.9 microM and the V(max) was 8.14 nmol/mg protein/min. AANAT of D. nigrofasciatus was light-sensitive. The activity was higher at night-time than at day-time as in vertebrates. To investigate most effective wavelengths on AANAT activity, a series of monochromatic lights was applied (350, 400, 450, 500, 550, 600 and 650 nm). AANAT showed the highest sensitivity to around 450 nm and 550 nm. 450 nm light was more effective than 550 nm light. Therefore, the most effective light affecting AANAT activity is blue light, which corresponds to the absorption spectrum of blue wave (BW)-opsin.

Chlorine and sulfur determination in extra-heavy crude oil by inductively coupled plasma optical emission spectrometry after microwave-induced combustion

NASA Astrophysics Data System (ADS)

Pereira, Juliana S. F.; Mello, Paola A.; Moraes, Diogo P.; Duarte, Fábio A.; Dressler, Valderi L.; Knapp, Guenter; Flores, Érico M. M.

2009-06-01

In this study, microwave-induced combustion (MIC) of extra-heavy crude oil is proposed for further chlorine and sulfur determination by inductively coupled plasma optical emission spectrometry (ICP OES). Combustion was carried out under oxygen pressure (20 bar) in quartz vessels using ammonium nitrate (50 µl of 6 mol l - 1 solution) as ignition aid. Samples were wrapped with polyethylene film and placed on a quartz holder positioned inside the quartz vessels. The need for an additional reflux step after combustion and the type and concentration of absorbing solution (water, 0.02 to 0.9 mmol l - 1 H 2O 2, 10 to 100 mmol l - 1 (NH 4) 2CO 3 or 0.1 to 14 mol l - 1 HNO 3) were studied. The influence of sample mass, O 2 pressure and maximum pressure attained during the combustion process were investigated. Recoveries from 92 to 102% were obtained for Cl and S for all absorbing solutions. For comparison, Cl and S determination was also performed by ion chromatography (IC) using 25 mmol l - 1 (NH 4) 2CO 3 as absorbing solution. Using MIC with a reflux step the agreement was better than 95% for certified reference materials of similar composition (crude oil, petroleum coke, coal and residual fuel oil). Microwave-assisted digestion and water extraction in high pressure closed vessels were also evaluated. Using these procedures the maximum recoveries were 30 and 98% for Cl and S, respectively, using microwave-assisted digestion and 70% for Cl and less than 1% for S by water extraction procedure. Limits of detection by ICP OES were 12 and 5 µg g - 1 for Cl and S, respectively, and the corresponding values by IC were 1.2 and 8 µg g - 1 . Using MIC it was possible to digest simultaneously up to eight samples resulting in a solution suitable for the determination of both analytes with a single combustion step.

On-line coupling of counter-current chromatography and macroporous resin chromatography for continuous isolation of arctiin from the fruit of Arctium lappa L.

PubMed

Guo, Mengzhe; Liang, Junling; Wu, Shihua

2010-08-13

In this work, we have developed a novel hybrid two-dimensional counter-current chromatography and liquid chromatography (2D CCC x LC) system for the continuous purification of arctiin from crude extract of Arctium lappa. The first dimensional CCC column has been designed to fractionalize crude complex extract into pure arctiin effluent using a one-component organic/salt-containing system, and the second dimensional LC column has been packed with macroporous resin for on-line adsorption, desalination and desorption of arctiin which was effluent purified from the first CCC dimension. Thus, the crude arctiin mixture has been purified efficiently and conveniently by on-line CCC x LC in spite of the use of a salt-containing solvent system in CCC separation. As a result, high purity (more than 97%) of arctiin has been isolated by repeated injections both using the ethyl acetate-8% sodium chloride aqueous solution and butanol-1% sodium chloride aqueous solution. By contrast with the traditional CCC processes using multi-component organic/aqueous solvent systems, the present on-line CCC x LC process only used a one-component organic solvent and thus the solvent is easier to recover and regenerate. All of used solvents such as ethyl acetate, n-butanol and NaCl aqueous solution are low toxicity and environment-friendly. Moreover, the lower phase of salt-containing aqueous solution used as mobile phase, only contained minor organic solvent, which will save much organic solvent in continuous separation. In summary, our results indicated that the on-line hybrid 2D CCC x LC system using one-component organic/salt-containing aqueous solution is very promising and powerful tool for high-throughput purification of arctiin from fruits of A. lappa. 2010 Elsevier B.V. All rights reserved.

Antimicrobial activity of broccoli (Brassica oleracea var. italica) cultivar Avenger against pathogenic bacteria, phytopathogenic filamentous fungi and yeast.

PubMed

Pacheco-Cano, R D; Salcedo-Hernández, R; López-Meza, J E; Bideshi, D K; Barboza-Corona, J E

2018-01-01

The objective of this study was to show whether the edible part of broccoli has antibacterial and antifungal activity against micro-organism of importance in human health and vegetable spoilage, and to test if this effect was partially due to antimicrobial peptides (AMPs). Crude extracts were obtained from florets and stems of broccoli cultivar Avenger and the inhibitory effect was demonstrated against pathogenic bacteria (Bacillus cereus, Staphylococcus xylosus, Staphylococcus aureus, Shigella flexneri, Shigella sonnei, Proteus vulgaris), phytopathogenic fungi (Colletotrichum gloeosporioides, Asperigillus niger) and yeasts (Candida albicans and Rhodotorula sp.). It was shown that samples treated with proteolytic enzymes had a reduction of approximately 60% in antibacterial activity against Staph. xylosus, suggesting that proteinaceous compounds might play a role in the inhibitory effect. Antimicrobial components in crude extracts were thermoresistant and the highest activity was observed under acidic conditions. It was shown that antifungal activity of broccoli's crude extracts might not be attributed to chitinases. Organic broccoli cultivar Avenger has antimicrobial activity against pathogenic bacteria, yeast and phytophatogenic fungi. Data suggest that this effect is partially due to AMPs. Broccoli's crude extracts have activity not only against pathogenic bacteria but also against phytophatogenic fungi of importance in agriculture. We suggest for first time that the inhibitory effect is probably due to AMPs. © 2017 The Society for Applied Microbiology.

A cell-free framework for rapid biosynthetic pathway prototyping and enzyme discovery.

PubMed

Karim, Ashty S; Jewett, Michael C

2016-07-01

Speeding up design-build-test (DBT) cycles is a fundamental challenge facing biochemical engineering. To address this challenge, we report a new cell-free protein synthesis driven metabolic engineering (CFPS-ME) framework for rapid biosynthetic pathway prototyping. In our framework, cell-free cocktails for synthesizing target small molecules are assembled in a mix-and-match fashion from crude cell lysates either containing selectively enriched pathway enzymes from heterologous overexpression or directly producing pathway enzymes in lysates by CFPS. As a model, we apply our approach to n-butanol biosynthesis showing that Escherichia coli lysates support a highly active 17-step CoA-dependent n-butanol pathway in vitro. The elevated degree of flexibility in the cell-free environment allows us to manipulate physiochemical conditions, access enzymatic nodes, discover new enzymes, and prototype enzyme sets with linear DNA templates to study pathway performance. We anticipate that CFPS-ME will facilitate efforts to define, manipulate, and understand metabolic pathways for accelerated DBT cycles without the need to reengineer organisms. Copyright © 2016 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

Laccase produced by a thermotolerant strain of Trametes trogii LK13

PubMed Central

Yan, Jinping; Chen, Yuhui; Niu, Jiezhen; Chen, Daidi; Chagan, Irbis

2015-01-01

Thermophilic and thermotolerant micro-organisms strains have served as the natural source of industrially relevant and thermostable enzymes. Although some strains of the Trametes genus are thermotolerant, few Trametes strains were studied at the temperature above 30 °C until now. In this paper, the laccase activity and the mycelial growth rate for Trametes trogii LK13 are superior at 37 °C. Thermostability and organic cosolvent tolerance assays of the laccase produced at 37 °C indicated that the enzyme possessed fair thermostability with 50% of its initial activity at 80 °C for 5 min, and could remain 50% enzyme activity treated with organic cosolvent at the concentration range of 25%–50% (v/v). Furthermore, the test on production of laccase and lignocellulolytic enzymes showed the crude enzymes possessed high laccase level (1000 U g −1 ) along with low cellulose (2 U g −1 ) and xylanase (140 U g −1 ) activity. Thus, T. trogii LK13 is a potential strain to be applied in many biotechnological processes. PMID:26221089

Salt dependent resistance against chemical denaturation of alkaline protease from a newly isolated haloalkaliphilic Bacillus sp.

PubMed

Dodia, M S; Bhimani, H G; Rawal, C M; Joshi, R H; Singh, S P

2008-09-01

Only few enzymes from haloalkaliphiles are biochemically characterized for their kinetic behaviour and stability. In view of this realization, an alkaline protease from Bacillus sp. AH-6, displaying salt-dependent resistance against chemical denaturation by Urea and Guanidium hydrochloride was investigated for denaturation and in vitro protein folding. The crude enzyme was highly resistant against urea (8 M) denaturation up to 72 h; however, on purification, it turned sensitive and got denatured within 2 h. Interestingly, the purified enzyme regained the resistance in the presence of NaCl. Effective refolding of the purified enzyme was achieved with glycerol; however, other approaches such as lower protein concentrations, rapid dilution and slow removal of the denaturant did not further add to refolding. The results are important from the viewpoint that only few enzymes from haloalkaliphilic bacteria are characterized. Since the resistance against chemical denaturation is a rare phenomenon, the findings would enrich the knowledge on protein stability and denaturation. Besides, such biocatalysts would definitely have novel applications under harsh chemical environments.

Characterization of a novel 8R,11S-linoleate diol synthase from Penicillium chrysogenum by identification of its enzymatic products[S

PubMed Central

Shin, Kyung-Chul; Seo, Min-Ju; Oh, Deok-Kun

2016-01-01

To identify novel fatty acid diol synthases, putative candidate sequences from Penicillium species were analyzed, and hydroxy fatty acid production by crude Penicillium enzyme extracts was assessed. Penicillium chrysogenum was found to produce an unknown dihydroxy fatty acid, a candidate gene implicated in this production was cloned and expressed, and the expressed enzyme was purified. The product obtained by the reaction of the purified enzyme with linoleic acid was identified as 8R,11S-dihydroxy-9,12(Z,Z)-octadecadienoic acid (8R,11S-DiHODE). The catalytic efficiency of this enzyme toward linoleic acid was the highest among the unsaturated fatty acids tested, indicating that this enzyme was a novel 8R,11S-linoleate diol synthase (8R,11S-LDS). A sexual stage in the life cycle of P. chrysogenum has recently been discovered, and 8R,11S-DiHODE produced by 8R,11S-LDS may constitute a precocious sexual inducer factor, responsible for regulating the sexual and asexual cycles of this fungus. PMID:26681780

Purification and Characterization of 1-Aminocyclopropane-1-Carboxylate Synthase from Apple Fruits 1

PubMed Central

Yip, Wing-Kin; Dong, Jian-Guo; Yang, Shang Fa

1991-01-01

1-Aminocyclopropane-1-carboxylate (ACC) synthase, a key enzyme in ethylene biosynthesis, was isolated and partially purified from apple (Malus sylvestris Mill.) fruits. Unlike ACC synthase isolated from other sources, apple ACC synthase is associated with the pellet fraction and can be solubilized in active form with Triton X-100. Following five purification steps, the solubilized enzyme was purified over 5000-fold to a specific activity of 100 micromoles per milligram protein per hour, and its purity was estimated to be 20 to 30%. Using this preparation, specific monoclonal antibodies were raised. Monoclonal antibodies against ACC synthase immunoglobulin were coupled to protein-A agarose to make an immunoaffinity column, which effectively purified the enzyme from a relatively crude enzyme preparation (100 units per milligram protein). As with the tomato enzyme, apple ACC synthase was inactivated and radiolabeled by its substrate S-adenosyl-l-methionine. Apple ACC synthase was identified to be a 48-kilodalton protein based on the observation that it was specifically bound to immunoaffinity column and it was specifically radiolabeled by its substrate S-adenosyl-l-methionine. Images Figure 4 Figure 6 PMID:16667960

Photodegradation of crude oil: liquid injection and headspace solid-phase microextraction for crude oil analysis by gas chromatography with mass spectrometer detector.

PubMed

D'Auria, Maurizio; Racioppi, Rocco; Velluzzi, Vincenzina

2008-04-01

The fate of crude oil under irradiation is studied. After UV irradiation, the fraction present in the highest percentage shifts from the C8-C9 fractions to C13, using gas chromatography-mass spectrometry (GC-MS) analysis in solution. An increase of the relative amount of the C13-C25 fraction is observed, while a decrease in the relative amount of the C7-C12 fractions is present. In headspace solid-phase microextraction (HS-SPME) analysis, the C8-C10 fractions represent 53% of all the compounds detected. A decrease in the relative amount of the C8-C10 fractions is observed, while C11-C15 fractions increase. The irradiation of crude oil with a solar simulator gives a mixture the analysis of which using GC-MS in solution furnishes the same type of results: the relative amounts of linear alkanes and aromatic compounds increase, while a sharp decrease in the relative amounts of branched and cyclic alkanes is observed. In the SPME analysis, a decreased relative amount of branched alkanes and alkenes, and an increase in the relative amounts of cyclic alkanes and aromatic compounds are observed. Analysis of the distribution of the compounds in all the types of compound shows that a dynamic equilibrium between different compounds and different types of compounds is present. To confirm the presence of a dynamic equilibrium, the irradiation of methylcyclohexane in the presence of 2-methylnaphthalene shows the presence in the reaction mixture of a small amount of tetradecane.

Exploring the inhibitory activity of Withaferin-A against Pteridine reductase-1 of L. donovani.

PubMed

Chandrasekaran, Sambamurthy; Veronica, Jalaja; Gundampati, Ravi Kumar; Sundar, Shyam; Maurya, Radheshyam

2016-12-01

Withaferin A is an abundant withanolide present in Withania somnifera leaves and to some extent in roots. It has been known for its profound anti-cancer properties, but its role in counteracting the Leishmania donovani infection has to be explored. Pteridine reductase 1 (PTR1) is involved in pteridine salvage and an important enzyme for the parasite growth, which could be targeted for the development of an efficient antileishmanial drug. We employed molecular docking studies to identify the binding mode of withaferin A with PTR1 in silico. We further cloned, expressed, and purified PTR1 of L. donovani and performed the enzyme kinetics using the Michaelis-Menten equation and enzyme inhibition studies with withaferin A by plotting the Lineweaver-Burk graph, which followed an uncompetitive mode of inhibition. We also showed the inhibition of the enzyme in the crude lysate of treated parasites. Thus, our study contributes towards understanding the mode of action of withaferin A against L. donovani parasite.

Evaluation of microwave-assisted pretreatment of lignocellulosic biomass immersed in alkaline glycerol for fermentable sugars production.

PubMed

Diaz, Ana Belen; Moretti, Marcia Maria de Souza; Bezerra-Bussoli, Carolina; Carreira Nunes, Christiane da Costa; Blandino, Ana; da Silva, Roberto; Gomes, Eleni

2015-06-01

A pretreatment with microwave irradiation was applied to enhance enzyme hydrolysis of corn straw and rice husk immersed in water, aqueous glycerol or alkaline glycerol. Native and pretreated solids underwent enzyme hydrolysis using the extract obtained from the fermentation of Myceliophthora heterothallica, comparing its efficiency with that of the commercial cellulose cocktail Celluclast®. The highest saccharification yields, for both corn straw and rice husk, were attained when biomass was pretreated in alkaline glycerol, method that has not been previously reported in literature. Moreover, FTIR, TG and SEM analysis revealed a more significant modification in the structure of corn straw subjected to this pretreatment. Highest global yields were attained with the crude enzyme extract, which might be the result of its content in a great variety of hydrolytic enzymes, as revealed zymogram analysis. Moreover, its hydrolysis efficiency can be improved by its supplementation with commercial β-glucosidase. Copyright © 2015 Elsevier Ltd. All rights reserved.

Penicillium subrubescens is a promising alternative for Aspergillus niger in enzymatic plant biomass saccharification.

PubMed

Mäkelä, Miia R; Mansouri, Sadegh; Wiebenga, Ad; Rytioja, Johanna; de Vries, Ronald P; Hildén, Kristiina S

2016-12-25

In industrial applications, efficient mixtures of polysaccharide-degrading enzymes are needed to convert plant biomass into fermentable sugars. Most of the commercially produced lignocellulolytic enzymes are from a limited number of filamentous fungi, such as Trichoderma and Aspergillus species. In contrast, the plant biomass-degrading capacity of Penicillia has been less explored. We performed growth profiling of several Penicillia on diverse plant biomass-related substrates demonstrating the capacity particularly of Penicillium subrubescens to degrade crude lignocellulose feedstock, as well as polysaccharides, and metabolise their monomeric components. We focussed on the lignocellulolytic potential of P. subrubescens FBCC1632, which produced a variable set of (hemi-)cellulolytic activities on plant biomass substrates with activity levels comparable to those of Aspergillus niger. The good ability of the extracellular enzyme mixtures produced by P. subrubescens to saccharify complex plant biomasses, wheat bran and sugar beet pulp, indicated a high potential for this strain as a producer of industrial enzyme cocktails. Copyright © 2016 Elsevier B.V. All rights reserved.

Effects of dietary stachyose on growth performance, digestive enzyme activities and intestinal morphology of juvenile turbot ( Scophthalmus maximus L)

NASA Astrophysics Data System (ADS)

Hu, Haibin; Zhang, Yanjiao; Mai, Kangsen; Ai, Qinghui; Xu, Wei; Zhang, Wenbing; Li, Yanxian; Liu, Jintao

2015-10-01

A 12-week feeding trial was conducted to evaluate the effects of dietary stachyose on the growth performance, digestive enzymes activities and intestinal structures of juvenile turbot ( Scophthalmus maximus L). Five isonitrogenous (49.58% crude protein) and isolipidic (10.50% crude lipid) diets were formulated to contain 0 (Control), 0.625% (S-0.625), 1.25% (S-1.25), 2.5% (S-2.5) and 5% (S-5) stachyose, respectively. With the increase of stachyose level, the growth performance and feed utilization of turbot, such as the specific growth rate, final mean body weight, weight gain rate and feed efficiency, increased significantly ( P< 0.05) and then stabilized. The feed intake of fish fed S-5 was significantly higher ( P< 0.05) than that of fish in other groups. The activities of trypsin, intestinal caseinolytic, stomach and intestinal amylase were significantly influenced by stachyose ( P<0.05). The highest values of trypsin and intestinal caseinolytic activities were observed in group S-1.25, while the highest activity of stomach amylase and the lowest activity of intestine amylase were observed in group S-5. No lesion or damage was found on the distal intestine structures of fish from all treatments, while the height of simple folds in the distal intestine was significantly increased ( P< 0.05) when 1.25% or 2.5% stachyose was added in the diets. These results indicated that moderate level of stachyose (1.25%) improves the growth performance, feed utilization, digestive enzyme activities and the distal intestine structures of juvenile turbot.

Controlling Vapor Pressure In Hanging-Drop Crystallization

NASA Technical Reports Server (NTRS)

Carter, Daniel C.; Smith, Robbie

1988-01-01

Rate of evaporation adjusted to produce larger crystals. Device helps to control vapor pressure of water and other solvents in vicinity of hanging drop of solution containing dissolved enzyme protein. Well of porous frit (sintered glass) holds solution in proximity to drop of solution containing protein or enzyme. Vapor from solution in frit controls evaporation of solvent from drop to control precipitation of protein or enzyme. With device, rate of nucleation limited to decrease number and increase size (and perhaps quality) of crystals - large crystals of higher quality needed for x-ray diffraction studies of macromolecules.

Distinct Kynureninase and Hydroxykynureninase Activities in Microorganisms: Occurrence and Properties of a Single Physiologically Discrete Enzyme in Yeast

PubMed Central

Shetty, A. S.; Gaertner, F. H.

1973-01-01

(i) Saccharomyces cerevisiae grown in the presence of 1.0 mM l-tryptophan slowly excreted fluorescent material that was chromatographically identifiable as 3-hydroxyanthranilate but did not excrete detectable amounts of anthranilate nor rapidly deplete the medium of l-tryptophan. Under similar growth conditions, Neurospora crassa rapidly excretes anthranilate and rapidly depletes the medium of l-tryptophan. (ii) Chromatographic analysis of crude extracts from yeast revealed a single kynureninase-type enzyme whose synthesis was not measurably affected by the presence of tryptophan in the medium. Previous studies have provided evidence for two kynureninase-type enzymes in N. crassa, an inducible kynureninase and a constitutive hydroxykynureninase. (iii) Kinetic analysis of the partially purified yeast enzyme provided Michaelis constants for l-3-hydroxykynurenine and l-kynurenine of 6.7 × 10−6 and 5.4 × 10−4 M, respectively. This and other kinetic properties of the yeast enzyme are comparable to those reported for the constitutive enzyme from N. crassa. (iv) These findings suggest that S. cerevisiae has in common with N. crassa the biosynthetic enzyme hydroxykynureninase but lacks the catabolic enzyme kynureninase. Therefore, it can be predicted that, unlike N. crassa, S. cerevisiae does not carry out the tryptophan-anthranilate cycle. Distinct kynureninase-type enzymes may exist in other microorganisms and in mammals. PMID:4266242

Optimization of enzymatic hydrolysis and fermentation conditions for improved bioethanol production from potato peel residues.

PubMed

Ben Taher, Imen; Fickers, Patrick; Chniti, Sofien; Hassouna, Mnasser

2017-03-01

The aim of this work was the optimization of the enzyme hydrolysis of potato peel residues (PPR) for bioethanol production. The process included a pretreatment step followed by an enzyme hydrolysis using crude enzyme system composed of cellulase, amylase and hemicellulase, produced by a mixed culture of Aspergillus niger and Trichoderma reesei. Hydrothermal, alkali and acid pretreatments were considered with regards to the enhancement of enzyme hydrolysis of potato peel residues. The obtained results showed that hydrothermal pretreatment lead to a higher enzyme hydrolysis yield compared to both acid and alkali pretreatments. Enzyme hydrolysis was also optimized for parameters such as temperature, pH, substrate loading and surfactant loading using a response surface methodology. Under optimized conditions, 77 g L -1 of reducing sugars were obtained. Yeast fermentation of the released reducing sugars led to an ethanol titer of 30 g L -1 after supplementation of the culture medium with ammonium sulfate. Moreover, a comparative study between acid and enzyme hydrolysis of potato peel residues was investigated. Results showed that enzyme hydrolysis offers higher yield of bioethanol production than acid hydrolysis. These results highlight the potential of second generation bioethanol production from potato peel residues treated with onsite produced hydrolytic enzymes. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:397-406, 2017. © 2017 American Institute of Chemical Engineers.

Assembly and multiple gene expression of thermophilic enzymes in Escherichia coli for in vitro metabolic engineering.

PubMed

Ninh, Pham Huynh; Honda, Kohsuke; Sakai, Takaaki; Okano, Kenji; Ohtake, Hisao

2015-01-01

In vitro reconstitution of an artificial metabolic pathway is an emerging approach for the biocatalytic production of industrial chemicals. However, several enzymes have to be separately prepared (and purified) for the construction of an in vitro metabolic pathway, thereby limiting the practical applicability of this approach. In this study, genes encoding the nine thermophilic enzymes involved in a non-ATP-forming chimeric glycolytic pathway were assembled in an artificial operon and co-expressed in a single recombinant Escherichia coli strain. Gene expression levels of the thermophilic enzymes were controlled by their sequential order in the artificial operon. The specific activities of the recombinant enzymes in the cell-free extract of the multiple-gene-expression E. coli were 5.0-1,370 times higher than those in an enzyme cocktail prepared from a mixture of single-gene-expression strains, in each of which a single one of the nine thermophilic enzymes was overproduced. Heat treatment of a crude extract of the multiple-gene-expression cells led to the denaturation of indigenous proteins and one-step preparation of an in vitro synthetic pathway comprising only a limited number of thermotolerant enzymes. Coupling this in vitro pathway with other thermophilic enzymes including the H2 O-forming NADH oxidase or the malate/lactate dehydrogenase facilitated one-pot conversion of glucose to pyruvate or lactate, respectively. © 2014 Wiley Periodicals, Inc.

SUSTAINABLE PACKAGING SOLUTIONS BASED ON BIODEGRADABLE PLASTICS

EPA Science Inventory

Packaging is one of the largest market segments for the polymer industry. Food packaging industry is currently dominated by crude oil-derived, non-biodegradable polyolefin and polyesters. Due to their environmental persistence (non-biodegradability) leading to accumulatio...

An experimental study on the bio-surfactant-assisted remediation of crude oil and salt contaminated soils.

PubMed

Zhang, Wen; Li, Jianbing; Huang, Guohe; Song, Weikun; Huang, Yuefei

2011-01-01

The effect of bio-surfactant (rhamnolipid) on the remediation of crude oil and salt contaminated soil was investigated in this study. The experimental results indicated that there was a distinct decline of total petroleum hydrocarbon (TPH) concentration within the soil when using rhamnolipid during a remediation period of 30 days, with maximum TPH reduction of 86.97%. The most effective remediation that was observed was with rhamnolipid at a concentration of 2 CMC in soil solution, and a first-order TPH degradation rate constant of 0.0866 d(-1). The results also illustrated that salts in soil had a negative impact on TPH reduction, and the degradation rate was negatively correlated with NaCl concentration in soil solution. The analysis of soil TPH fractions indicated that there was a significant reduction of C13-C30 during the remediation process when using bio-surfactant.

Alternative fuels

NASA Technical Reports Server (NTRS)

Grobman, J. S.; Butze, H. F.; Friedman, R.; Antoine, A. C.; Reynolds, T. W.

1977-01-01

Potential problems related to the use of alternative aviation turbine fuels are discussed and both ongoing and required research into these fuels is described. This discussion is limited to aviation turbine fuels composed of liquid hydrocarbons. The advantages and disadvantages of the various solutions to the problems are summarized. The first solution is to continue to develop the necessary technology at the refinery to produce specification jet fuels regardless of the crude source. The second solution is to minimize energy consumption at the refinery and keep fuel costs down by relaxing specifications.

[Inhibition effects of Houttuynia cordata Thunb. on Microcystis aeruginosa].

PubMed

Liu, Lu; Li, Cheng; Xia, Wentong; Yang, Xiaohui; Zhang, Tingting

2014-05-01

To research the inhibitory effect of Houttuynia cordata Thunb. on Microcystis aeruginosa. M. aeruginosat were treated respectively by H. cordata leaching solution or H. cordata extracts. H. cordata leaching solution extracted by water and the H. cordata extracts extracted by organic solvent (acetone, ethyl acetate, petroleum ether and ethanol, respectively). The inhibition ratios were calculated according to the M. aeruginosa densities, and the allelochemicals of the extract that had the best inhibitiory effect on M. aeruginosa were identified by GC-MS analysis. It was proved that leaching solution of H. cordata and four crude extracts had good inhibitory effect on M. aeruginosa. The inhibitory effects of the four crude extracts were the fraction extracted by ethyl acetate, the fraction extracted by ethanol, the fraction extracted by acetone and the fraction extracted by petroleum ether form strong to weak in turn. Then, the allelochemicals of the fraction extracted by ethyl acetate were indentified, mainly including acetonyldimethylcarbinol, 2,2-dimethyl-3-hexanone, 6-chlorohexanoic and 4-cyanophenyl ester. H. cordata has strong inhibitory effect on water-blooming cyanobacteria and the potential to develop into an ecological M. aeruginosa inhibiting agent.

Guaiacol peroxidase zymography for the undergraduate laboratory.

PubMed

Wilkesman, Jeff; Castro, Diana; Contreras, Lellys M; Kurz, Liliana

2014-01-01

This laboratory exercise presents a novel way to introduce undergraduate students to the specific detection of enzymatic activity by electrophoresis. First, students prepare a crude peroxidase extract and then analyze the homogenate via electrophoresis. Zymography, that is, a SDS-PAGE method to detect enzyme activity, is used to specifically detect peroxidase activity and furthermore, to analyze the total protein profile. After the assay, students may estimate the apparent molecular mass of the enzyme and discuss its structure. After the 4-h experiment, students gain knowledge concerning biological sample preparation, gel preparation, electrophoresis, and the importance of specific staining procedures for the detection of enzymatic activity. Copyright © 2014 The International Union of Biochemistry and Molecular Biology.

[Polyketone Reaction in Biosynthetic Pathways of 2, 3, 5, 4'-Tetrahydroxy Stilhene-2-O-β-D-glucoside in Polygonum multiflorum by Biocatalysis].

PubMed

Lei, Lei; Xia, Wan-xia; Shao, Li; Zhao, Shu-jin

2015-10-01

2, 3, 5, 4'-Tetrahydroxy stilbene-2-O-β-D-glucoside (THSG), the active ingredient of Polygonum multiflorum, its polyketone reaction in the biosynthesis pathways was studied by biocatalysis method. The substrates 4-coumaroyl-CoA and malonyl-CoA were catalyzed in vitro by the crude enzyme extracted from Polygonum multiflorum callus, then the products were verified by HPLC and LC-MS methods. And the crude enzyme was analyzed by ammonium sulfate precipitation method and SDS-PAGE. HPLC chromatogram showed the same retention time of both the product and resveratrol standards; LC-MS spectra showed that the m/z of product was 227, which was consistent with resveratrol standards under the mode of negative ion; Ammonium sulfate (AS) precipitation method showed AS of 40% - 70% had catalytic activity,and 50% - 60% was the optimum; SDS-PAGE showed protein bands were obviously different among different AS concentration between 20% - 80%, the protein band of 42 kDa was found in AS of 50% - 60%, which had the same molecular weight with stilbene synthase. The product of polyketone reaction in the biosynthesis of THSG is resveratrol rather than THSG, so it is speculated that THSG is the conversion product of resveratrol instead of the direct product of the polyketone reaction.

White-rot fungus Ganoderma sp.En3 had a strong ability to decolorize and tolerate the anthraquinone, indigo and triphenylmethane dye with high concentrations.

PubMed

Lu, Ruoying; Ma, Li; He, Feng; Yu, Dong; Fan, Ruozhi; Zhang, Yangming; Long, Zheping; Zhang, Xiaoyu; Yang, Yang

2016-03-01

The ability of the white-rot fungus Ganoderma sp.En3 to decolorize different kinds of dyes widely applied in the textile and dyeing industry, including the anthraquinone dye Remazol Brilliant Blue R (RBBR), indigo dye indigo carmine and triphenylmethane dye methyl green, was evaluated in this study. Ganoderma sp.En3 had a strong capability of decolorizing high concentrations of RBBR, indigo carmine and methyl green. Obvious reduction of Chemical Oxygen Demand was observed after decolorization of different dyes. Ganoderma sp.En3 had a strong ability to tolerate RBBR, indigo carmine and methyl green with high concentrations. High concentrations of RBBR, indigo carmine and methyl green could also be efficiently decolorized by the crude enzyme of Ganoderma sp.En3. Different redox mediators such as syringaldehyde, acetosyringone and acetovanillone could enhance the decolorization capability for higher concentration of indigo carmine and methyl green. Different metal ions had little effect on the ability of the crude enzyme to decolorize indigo carmine and methyl green. Our study suggested that Ganoderma sp.En3 had a strong capability for decolorizing and tolerating high concentrations of different types of dyes such as RBBR, indigo carmine and methyl green.

Characterising the enzymatic profile of crude tentacle extracts from the South Atlantic jellyfish Olindias sambaquiensis (Cnidaria: Hydrozoa).

PubMed

Knittel, Paloma S; Long, Paul F; Brammall, Lucas; Marques, Antonio C; Almeida, Michelle T; Padilla, Gabriel; Moura-da-Silva, Ana M

2016-09-01

Jellyfish venoms are of medical and biotechnological importance, with toxins displaying antimicrobial, analgesic and anti-tumor activities. Although proteolytic enzymes have also been described, detailed characterisation of these proteins is scant in Olindias spp. High throughput mass spectrometry profiling of cnidarian venoms has become increasingly popular since the first description of the proteomic profile of putative toxins isolated from nematocysts of the hydrozoan jellyfish Olindias sambaquiensis describing the presence of orthologous enzymes as presented in venoms of advanced species as snakes. Rigorous bioinformatics analyses can aid functional annotation, but biochemical assays are prerequisite to unambiguously assign toxic function to a peptide or protein. Here we present results that experimentally confirm previously predicted proteomic analysis that crude venom extracts from tentacles of O. sambaquiensis are composed of polypeptides with metalloproteinase, serine proteinase and phospholipases A2 activities. Surprisingly, levels of serine proteinase and phospholipase A2 activities were comparable to those observed in venoms of Bothrops snakes which were used as positive controls in this study. Hence, these data offer new opportunities to explore serine proteinase and phospholipase A2 activities in the clinical sequelae following O. sambaquiensis envenomation, with future possible biopharmaceutical applications. Copyright © 2016 Elsevier Ltd. All rights reserved.

Effects of cyanobacterial toxins and cyanobacterial cell-free crude extract on germination of alfalfa (Medicago sativa) and induction of oxidative stress.

PubMed

Pflugmacher, Stephan; Jung, Katharina; Lundvall, Linn; Neumann, Stefanie; Peuthert, Anja

2006-09-01

Cyanobacterial toxins have adverse effects on both terrestrial and aquatic plants. Microcystins are cyclic heptapeptides and an important group of cyanotoxins. When lake water contaminated with cyanobacterial blooms is used for spray irrigation, these toxins can come in contact with agricultural plants. During the exposure to these toxins, reactive oxygen species can form. These reactive oxygen species have a strong reactivity and are able to interact with other cellular compounds (lipids, protein, and DNA). Plants have antioxidative systems that will limit the negative effects caused by reactive oxygen species. These systems consist of enzymes, such as superoxide dismutase, catalase, and ascorbate peroxidase, and nonenzymatic substances, such as reduced glutathione or vitamins. The aim of the present study was to investigate the effects of cyanobacterial toxins (microcystins and anatoxin-a) and cyanobacterial cell-free crude extract on alfalfa (Medicago sativa) seedlings. Inhibition of germination and root growth was observed with toxin concentrations of 5.0 microg/L. Also, oxidative damage, such as lipid peroxidation, was detected after the exposure of alfalfa seedlings to the toxin. Reactive oxygen detoxifying enzymes were elevated, showing a marked response in alfalfa to oxidative stress caused by the exposure to cyanobacterial metabolites that might influence the growth and development of these plants negatively.

Micellar Polymer Encapsulation of Enzymes.

PubMed

Besic, Sabina; Minteer, Shelley D

2017-01-01

Although enzymes are highly efficient and selective catalysts, there have been problems incorporating them into fuel cells. Early enzyme-based fuel cells contained enzymes in solution rather than immobilized on the electrode surface. One problem utilizing an enzyme in solution is an issue of transport associated with long diffusion lengths between the site of bioelectrocatalysis and the electrode. This issue drastically decreases the theoretical overall power output due to the poor electron conductivity. On the other hand, enzymes immobilized at the electrode surface have eliminated the issue of poor electron conduction due to close proximity of electron transfer between electrode and the biocatalyst. Another problem is inefficient and short term stability of catalytic activity within the enzyme that is suspended in free flowing solution. Enzymes in solutions are only stable for hours to days, whereas immobilized enzymes can be stable for weeks to months and now even years. Over the last decade, there has been substantial research on immobilizing enzymes at electrode surfaces for biofuel cell and sensor applications. The most commonly used techniques are sandwich or wired. Sandwich techniques are powerful and successful for enzyme immobilization; however, the enzymes optimal activity is not retained due to the physical distress applied by the polymer limiting its applications as well as the non-uniform distribution of the enzyme and the diffusion of analyte through the polymer is slowed significantly. Wired techniques have shown to extend the lifetime of an enzyme at the electrode surface; however, this technique is very hard to master due to specific covalent bonding of enzyme and polymer which changes the three-dimensional configuration of enzyme and with that decreases the optimal catalytic activity. This chapter details encapsulation techniques where an enzyme will be immobilized within the pores/pockets of the hydrophobically modified micellar polymers such as Nafion ® and chitosan. This strategy has been shown to safely immobilize enzymes at electrode surfaces with storage and continuous operation lifetime of more than 2 years.

Purification and characterization of multiple forms of the pineapple-stem-derived cysteine proteinases ananain and comosain.

PubMed Central

Napper, A D; Bennett, S P; Borowski, M; Holdridge, M B; Leonard, M J; Rogers, E E; Duan, Y; Laursen, R A; Reinhold, B; Shames, S L

1994-01-01

A mixture of ananain (EC 3.4.22.31) and comosain purified from crude pineapple stem extract was found to contain numerous closely related enzyme forms. Chromatographic separation of the major enzyme forms was achieved after treatment of the mixture with thiol-modifying reagents: reversible modification with 2-hydroxyethyl disulphide provided enzyme for kinetic studies, and irreversible alkylation with bromotrifluoroacetone or iodoacetamide gave enzyme for structural analyses by 19F-n.m.r. and electrospray mass spectrometry respectively. Structural and kinetic analyses revealed comosain to be closely related to stem bromelain (EC 3.4.22.32), whereas ananain differed markedly from both comosain and stem bromelain. Nevertheless, differences were seen between comosain and stem bromelain in amino acid composition and kinetic specificity towards the epoxide inhibitor E-64. Differences between five isolatable alternative forms of ananain were characterized by amidolytic activity, thiol stoichiometry and accurate mass determinations. Three of the enzyme forms displayed ananain-like amidolytic activity, whereas the other two forms were inactive. Thiol-stoichiometry determinations revealed that the active enzyme forms contained one free thiol, whereas the inactive forms lacked the reactive thiol required for enzyme activity. M.s. provided direct evidence for oxidation of the active-site thiol to the corresponding sulphinic acid. Images Figure 3 Figure 4 PMID:8053898

Comparison of in vitro systems of protein digestion using either mammal or fish proteolytic enzymes.

PubMed

Moyano, F J; Savoie, L

2001-02-01

Hydrolysis of three different proteins by either crude fish digestive extracts or purified mammal proteases was assayed using two different in vitro systems. The closed system was a modification of the pH-stat method including a previous acid digestion. The open system used a digestion cell containing a semi-permeable membrane which allowed continuous separation of the final products of hydrolysis with a molecular cut-off of 1000 Da. Assays in both systems resulted a similar arrangement of the tested proteins in relation to their ability to be hydrolyzed, with casein>fish meal> or =soybean meal. With the exception of casein, no significant differences were found between results produced by any of the enzyme sources using the closed system. In constrast, significantly higher hydrolysis of all proteins was produced by mammal enzymes under conditions operating in the open system. Differences in the rate of release of amino acids measured in this latter system were related both to the type of protein and the origin of the enzymes. When using purified mammal enzymes, release of lysine or phenylalanine from casein and soybean was high, but low from fishmeal. Isoleucine and valine present in fishmeal were preferentially hydrolyzed by commercial enzymes, but glycine and proline by fish enzymes.

Analysis of class II (hydrolytic) and class I (beta-lyase) apurinic/apyrimidinic endonucleases with a synthetic DNA substrate.

PubMed Central

Levin, J D; Demple, B

1990-01-01

We have developed simple and sensitive assays that distinguish the main classes of apurinic/apyrimidinic (AP) endonucleases: Class I enzymes that cleave on the 3' side of AP sites by beta-elimination, and Class II enzymes that cleave by hydrolysis on the 5' side. The distinction of the two types depends on the use of a synthetic DNA polymer that contains AP sites with 5'-[32P]phosphate residues. Using this approach, we now show directly that Escherichia coli endonuclease IV and human AP endonuclease are Class II enzymes, as inferred previously on the basis of indirect assays. The assay method does not exhibit significant interference by nonspecific nucleases or primary amines, which allows the ready determination of different AP endonuclease activities in crude cell extracts. In this way, we show that virtually all of the Class II AP endonuclease activity in E. coli can be accounted for by two enzymes: exonuclease III and endonuclease IV. In the yeast Saccharomyces cerevisiae, the Class II AP endonuclease activity is totally dependent on a single enzyme, the Apn1 protein, but there are probably multiple Class I enzymes. The versatility and ease of our approach should be useful for characterizing this important class of DNA repair enzymes in diverse systems. PMID:1698278

Membrane separation of hydrocarbons using cycloparaffinic solvents

DOEpatents

Kulkarni, S.S.; Chang, Y.A.; Gatsis, J.G.; Funk, E.W.

1988-06-14

Heavy crude oils which contain metal contaminants such as nickel, vanadium and iron may be separated from light hydrocarbon oils by passing a solution of the crude oil dissolved in a cycloparaffinic hydrocarbon solvent containing from about 5 to about 8 carbon atoms by passing through a polymeric membrane which is capable of maintaining its integrity in the presence of hydrocarbon compounds. The light hydrocarbon oils which possess relatively low molecular weights will be recovered as the permeate while the heavy oils which possess relatively high molecular weights as well as the metal contaminants will be recovered as the retentate.

Membrane separation of hydrocarbons using cycloparaffinic solvents

DOEpatents

Kulkarni, Sudhir S.; Chang, Y. Alice; Gatsis, John G.; Funk, Edward W.

1988-01-01

Heavy crude oils which contain metal contaminants such as nickel, vanadium and iron may be separated from light hydrocarbon oils by passing a solution of the crude oil dissolved in a cycloparaffinic hydrocarbon solvent containing from about 5 to about 8 carbon atoms by passing through a polymeric membrane which is capable of maintaining its integrity in the presence of hydrocarbon compounds. The light hydrocarbon oils which possess relatively low molecular weights will be recovered as the permeate while the heavy oils which possess relatively high molecular weights as well as the metal contaminants will be recovered as the retentate.

The effects of ionic liquid 1-ethyl-3-methylimidazolium trifluoromethanesulfonate on the production of 1,3-propanediol from crude glycerol by microbial consortium.

PubMed

Jiang, Lili; Dai, Jianying; Sun, Yaqin; Xiu, Zhilong

2018-04-12

Ionic liquids (ILs) as "green" solvents have been widely used owing to their excellent properties, e.g., for biodiesel production. Crude glycerol as a by-product in biodiesel production is an ideal feedstock for the microbial production of 1,3-propanediol (PDO), which is a versatile bulk chemical. PDO can be produced by microbial consortium with the advantages of high substrate tolerance and narrow by-product profile. In the present study, the effect of IL 1-ethyl-3-methylimidazolium trifluoromethanesulfonate ([Emim][TfO]) was evaluated on the capacity of PDO production from crude glycerol by microbial consortium DL38-BH. In the batch fermentation at 60 g/L crude glycerol and 10 g/L [Emim][TfO], the concentration and yield of PDO from glycerol increased from 23.14 g/L and 0.45 mol/mol to 31.17 g/L and 0.60 mol/mol, respectively. Our results showed that [Emim][TfO] decreased the ratio of intracellular NADH to NAD + and increased the concentration of 3-HPA during batch fermentation. The activities of three key enzymes in glycerol metabolism were stimulated by [Emim][TfO] during the batch fermentation by microbial consortium DL38-BH. Compared to the control, the proportion of Klebsiella genus which could convert glycerol to PDO increased significantly from 79.19% to 89.49% and the other genera that did not produce PDO were dramatically decreased (P < 0.05) at the end of batch fermentation. This work demonstrated that [Emim][TfO] significantly improved the concentration and yield of PDO from crude glycerol by adjusting microbial community during batch fermentation by microbial consortium.

Supercritical water reformation of crude glycerol solution for hydrogen production.

DOT National Transportation Integrated Search

2009-12-01

Glycerol, also known as glycerin, is a less desirable byproduct formed in the production of biodiesel via the transesterification otriglycerides and presents a nontrivial issue in terms of developing other beneficial end uses. With an inflated glycer...

Phytoremediation of petroleum hydrocarbons by using a freshwater fern species Azolla filiculoides Lam.

PubMed

Kösesakal, Taylan; Ünal, Muammer; Kulen, Oktay; Memon, Abdülrezzak; Yüksel, Bayram

2016-01-01

In this study, the phytoremediation capacity of Azolla filiculoides Lam. for the water resources contaminated with petroleum hydrocarbons was investigated. The plants were grown in nitrogen-free Hoagland nutrient solution containing 0.005%, 0.01%, 0.05%, 0.1%, 0.2%, 0.3%, 0.4%, and 0.5% crude oil under greenhouse conditions for 15 days. Although the growth rate of the plants were not negatively influenced by the presence of crude oil in the media for the concentration of 0.005% and 0.01% v/v, a gradual impeding effect of crude oil in the growth media has been observed at concentrations 0.05-0.1%. More than 0.1% crude oil in the growth medium ostensibly retarded the growth. For example, 0.2% oil in the media reduced growth approximately 50% relative to the control, and the presence of crude oil at concentrations 0.3% or more were lethal. The data about the percentage of plant growth, fresh weight increase and root growth clearly indicated that the tolerance level of A. filiculoides plants to crude oil ranges between 0.1% and 0.2%. In comparison to control samples, the biodegradation rate of total aliphatic and aromatic (phenathrene) hydrocarbons at 0.05-0.2% oil concentrations, was 94-73% and 81-77%, respectively. On the other hand, in case of further increases in oil concentration in media, i.e.; 0.3-0.5%, the biodegradation rate was still higher in the experimental samples, respectively 71-63% and 75-71%. The high biodegradation rates of petroleum hydrocarbons in the experimental samples suggested that A. filiculoides plants could be a promising candidate to be used for the phytoremediation of low crude oil contaminated precious freshwater resources.

Thermostability of glucose oxidase in silica gel obtained by sol-gel method and in solution studied by fluorimetric method.

PubMed

Przybyt, Małgorzata; Miller, Ewa; Szreder, Tomasz

2011-04-04

The thermostability of glucose oxidase entrapped in silica gel obtained by sol-gel method was studied by thermostimulated fluorescence of FAD at pH 5 and 7 and compared with that of the native enzyme in the solution and at the presence of ethanol. The unfolding temperatures were found to be lower for the enzyme immobilised in gel as compared with the native enzyme but higher as for the enzyme at the presence of ethanol. In gel, the thermal denaturation of glucose oxidase is independent on pH while in solution the enzyme is more stable at pH 5. The investigation the enzyme in different environment by steady-state fluorescence of FAD and tryptophan, synchronous fluorescence and time-resolved fluorescence of tryptophan indicates that the state of the molecule (tertiary structure and molecular dynamics) is different in gel and in solution. The ethanol produced during gel precursor hydrolysis is not the main factor influencing the thermostability of the enzyme but more important are interactions of the protein with the gel lattice. Copyright © 2011 Elsevier B.V. All rights reserved.

Fungal Biomass Protein Production from Trichoderma harzianum Using Rice Polishing

PubMed Central

Mustafa, Ghulam; Arshad, Muhammad

2017-01-01

Industrially important enzymes and microbial biomass proteins have been produced from fungi for more than 50 years. High levels of crude protein as much as 45% are present in fungal biomass with balanced essential amino acids. The aim of this study was to access the potential of Trichoderma harzianum to produce fungal biomass protein from rice polishings. Maximum biomass yield was obtained at 5% (w/v) rice polishings after 72 h of incubation at 28°C at pH 4. Carbon and nitrogen ratio of 20 : 1 gave significantly higher production of fungal biomass protein. The FBP in the 75 L fermenter contained 49.50% crude protein, 32.00% true protein, 19.45% crude fiber, 9.62% ash, 11.5% cellulose content, and 0.325% RNA content. The profile of amino acids of final FBP exhibited that all essential amino acids were present in great quantities. The FBP produced by this fungus has been shown to be of good nutritional value for supplementation to poultry. The results presented in this study have practical implications in that the fungus T. harzianum could be used successfully to produce fungal biomass protein using rice polishings. PMID:28367444

Fungal Biomass Protein Production from Trichoderma harzianum Using Rice Polishing.

PubMed

Ahmed, Sibtain; Mustafa, Ghulam; Arshad, Muhammad; Rajoka, Muhammad Ibrahim

2017-01-01

Industrially important enzymes and microbial biomass proteins have been produced from fungi for more than 50 years. High levels of crude protein as much as 45% are present in fungal biomass with balanced essential amino acids. The aim of this study was to access the potential of Trichoderma harzianum to produce fungal biomass protein from rice polishings. Maximum biomass yield was obtained at 5% (w/v) rice polishings after 72 h of incubation at 28°C at pH 4. Carbon and nitrogen ratio of 20 : 1 gave significantly higher production of fungal biomass protein. The FBP in the 75 L fermenter contained 49.50% crude protein, 32.00% true protein, 19.45% crude fiber, 9.62% ash, 11.5% cellulose content, and 0.325% RNA content. The profile of amino acids of final FBP exhibited that all essential amino acids were present in great quantities. The FBP produced by this fungus has been shown to be of good nutritional value for supplementation to poultry. The results presented in this study have practical implications in that the fungus T. harzianum could be used successfully to produce fungal biomass protein using rice polishings.

Thrombin-like enzymes from snake venom: Structural characterization and mechanism of action.

PubMed

Ullah, Anwar; Masood, Rehana; Ali, Ijaz; Ullah, Kifayat; Ali, Hamid; Akbar, Haji; Betzel, Christian

2018-07-15

Snake venom thrombin-like enzymes (SVTLEs) constitute the major portion (10-24%) of snake venom and these are the second most abundant enzymes present in the crude venom. During envenomation, these enzymes had shown prominently the various pathological effects, such as disturbance in hemostatic system, fibrinogenolysis, fibrinolysis, platelet aggregation, thrombosis, neurologic disorders, activation of coagulation factors, coagulant, procoagulant etc. These enzymes also been used as a therapeutic agent for the treatment of various diseases such as congestive heart failure, ischemic stroke, thrombotic disorders etc. Although the crystal structures of five SVTLEs are available in the Protein Data Bank (PDB), there is no single article present in the literature that has described all of them. The current work describes the structural aspects, structure-based mechanism of action, processing and inhibition of these enzymes. The sequence analysis indicates that these enzymes show a high sequence identity (57-85%) with each other and low sequence identity with trypsin (36-43%), human alpha-thrombin (29-36%) and other snake venom serine proteinases (57-85%). Three-dimensional structural analysis indicates that the loops surrounding the active site are variable both in amino acids composition and length that may convey variable substrate specificity to these enzymes. The surface charge distributions also vary in these enzymes. Docking analysis with suramin shows that this inhibitor preferably binds to the C-terminal region of these enzymes and causes the destabilization of their three-dimensional structure. Copyright © 2018 Elsevier B.V. All rights reserved.

Functional characterization and target discovery of glycoside hydrolases from the digestome of the lower termite Coptotermes gestroi

PubMed Central

2011-01-01

Background Lignocellulosic materials have been moved towards the forefront of the biofuel industry as a sustainable resource. However, saccharification and the production of bioproducts derived from plant cell wall biomass are complex and lengthy processes. The understanding of termite gut biology and feeding strategies may improve the current state of biomass conversion technology and bioproduct production. Results The study herein shows comprehensive functional characterization of crude body extracts from Coptotermes gestroi along with global proteomic analysis of the termite's digestome, targeting the identification of glycoside hydrolases and accessory proteins responsible for plant biomass conversion. The crude protein extract from C. gestroi was enzymatically efficient over a broad pH range on a series of natural polysaccharides, formed by glucose-, xylose-, mannan- and/or arabinose-containing polymers, linked by various types of glycosidic bonds, as well as ramification types. Our proteomic approach successfully identified a large number of relevant polypeptides in the C. gestroi digestome. A total of 55 different proteins were identified and classified into 29 CAZy families. Based on the total number of peptides identified, the majority of components found in the C. gestroi digestome were cellulose-degrading enzymes. Xylanolytic enzymes, mannan- hydrolytic enzymes, pectinases and starch-degrading and debranching enzymes were also identified. Our strategy enabled validation of liquid chromatography with tandem mass spectrometry recognized proteins, by enzymatic functional assays and by following the degradation products of specific 8-amino-1,3,6-pyrenetrisulfonic acid labeled oligosaccharides through capillary zone electrophoresis. Conclusions Here we describe the first global study on the enzymatic repertoire involved in plant polysaccharide degradation by the lower termite C. gestroi. The biochemical characterization of whole body termite extracts evidenced their ability to cleave all types of glycosidic bonds present in plant polysaccharides. The comprehensive proteomic analysis, revealed a complete collection of hydrolytic enzymes including cellulases (GH1, GH3, GH5, GH7, GH9 and CBM 6), hemicellulases (GH2, GH10, GH11, GH16, GH43 and CBM 27) and pectinases (GH28 and GH29). PMID:22081966

[High-level expression of heterologous protein based on increased copy number in Saccharomyces cerevisiae].

PubMed

Zhang, Xinjie; He, Peng; Tao, Yong; Yang, Yi

2013-11-04

High-level expression system of heterologous protein mediated by internal ribosome entry site (IRES) in Saccharomyces cerevisiae was constructed, which could be used for other applications of S. cerevisiae in metabolic engineering. We constructed co-expression cassette (promoter-mCherry-TIF4631 IRES-URA3) containing promoters Pilv5, Padh2 and Ptdh3 and recombined the co-expression cassette into the genome of W303-1B-A. The URA3+ transformants were selected. By comparing the difference in the mean florescence value of mCherry in transformants, the effect of three promoters was detected in the co-expression cassette. The copy numbers of the interested genes in the genome were determined by Real-Time PCR. We analyzed genetic stability by continuous subculturing transformants in the absence of selection pressure. To verify the application of co-expression cassette, the ORF of mCherry was replaced by beta-galactosidase (LACZ) and xylose reductase (XYL1). The enzyme activities and production of beta-galactosidase and xylose reductase were detected. mCherry has been expressed in the highest-level in transformants with co-expression cassette containing Pilv5 promoter. The highest copy number of DNA fragment integrating in the genome was 47 in transformants containing Pilv5. The engineering strains showed good genetic stability. Xylose reductase was successfully expressed in the co-expression cassette containing Pilv5 promoter and TIF4631 IRES. The highest enzyme activity was 0. 209 U/mg crude protein in the transformants WIX-10. Beta-galactosidase was also expressed successfully. The transformants that had the highest enzyme activity was WIL-1 and the enzyme activity was 12.58 U/mg crude protein. The system mediated by Pilv5 promoter and TIF4631 IRES could express heterologous protein efficiently in S. cerevisiae. This study offered a new strategy for expression of heterologous protein in S. cerevisiae and provided sufficient experimental evidence for metabolic engineering application of this system in yeast.

Physicochemical Properties Analysis and Secretome of Aspergillus niger in Fermented Rapeseed Meal

PubMed Central

Shi, Changyou; He, Jun; Yu, Jie; Yu, Bing; Mao, Xiangbing; Zheng, Ping; Huang, Zhiqing; Chen, Daiwen

2016-01-01

The nutrient digestibility and feeding value of rapeseed meal (RSM) for non-ruminant animals is poor due to the presence of anti-nutritional substances such as glucosinolate, phytic acid, crude fiber etc. In the present study, a solid state fermentation (SSF) using Aspergillus niger was carried out with the purpose of improving the nutritional quality of RSM. The chemical composition and physicochemical properties of RSM before and after fermentation were compared. To further understand possible mechanism of solid state fermentation, the composition of extracellular enzymes secreted by Aspergillus niger during fermentation was analysed using two-dimentional difference gel electrophoresis (2D-DIGE) combined with matrix assisted laser desorption ionization—time of flight—mass spectrometer (MALDI-TOF-MS). Results of the present study indicated that SSF had significant effects on chemical composition of RSM. The fermented rapeseed meal (FRSM) contained more crude protein (CP) and amino acid (AA) (except His) than unfermented RSM. Notably, the small peptide in FRSM was 2.26 time larger than that in unfermented RSM. Concentrations of anti-nutritional substrates in FRSM including neutral detergent fiber (NDF), glucosinolates, isothiocyanate, oxazolidithione, and phytic acid declined (P < 0.05) by 13.47, 43.07, 55.64, 44.68 and 86.09%, respectively, compared with unfermented RSM. A. niger fermentation disrupted the surface structure, changed macromolecular organic compounds, and reduced the protein molecular weights of RSM substrate. Total proteins of raw RSM and FRSM were separated and 51 protein spots were selected for mass spectrometry according to 2D-DIGE map. In identified proteins, there were 15 extracellular hydrolases secreted by A. niger including glucoamylase, acid protease, beta-glucanase, arabinofuranosidase, xylanase, and phytase. Some antioxidant related enzymes also were identified. These findings suggested that A. niger is able to secrete many extracellular degradation enzymes (especially lignocellulosic hydrolyzing enzymes, acid proteases and phytase) during fermentation of RSM, thus altering chemical composition and physicochemical properties of RSM. PMID:27049858

Physicochemical Properties Analysis and Secretome of Aspergillus niger in Fermented Rapeseed Meal.

PubMed

Shi, Changyou; He, Jun; Yu, Jie; Yu, Bing; Mao, Xiangbing; Zheng, Ping; Huang, Zhiqing; Chen, Daiwen

2016-01-01

The nutrient digestibility and feeding value of rapeseed meal (RSM) for non-ruminant animals is poor due to the presence of anti-nutritional substances such as glucosinolate, phytic acid, crude fiber etc. In the present study, a solid state fermentation (SSF) using Aspergillus niger was carried out with the purpose of improving the nutritional quality of RSM. The chemical composition and physicochemical properties of RSM before and after fermentation were compared. To further understand possible mechanism of solid state fermentation, the composition of extracellular enzymes secreted by Aspergillus niger during fermentation was analysed using two-dimentional difference gel electrophoresis (2D-DIGE) combined with matrix assisted laser desorption ionization-time of flight-mass spectrometer (MALDI-TOF-MS). Results of the present study indicated that SSF had significant effects on chemical composition of RSM. The fermented rapeseed meal (FRSM) contained more crude protein (CP) and amino acid (AA) (except His) than unfermented RSM. Notably, the small peptide in FRSM was 2.26 time larger than that in unfermented RSM. Concentrations of anti-nutritional substrates in FRSM including neutral detergent fiber (NDF), glucosinolates, isothiocyanate, oxazolidithione, and phytic acid declined (P < 0.05) by 13.47, 43.07, 55.64, 44.68 and 86.09%, respectively, compared with unfermented RSM. A. niger fermentation disrupted the surface structure, changed macromolecular organic compounds, and reduced the protein molecular weights of RSM substrate. Total proteins of raw RSM and FRSM were separated and 51 protein spots were selected for mass spectrometry according to 2D-DIGE map. In identified proteins, there were 15 extracellular hydrolases secreted by A. niger including glucoamylase, acid protease, beta-glucanase, arabinofuranosidase, xylanase, and phytase. Some antioxidant related enzymes also were identified. These findings suggested that A. niger is able to secrete many extracellular degradation enzymes (especially lignocellulosic hydrolyzing enzymes, acid proteases and phytase) during fermentation of RSM, thus altering chemical composition and physicochemical properties of RSM.

URANIUM EXTRACTION

DOEpatents

Harrington, C.D.; Opie, J.V.

1958-07-01

The recovery of uranium values from uranium ore such as pitchblende is described. The ore is first dissolved in nitric acid, and a water soluble nitrate is added as a salting out agent. The resulting feed solution is then contacted with diethyl ether, whereby the bulk of the uranyl nitrate and a portion of the impurities are taken up by the ether. This acid ether extract is then separated from the aqueous raffinate, and contacted with water causing back extractioa of the uranyl nitrate and impurities into the water to form a crude liquor. After separation from the ether extract, this crude liquor is heated to about 118 deg C to obtain molten uranyl nitrate hexahydratc. After being slightly cooled the uranyl nitrate hexahydrate is contacted with acid free diethyl ether whereby the bulk of the uranyl nitrate is dissolved into the ethcr to form a neutral ether solution while most of the impurities remain in the aqueous waste. After separation from the aqueous waste, the resultant ether solution is washed with about l0% of its volume of water to free it of any dissolved impurities and is then contacted with at least one half its volume of water whereby the uranyl nitrate is extracted into the water to form an aqueous product solution.

Removal and biodegradation of different petroleum hydrocarbons using the filamentous fungus Aspergillus sp. RFC-1.

PubMed

Al-Hawash, Adnan B; Zhang, Xiaoyu; Ma, Fuying

2018-03-25

Petroleum pollution inevitably occurs at any stage of oil production and exerts a negative impact on the environment. Some microorganisms can degrade petroleum hydrocarbons (PHs). Polluted sludge of Rumaila oil field was use to isolate the highly efficient hydrocarbon-degrading fungal strain. Aspergillus sp. RFC-1 was obtained and its degradation ability for petroleum hydrocarbons was evaluated through surface adsorption, cell uptake, hydrophobicity, surface tension, biosurfactant production, and emulsification activity. In addition, the degradation mechanism was investigated. The results indicated the strain RFC-1 showed high removal activity for PHs, including biodegradation, adsorption, and emulsifiability. On the day 7 of incubation, the removal efficiencies of crude oil, naphthalene (NAP), phenanthrene (PHE), and pyrene (PYR) reached 60.3%, 97.4%, 84.9%, and 90.7%, respectively. Biodegradation efficiencies of crude oil, NAP, PHE, and PYR were 51.8%, 84.6%, 50.3%, and 55.1%, respectively. Surface adsorption and cell absorption by live mycelial pellets followed a decreasing order: PYR ≥ PHE > NAP > crude oil. Adsorption by heat-killed mycelial pellets increased within 40 and 10 min for crude oil and PAHs, respectively, and remained constant thereafter. Effects of cell surface hydrophobicity, surface tension, and emulsification index were discussed. Intra- and extracellular enzymes of strain RFC-1 played important roles in PHs degradation. The strain RFC-1 is a prospective strain for removing PHs from aqueous environments. © 2018 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.

Alternaria toxin-induced resistance in rose plants against rose aphid (Macrosiphum rosivorum): effect of tenuazonic acid.

PubMed

Yang, Fa-zhong; Yang, Bin; Li, Bei-bei; Xiao, Chun

2015-04-01

Many different types of toxins are produced by the fungus, Alternaria alternata (Fr.) Keissler. Little is known, however, regarding the influence of these toxins on insects. In this study, we investigated the toxin-induced inhibitory effects of the toxin produced by A. alternata on the rose aphid, Macrosiphum rosivorum, when the toxin was applied to leaves of the rose, Rosa chinensis. The results demonstrated that the purified crude toxin was non-harmful to rose plants and rose aphids, but had an intensive inhibitory effect on the multiplication of aphids. The inhibitory index against rose aphids reached 87.99% when rose plants were sprayed with the toxin solution at a low concentration. Further results from bioassays with aphids and high performance liquid chromatography (HPLC) analyses demonstrated that tenuazonic acid (TeA) was one of the most important resistance-related active components in the crude toxin. The content of TeA was 0.1199% in the crude toxin under the HPLC method. Similar to the crude toxin, the inhibitory index of pure TeA reached 83.60% 15 d after the rose plants were sprayed with pure TeA solution at the lower concentration of 0.060 μg/ml, while the contents of residual TeA on the surface and in the inner portion of the rose plants were only 0.04 and 0.00 ng/g fresh weight of TeA-treated rose twigs, respectively, 7 d after the treatment. Our results show that TeA, an active component in the A. alternata toxin, can induce the indirect plant-mediated responses in rose plants to intensively enhance the plant's resistances against rose aphids, and the results are very helpful to understand the plant-mediated interaction between fungi and insects on their shared host plants.

Purification and characterization of α-Amylase from Miswak Salvadora persica.

PubMed

Mohamed, Saleh A; Almulaiky, Yaaser Q; Ahmed, Youssri M; Al-Bar, Omar A M; Ibrahim, Ibrahim H

2014-04-01

The miswak (Salvadora persica) is a natural toothbrush. It is well known that very little information has been reported on enzymes in miswak as medicinal plant. Recently, we study peroxidase in miswak. In the present study, the main goal of this work is to purify and characterize α-amylase from miswak. The second goal is to study the storage stability of α-amylase in toothpaste. The purification method included chromatography of miswak α-amylase on DEAE-Sepharose column and Sephacryl S-200 column. Molecular weight was determined by gel filtration and SDS-PAGE. Five α-amylases A1, A4a, A4b, A5a and A5b from miswak were purified and they had molecular weights of 14, 74, 16, 30 and 20 kDa, respectively. α-Amylases had optimum pH from 6 to 8. Affinity of the substrates toward all enzymes was studied. Miswak α-amylases A1, A4a, A4b, A5a and A5b had Km values for starch and glycogen of 3.7, 3.7, 7.1, 0.52, 4.3 mg/ml and 5.95, 5.9 4.16, 6.3, 6.49 mg/ml, respectively. The optimum temperature for five enzymes ranged 40°C- 60°C. Miswak α-amylases were stable up to 40°C- 60°C after incubation for 30 min. Ca+2 activated all the miswak α-amylases, while Ni2+, Co+2 and Zn+2 activated or inhibited some of these enzymes. The metal chelators, EDTA, sodium citrate and sodium oxalate had inhibitory effects on miswak α-amylases. PMSF, p-HMB, DTNB and 1,10 phenanthroline caused inhibitory effect on α-amylases. The analysis of hydrolytic products after starch hydrolysis by miswak α-amylases on paper chromatography revealed that glucose, maltose, maltotriose and oligosaccharide were the major products. Crude miswak α-amylase in the toothpaste retained 55% of its original activity after 10 months of storage at room temperature. From these findings, α-amylases from miswak can be considered as beneficial enzymes for pharmaceuticals. Therefore, we study the storage stability of the crude α-amylase of miswak, which contained the five α-amylases, in toothpaste. The enzyme in the toothpaste retained 55% of its original activity after 10 months of storage at room temperature.

Purification and characterization of α-Amylase from Miswak Salvadora persica

PubMed Central

2014-01-01

Background The miswak (Salvadora persica) is a natural toothbrush. It is well known that very little information has been reported on enzymes in miswak as medicinal plant. Recently, we study peroxidase in miswak. In the present study, the main goal of this work is to purify and characterize α-amylase from miswak. The second goal is to study the storage stability of α-amylase in toothpaste. Method The purification method included chromatographaphy of miswak α-amylase on DEAE-Sepharose column and Sephacryl S-200 column. Molecular weight was determined by gel filtration and SDS-PAGE. Results Five α-amylases A1, A4a, A4b, A5a and A5b from miswak were purified and they had molecular weights of 14, 74, 16, 30 and 20 kDa, respectively. α-Amylases had optimum pH from 6 to 8. Affinity of the substrates toward all enzymes was studied. Miswak α-amylases A1, A4a, A4b, A5a and A5b had Km values for starch and glycogen of 3.7, 3.7, 7.1, 0.52, 4.3 mg/ml and 5.95, 5.9 4.16, 6.3, 6.49 mg/ml, respectively. The optimum temperature for five enzymes ranged 40°C- 60°C. Miswak α-amylases were stable up to 40°C- 60°C after incubation for 30 min. Ca+2 activated all the miswak α-amylases, while Ni2+, Co+2 and Zn+2 activated or inhibited some of these enzymes. The metal chelators, EDTA, sodium citrate and sodium oxalate had inhibitory effects on miswak α-amylases. PMSF, p-HMB, DTNB and 1,10 phenanthroline caused inhibitory effect on α-amylases. The analysis of hydrolytic products after starch hydrolysis by miswak α-amylases on paper chromatography revealed that glucose, maltose, maltotriose and oligosaccharide were the major products. Crude miswak α-amylase in the toothpaste retained 55% of its original activity after 10 months of storage at room temperature. Conclusions From these findings, α-amylases from miswak can be considered as beneficial enzymes for pharmaceuticals. Therefore, we study the storage stability of the crude α-amylase of miswak, which contained the five α-amylases, in toothpaste. The enzyme in the toothpaste retained 55% of its original activity after 10 months of storage at room temperature. PMID:24690287

Amperometric biosensors for the determination of heavy metals

NASA Astrophysics Data System (ADS)

Compagnone, Dario; Palleschi, Giuseppe; Varallo, Giuseppe; Imperiali, PierLuigi

1995-10-01

A bioelectrochemical method for the determination of heavy metal ions has been developed. This method is based on the inhibition effect of metal ions on the enzymatic activity of oxidase enzymes. The enzymatic activity was determined with an amperometric hydrogen peroxide probe. The inhibition effect on enzymes in solution and covalently immobilized on polymeric supports has been evaluated. Hg(II) was the metal ion that inhibited almost all the enzymes, particularly glycerol-3-P oxidase. Hg(II) was detected in the 0.05/0.5 ppm range with the enzyme in solution. Calibration curves for Hg(II) were also obtained with the other oxidase enzymes in the 0.5/10 ppm range. The other metal ions tested inhibited the enzymes more specifically. The metal ion/enzyme systems which gave the best inhibition were Se(IV)/glutathione oxidase, Ni(II)/sarcosine oxidase, V(V)/glutathione oxidase, Cu(II)/alcohol oxidase from Pichia Pastoris and Cd(II)/D-aminoacid oxidase. All these metal ions were detected in the 0.1/10 ppm range using the enzymes in solution or covalently immobilized.

Isolation and purification of glutathione S-transferases from Brachionus plicatilis and B. calyciflorus (Rotifera).

PubMed

Bowman, B P; Snell, T W; Cochrane, B J

1990-01-01

1. The enzyme glutathione S-transferase (GST), a critical element in xenobiotic metabolism, was isolated from the marine rotifer Brachionus plicatilis and its freshwater congener B. calyciflorus. 2. In B. plicatilis, GST comprised 4.2% of cytosolic protein and was present as three separate isozymes with mol. wts 30,000, 31,400 and 33,700. Specific activity of crude homogenates was 56 nmol min-1 mg-1 protein, while that of affinity chromatography purified GST was 1850. 3. In B. calyciflorus, GST was present as two isozymes with mol. wts of 26,300 and 28,500, representing 1.0% of cytosolic protein. Crude GST specific activity was 1750 nmol min-1 mg-1 protein and purified was 72,400. 4. Rotifer GSTs are unusual because they are monomers whereas all other animals thus far investigated posses dimeric GSTs.

Role of laccase from Coriolus versicolor MTCC-138 in selective oxidation of aromatic methyl group.

PubMed

Chaurasia, Pankaj Kumar; Singh, Sunil Kumar; Bharati, Shashi Lata

2014-01-01

Now a day, laccases are the most promising enzymes in the area of biotechnology and synthesis. One of the best applications of laccases is the selective oxidation of aromatic methyl group to aldehyde group. Such transformations are valuable because it is difficult to stop the reaction at aldehyde stage. Chemical methods used for such biotransformations areexpensive and give poor yields. But, the laccase-catalyzed biotransformations of such type are non-expensive and yield is excellent. Authors have used crude laccase obtained from the liquid culture growth medium of fungal strain Coriolus versicolor MTCC-138 for the biotransformations of toluene, 3-nitrotoluene, and 4-chlorotoluene to benzaldehyde, 3-nitrobenzaldehyde, and 4-chlorobenzaldehyde, respectively, instead of purified laccase because purification process requires much time and cost. This communication reports that crude laccase can also be used in the place of purified laccase as effective biocatalyst.

Comparison of various solutions to dissolve critical care diet clots.

PubMed

Parker, Valerie J; Freeman, Lisa M

2013-01-01

Enteral feeding tubes are frequently placed in animals to provide assisted nutritional support; however, one major reported complication is clogging of the tubes. The goal of this study was to determine which solution is most effective at dissolving in vitro clots made using a veterinary canned critical care diet. Various solutions were tested for their ability to dissolve enteral feed clots, including water, meat tenderizers in water, predetermined amounts of pancreatic enzymes (with and without sodium bicarbonate) in water, carbonated beverages, and cranberry juice. The solution that resulted in the greatest dissolution was ¼ teaspoon pancreatic enzymes and 325 mg sodium bicarbonate in 5 mL water, which was significantly better than all other solutions (water: P = 0.03; ¼ teaspoon pancreatic enzymes in water: P = 0.002; all others: P < 0.001). Water was significantly better than all carbonated beverages and cranberry juice (P < 0.001). The least successful solution was ½ teaspoon pancreatic enzymes and sodium bicarbonate in water. Despite anecdotal reports of using carbonated beverages, cranberry juice, and ½ teaspoon pancreatic enzymes to unclog feeding tubes, all were significantly less effective than water. In vivo studies to evaluate the effectiveness of methods to unclog feeding tubes are warranted to further investigate these findings. © Veterinary Emergency and Critical Care Society 2013.

Isolation, characterization and optimization of culture parameters for production of an alkaline protease isolated from Aspergillus tamarii.

PubMed

Anandan, Dayanandan; Marmer, William N; Dudley, Robert L

2007-05-01

Aspergillus tamarii expresses an extracellular alkaline protease that we show to be effective in removing hair from cattle hide. Large quantities of the enzyme will be required for the optimization of the enzymatic dehairing process so the growth conditions for maximum protease expression by A. tamarii were optimized for both solid-state culture on wheat bran and for broth culture. Optimal protease expression occurred, for both cultural media, at initial pH 9; the culture was incubated at 30 degrees C for 96 h using a 5% inoculum. The crude enzyme was isolated, purified and characterized using MALDI TOF TOF. The alkaline protease was homologous to the alkaline protease expressed by Aspergillus viridinutans.

The effectiveness of crude papain enzyme supplement for tilapia’s (Oreochromis niloticus) growth at the floating nets of Cirata Reservoir

NASA Astrophysics Data System (ADS)

Rostika, R.; Sunarto; Sugiyanto, H. N.; Dewanti, L. P.

2018-03-01

Papain is an enzyme capable of hydrolyzing protease into a more simple elements i.e. the peptide to amino acids. The enzyme in the feed can increase the absorption of protein and digestion rate in the digestive tract of fish. This research examined the effective level of enzyme papain to increase the Feed Utilization Efficiency (FUE), Protein Efficiency Ratio (PER) and Average Daily Gain (ADG). This research used Completely Randomized Design (CRD) with five treatments i.e. treatment A (control), treatment B (1.5 %), treatment C (2.25 %), treatment D (3 %) and treatment E (3.75 %) in triplicate. Tilapia (Oreochromis niloticus) with the average initial weight of 17 g, and initial total lenght of 8–10 cm was fed three times daily at feeding rate of 5 % of the total body weight. The results showed that supplementation of papain in the feed significantly increased the activity of protease, FUE, PER and ADG. The optimal dose of the enzyme papain at 3.75 % was able to increase 48.31 % of FUE, 2.13 % of PER and 2.07 % of ADG.

Some Immunochemical Properties of Dextransucrase and Invertase from Streptococcus mutans

PubMed Central

Fukui, Kazuhiro; Fukui, Yoshio; Moriyama, Takafumi

1974-01-01

Dextransucrase and invertase of some strains of Streptococcus mutans were examined by immunodiffusion with antisera against enzymes purified from strain HS-6 (Bratthall's serotype a). Both antisera cross-reacted with crude enzyme preparations from the other serotype a (strains HS-1 and AHT) and d organisms (strains KIR, OMZ176, and OMZ65) but not with those from serotype b (strains FA-1 and BHT) or c organisms (strains GS-5, Ingbritt, and NCTC 10449). Based upon the antiserum used, the orders of antigenic similarity of the cross-reacting enzymes to the HS-6 enzymes were HS-6 > HS-1 > AHT = KIR = OMZ176 = OMZ65 for dextransucrase and HS-6 = HS-1 > AHT = KIR = OMZ176 = OMZ65 for invertase. It was found that the enzymes from serotype a organisms were not always antigenically homogeneous, as seen between strains HS-6, HS-1, or AHT for dextransucrase, and between the HS group and strain AHT for invertase. Antiserum against the HS-6 dextransucrase markedly inhibited the heterologous dextransucrases of serotype a organisms with the exception of strain HS-1 and d organisms, with or without the addition of dextran. Images PMID:16558114

Fermentation and aerobic metabolism of cellodextrins by yeasts. [Candida wickerhamii; C. guiliermondii; C. molischiana; Debaryomyces polymorphus; Pichia guilliermondii; Clavispora lusitaniae; Kluyveromyces lactis; Brettanomyces claussenii; Rhodotorula minuta; Dekkera intermedia

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

Freer, S.N.

1991-03-01

The fermentation and aerobic metabolism of cellodextrins by 14 yeast species or strains was monitored. When grown aerobically, Candida wickerhamii, C. guilliermondii, and C. molischiana metabolized cellodextrins of degree of polymerization 3 to 6. C. wicherhamii and C. molischiana also fermented these substrates, while C. guilliermondii fermented only cellodextrins of degree of polymerization {<=} 3. Debaryomyces polymorphus, Pichia guilliermondii, Clavispora lusitaniae, and one of two strains of Kluyveromyces lactis metabolized glucose, cellobiose, and cellotriose when grown aerobically. These yeasts also fermented these substrates, except for K. lactis, which fermented only glucose and cellobiose. The remaining species/strains tested, K. lactis, Brettanomycesmore » claussenii, Brettanomyces anomalus, Kluyveromyces dobzhanskii, Rhodotorula minuta, and Dekkera intermedia, both fermented and aerobically metabolized glucose and cellobiose. Crude enzyme preparations from all 14 yeast species or strains were tested for ability to hydrolyze cellotriose and cellotretose. Most of the yeasts produced an enzyme(s) capable of hydrolyzing cellotriose. However, with two exceptions, R. minuta and P. guilliermondii, only the yeasts that metabolized cellodextrins of degree of polymerization >3 produced an enzyme(s) that hydrolyzed cellotretose.« less

Purification and Characterization of an Aminopeptidase from Lactococcus lactis subsp. cremoris AM2.

PubMed

Neviani, E; Boquien, C Y; Monnet, V; Thanh, L P; Gripon, J C

1989-09-01

An aminopeptidase was purified from cell extracts of Lactococcus lactis subsp. cremoris AM2 by ion-exchange chromatography. After electrophoresis of the purified enzyme in the presence or absence of sodium dodecyl sulfate, one protein band was detected. The enzyme was a 300-kilodalton hexamer composed of identical subunits not linked by disulfide bridges. Activity was optimal at 40 degrees C and pH 7 and was inhibited by classical thiol group inhibitors. The aminopeptidase hydrolyzed naphthylamide-substituted amino acids, as well as dipeptides and tripeptides. Longer protein chains such as the B chain of insulin were hydrolyzed, but at a much slower rate. The Michaelis constant (K(m)) and the maximal rate of hydrolysis (V(max)) were, respectively, 4.5 mM and 3,600 pkat/mg for the substrate l-histidyl-beta-naphthylamide. Amino acid analysis showed that the enzyme contained low levels of hydrophobic residues. The partial N-terminal sequence of the first 19 residues of the mature enzyme was determined. Polyclonal antibodies were obtained from the purified enzyme, and after immunoblotting, there was no cross-reaction between these antibodies and other proteins in the crude extract.

Fast and economic immobilization methods described for non-commercial Pseudomonas lipases

PubMed Central

2014-01-01

Background There is an increasing interest to seek new enzyme preparations for the development of new products derived from bioprocesses to obtain alternative bio-based materials. In this context, four non-commercial lipases from Pseudomonas species were prepared, immobilized on different low-cost supports, and examined for potential biotechnological applications. Results To reduce costs of eventual scaling-up, the new lipases were obtained directly from crude cell extracts or from growth culture supernatants, and immobilized by simple adsorption on Accurel EP100, Accurel MP1000 and Celite®545. The enzymes evaluated were LipA and LipC from Pseudomonas sp. 42A2, a thermostable mutant of LipC, and LipI.3 from Pseudomonas CR611, which were produced in either homologous or heterologous hosts. Best immobilization results were obtained on Accurel EP100 for LipA and on Accurel MP1000 for LipC and its thermostable variant. Lip I.3, requiring a refolding step, was poorly immobilized on all supports tested (best results for Accurel MP1000). To test the behavior of immobilized lipases, they were assayed in triolein transesterification, where the best results were observed for lipases immobilized on Accurel MP1000. Conclusions The suggested protocol does not require protein purification and uses crude enzymes immobilized by a fast adsorption technique on low-cost supports, which makes the method suitable for an eventual scaling up aimed at biotechnological applications. Therefore, a fast, simple and economic method for lipase preparation and immobilization has been set up. The low price of the supports tested and the simplicity of the procedure, skipping the tedious and expensive purification steps, will contribute to cost reduction in biotechnological lipase-catalyzed processes. PMID:24755191

Reversible and strong immobilization of proteins by ionic exchange on supports coated with sulfate-dextran.

PubMed

Fuentes, Manuel; Pessela, Benevides C C; Maquiese, Jorgette V; Ortiz, Claudia; Segura, Rosa L; Palomo, Jose M; Abian, Olga; Torres, Rodrigo; Mateo, Cesar; Fernández-Lafuente, Roberto; Guisán, J M

2004-01-01

New and strong ionic exchange resins have been prepared by the simple and rapid ionic adsorption of anionic polymers (sulfate-dextran) on porous supports activated with the opposite ionic group (DEAE/MANAE). Ionic exchange properties of such composites were strongly dependent on the size of the ionic polymers as well as on the conditions of the ionic coating of the solids with the ionic polymers (optimal conditions were 400 mg of sulfate-dextran 5000 kDa per gram of support). Around 80% of the proteins contained in crude extracts from Escherichia coli and Acetobacter turbidans could be adsorbed on these porous composites even at pH 7. This interaction was stronger than that using conventional carboxymethyl cellulose (CMC) and even others such as supports coated with aspartic-dextran polymer. By means of the sequential use of the new supports and supports coated with polyethyleneimine (PEI), all proteins from crude extracts could be immobilized. In fact, a large percentage (over 50%) could be immobilized on both supports. Finally, some industrially relevant enzymes (beta-galactosidases from Aspergillus oryzae, Kluyveromyces lactis, and Thermussp. strain T2, lipases from Candida antarctica A and B, Candida rugosa, Rhizomucor miehei, and Rhyzopus oryzae and bovine pancreas trypsin and chymotrypsin) have been immobilized on these supports with very high activity recoveries and immobilization rates. After enzyme inactivation, the protein could be fully desorbed from the support, and then the support could be reused for several cycles. Moreover, in some instances the enzyme stability was significantly improved, mainly in the presence of organic solvents, perhaps as a consequence of the highly hydrophilic microenvironment of the support.

Ultrasound assisted three phase partitioning of a fibrinolytic enzyme.

PubMed

Avhad, Devchand N; Niphadkar, Sonali S; Rathod, Virendra K

2014-03-01

The present investigation is aimed at ultrasound assisted three phase partitioning (UATPP) of a fibrinolytic enzyme from Bacillus sphaericus MTCC 3672. Three phase partitioning integrates the concentration and partial purification step of downstream processing of a biomolecule. Three phase system is formed with simultaneous addition of ammonium sulfate to crude broth and followed by t-butanol. UATPP of a fibrinolytic enzyme was studied by varying different process parameters such as ammonium sulfate saturation concentration, pH, broth to t-butanol ratio, temperature, ultrasound frequency, ultrasonication power, and duty cycle. The optimized parameters yielding maximum purity of 16.15-fold of fibrinolytic enzyme with 65% recovery comprised of 80% ammonium sulfate saturation, pH 9, temperature 30 °C, broth to t-butanol ratio 0.5 (v/v), at 25 kHz frequency and 150 W ultrasonication power with 40% duty cycle for 5 min irradiation time. SDS PAGE analysis of partitioned enzyme shows partial purification with a molecular weight in the range of 55-70 kDa. Enhanced mass transfer of UATPP resulted in higher fold purity of fibrinolytic enzyme with reduced time of operation from 1 h to 5 min as compared to conventional TPP. Outcome of our findings highlighted the use of UATPP as an efficient biosepartion technique. Copyright © 2013 Elsevier B.V. All rights reserved.

Screening and isolation of halophilic bacteria producing industrially important enzymes.

PubMed

Kumar, Sumit; Karan, Ram; Kapoor, Sanjay; S P, Singh; S K, Khare

2012-10-01

Halophiles are excellent sources of enzymes that are not only salt stable but also can withstand and carry out reactions efficiently under extreme conditions. The aim of the study was to isolate and study the diversity among halophilic bacteria producing enzymes of industrial value. Screening of halophiles from various saline habitats of India led to isolation of 108 halophilic bacteria producing industrially important hydrolases (amylases, lipases and proteases). Characterization of 21 potential isolates by morphological, biochemical and 16S rRNA gene analysis found them related to Marinobacter, Virgibacillus, Halobacillus, Geomicrobium, Chromohalobacter, Oceanobacillus, Bacillus, Halomonas and Staphylococcus genera. They belonged to moderately halophilic group of bacteria exhibiting salt requirement in the range of 3-20%. There is significant diversity among halophiles from saline habitats of India. Preliminary characterization of crude hydrolases established them to be active and stable under more than one extreme condition of high salt, pH, temperature and presence of organic solvents. It is concluded that these halophilic isolates are not only diverse in phylogeny but also in their enzyme characteristics. Their enzymes may be potentially useful for catalysis under harsh operational conditions encountered in industrial processes. The solvent stability among halophilic enzymes seems a generic novel feature making them potentially useful in non-aqueous enzymology.

Screening and isolation of halophilic bacteria producing industrially important enzymes

PubMed Central

Kumar, Sumit; Karan, Ram; Kapoor, Sanjay; S.P., Singh; S.K., Khare

2012-01-01

Halophiles are excellent sources of enzymes that are not only salt stable but also can withstand and carry out reactions efficiently under extreme conditions. The aim of the study was to isolate and study the diversity among halophilic bacteria producing enzymes of industrial value. Screening of halophiles from various saline habitats of India led to isolation of 108 halophilic bacteria producing industrially important hydrolases (amylases, lipases and proteases). Characterization of 21 potential isolates by morphological, biochemical and 16S rRNA gene analysis found them related to Marinobacter, Virgibacillus, Halobacillus, Geomicrobium, Chromohalobacter, Oceanobacillus, Bacillus, Halomonas and Staphylococcus genera. They belonged to moderately halophilic group of bacteria exhibiting salt requirement in the range of 3–20%. There is significant diversity among halophiles from saline habitats of India. Preliminary characterization of crude hydrolases established them to be active and stable under more than one extreme condition of high salt, pH, temperature and presence of organic solvents. It is concluded that these halophilic isolates are not only diverse in phylogeny but also in their enzyme characteristics. Their enzymes may be potentially useful for catalysis under harsh operational conditions encountered in industrial processes. The solvent stability among halophilic enzymes seems a generic novel feature making them potentially useful in non-aqueous enzymology. PMID:24031991

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.

Biodegradation of Ochratoxin A by Aspergillus tubingensis Isolated from Meju.

PubMed

Cho, Sung Min; Jeong, Seong Eun; Lee, Kyu Ri; Sudhani, Hemanth P K; Kim, Myunghee; Hong, Sung-Yong; Chung, Soo Hyun

2016-10-28

Ochratoxin A (OTA), a mycotoxin, contaminates agricultural products and poses a serious threat to public health worldwide. Microbiological methods are known to be a promising approach for OTA biodegradation because physical and chemical methods have practical limitations. In the present study, a total of 130 fungal isolates obtained from 65 traditional Korean meju (a fermented starter for fermentation of soybeans) samples were examined for OTA-biodegradation activity using thin-layer chromatography. Two fungal isolates were selected for OTA-biodegradation activity and were identified as Aspergillus tubingensis M036 and M074 through sequence analysis of the beta-tubulin gene. After culturing both A. tubingensis isolates in Soytone-Czapek medium containing OTA (40 ng/ml), OTA-biodegradation activity was analyzed using high-performance liquid chromatography (HPLC). Both A. tubingensis strains degraded OTA by more than 95.0% after 14 days, and the HPLC analysis showed that the OTA biodegradation by the A. tubingensis strains led to the production of ochratoxin α, which is much less toxic than OTA. Moreover, crude enzymes from the cultures of A. tubingensis M036 and M074 led to OTA biodegradation of 97.5% and 91.3% at pH 5, and 80.3% and 75.3% at pH 7, respectively, in a buffer solution containing OTA (40 ng/ml) after 24 h. In addition, the OTA-biodegrading fungi did not exhibit OTA production activity. Our data suggest that A. tubingensis isolates and their enzymes have the potential for practical application to reduce levels of OTA in food and feed.

β-1,3-1,4-glucanase gene from Bacillus velezensis ZJ20 exerts antifungal effect on plant pathogenic fungi.

PubMed

Xu, Ting; Zhu, Tianhui; Li, Shujiang

2016-02-01

Bacillus velezensis is a known antifungal bacteria. To understand the role of β-1, 3-1, 4-glucanase played on B. velezensis about the mechanism which exerts effect on fungi, we isolated and cloned the β-1, 3-1, 4-glucanase gene (Bglu1) from B. velezensis ZJ20. The Bglu1 open reading frame was 732 bp that encoded a protein with 243 amino acids and a calculated molecular weight of 27.3 kDa. The same gene without the signal peptide, termed Bglu2, was also cloned and expressed in E. coli BL21. Among the two variants, only Bglu2 protein was expressed. Purified Bglu2 could be eluted with imidazole solution at concentrations ranging from 100 to 500 mM although the highest expression was observed at 150 and 200 mM and the purest was at 500 mM. In addition, activity of the crude enzyme was 1527 U ml(-1) and the highest activity of the purified enzyme was 1706 U ml(-1). The purified β-1, 3-1, 4-glucanase had activity on a wide range of pH and temperatures and displayed optimal activity at pH 5.0 and 35 °C. More importantly, the mycelial morphology of three pathogenic fungi was destroyed by the purified β-1, 3-1, 4-glucanase. In conclusion, β-1, 3-1, 4-glucanase from B. velezensis ZJ20 can be highly expressed in E. coli BL21 and the recombinant protein is pathogenic to fungi.

Photooxidation products of polycyclic aromatic compounds containing sulfur.

PubMed

Bobinger, Stefan; Andersson, Jan T

2009-11-01

Photooxidation of crude oil components is an important process that removes pollutants from the environment. Polycyclic aromatic compounds (PACs) are known to be toxic to many life forms, but little is known about their photooxidation products in the aqueous phase. We here identify a large number of photoproducts from 11 benzothiophenes, a polycyclic aromatic sulfur heterocycle that is a major representative of PACs in crude oil. The investigated compounds contain two to four methyl groups and an ethyl or an n-octyl group. In water, the products arise through oxidation of alkyl side chains to aldehydes and carboxylic acids or through an opening in one of the aromatic rings. The product analysis was performed using gas chromatography with mass spectrometric or atomic emission detection. The main product is always a sulfobenzoic acid, which strongly lowers the pH of the solution. With long alkyl substituents, surfactants are formed, which may possess solubilizing properties in water. The larger the number of alkyl groups, the faster is the photooxidation. Several of the identified acidic compounds were also found when whole crude oil was photooxidized, showing that simulation with individual compounds reflects the situation in whole crude.

Biotreatment of oily wastewater by rhamnolipids in aerated active sludge system*

PubMed Central

Zhang, Hong-zi; Long, Xu-wei; Sha, Ru-yi; Zhang, Guo-liang; Meng, Qin

2009-01-01

Oily wastewater generated by various industries creates a major ecological problem throughout the world. The traditional methods for the oily wastewater treatment are inefficient and costly. Surfactants can promote the biodegradation of petroleum hydrocarbons by dispersing oil into aqueous environment. In the present study, we applied rhamnolipid-containing cell-free culture broth to enhance the biodegradation of crude oil and lubricating oil in a conventional aerobically-activated sludge system. At 20 °C, rhamnolipids (11.2 mg/L) increased the removal efficiency of crude oil from 17.7% (in the absence of rhamnolipids) to 63%. At 25 °C, the removal efficiency of crude oil was over 80% with the presence of rhamnolipids compared with 22.3% in the absence of rhamnolipids. Similarly, rhamnolipid treatment (22.5 mg/L) for 24 h at 20 °C significantly increased the removal rate of lubricating oil to 92% compared with 24% in the absence of rhamnolipids. The enhanced removal of hydrocarbons was mainly attributed to the improved solubility and the reduced interfacial tension by rhamnolipids. We conclude that a direct application of the crude rhamnolipid solution from cell culture is effective and economic in removing oily contaminants from wastewater. PMID:19882761

Application of dry elixir system to oriental traditional medicine: taste masking of peonjahwan by coated dry elixir.

PubMed

Choi, H G; Kim, C K

2000-02-01

Peonjahwan, an oriental traditional medicine composed of crude herbal drugs and animal tissues is bitter and poorly water-soluble. To mask the bitterness of peonjahwan and enhance the release of bilirubin, one of the crude active ingredients of peonjahwan, peonja dry elixir (PDE), was prepared using a spray-dryer after extracting the crude materials in ethanol-water solution. Coated peonja dry elixir (CPDE) was then prepared by coating the PDE with Eudragit acrylic resin. Panel assessed bitterness and release test of bilirubin from PDE and CPDE were carried out and compared with peonjahwan alone. PDE was found to have little effect upon the reduction of the bitterness of peonjahwan. However, the bitterness of CPDE was found to reduce to 1/4 of that of peonjahwan due to the encapsulation of crude active ingredients by the dextrin and Eudragit shell (P<0.05). The release rate of bilirubin from PDE and CPDE for 60 min increased about 3.5- and 2.5- fold, respectively, compared to peonjahwan at pH 1.2. It is concluded that CPDE, which masked the bitterness of peonjahwan and enhanced the release of bilirubin, is a preferable delivery system for peonjahwan.

Accord near for offshore California oil shipments

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

Not Available

There are faint glimmers of hope again for offshore California operators. After more than a decade of often bitter strife over offshore oil and gas development and transportation issues, state officials and oil producers may be moving toward compromise solutions. One such solution may be forthcoming on offshore development. But the real change came with the turnabout of the California Coastal Commission (CCC), which last month approved a permit for interim tankering of crude from Point Arguello oil field in the Santa Barbara Channel to Los Angeles. The dispute over how to ship offshore California crude to market has draggedmore » on since before Point Arguelo development plans were unveiled. The project's status has become a flashpoint in the U.S. debate over resource use and environmental concerns. The controversy flared anew in the wake of the 1989 Exxon Valdez tanker spill off Alaska, when CCC voided a Santa Barbara County permit for interim tankering, a move project operator Chevron Corp. linked to the Exxon Valdez accident. Faced with litigation, the state's economic devastation, and acrimonious debate over transporting California crude, Gov. Pete Wilson and other agencies approved the CCC permit. But there's a catch: A permanent pipeline must be built to handle full production within 3 years. The paper discusses permit concerns, the turnaround decision, the anger of environmental groups, and pipeline proposals.« less

Xylanase production by Burkholderia sp. DMAX strain under solid state fermentation using distillery spent wash.

PubMed

Mohana, Sarayu; Shah, Amita; Divecha, Jyoti; Madamwar, Datta

2008-11-01

Xylanase production by a newly isolated strain of Burkholderia sp. was studied under solid state fermentation using anaerobically treated distillery spent wash. Response surface methodology (RSM) involving Box-Behnken design was employed for optimizing xylanase production. The interactions between distillery effluent concentration, initial pH, moisture ratio and inoculum size were investigated and modeled. Under optimized conditions, xylanase production was found to be in the range of 5200-5600 U/g. The partially purified enzyme recovered after ammonium sulphate fractionation showed maximum activity at 50 degrees C and pH 8.6. Kinetic parameters like Km and Vmax for xylan were found to be 12.75 mg/ml and 165 micromol/mg/min. In the presence of metal ions such as Ca2+, Co2+, Mn2+, Ba2+, Mg2+ and protein disulphide reducing agents such as beta-mercaptoethanol and dithiotheritol (DTT) the activity of enzyme increased, where as strong inhibition of enzyme activity was observed in the presence of Cu2+, Ag+, Fe2+ and SDS. The crude enzyme hydrolysed lignocellulosic substrate, wheat bran as well as industrial pulp.

Effects of controlled gas environments in microbial enhancement of plant protein recovery

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

Mudgett, R.E.; Bajracharya, R.

Controlled gas environments were maintained by a novel aeration system in solid substrate fermentations for enhanced protein recovery from pressed alfalfa residues. High O/sub 2/ pressures stimulated biomass and enzyme production by an Aspergillus species, isolated from alfalfa, which produced cellulase and pectinase activities in growth-associated metabolism. High CO/sub 2/ pressures also stimulated enzyme production, but had less effect on biomass production, as established from the dissimilation of plant solids. Cellulase and pectinase activities were generally related to protein recoveries. Recoveries were greater than or equal to 50% higher than those obtained by mechanical extraction, with maximum recoveries of greatermore » than or equal to 70% of crude protein contents. Protein not recovered at high cellulase and pectinase activities were believed to be in structurally bound forms not amenable to recovery by nonproteolytic enzymes. Buffering at pH 8 and autoclaving of residues prior to fermentation had little effect on protein recoveries. Controlled gas environments are seen to offer an interesting potential for optimizing industrial fermentation processes for the production of microbial enzymes.« less

Suitability of the hydrocarbon-hydroxylating molybdenum-enzyme ethylbenzene dehydrogenase for industrial chiral alcohol production.

PubMed

Tataruch, M; Heider, J; Bryjak, J; Nowak, P; Knack, D; Czerniak, A; Liesiene, J; Szaleniec, M

2014-12-20

The molybdenum/iron-sulfur/heme protein ethylbenzene dehydrogenase (EbDH) was successfully applied to catalyze enantiospecific hydroxylation of alkylaromatic and alkylheterocyclic compounds. The optimization of the synthetic procedure involves use of the enzyme in a crude purification state that saves significant preparation effort and is more stable than purified EbDH without exhibiting unwanted side reactions. Moreover, immobilization of the enzyme on a crystalline cellulose support and changes in reaction conditions were introduced in order to increase the amounts of product formed (anaerobic atmosphere, electrochemical electron acceptor recycling or utilization of ferricyanide as alternative electron acceptor in high concentrations). We report here on an extension of effective enzyme activity from 4h to more than 10 days and final product yields of up to 0.4-0.5g/l, which represent a decent starting point for further optimization. Therefore, we expect that the hydrocarbon-hydroxylation capabilities of EbDH may be developed into a new process of industrial production of chiral alcohols. Copyright © 2014 Elsevier B.V. All rights reserved.

Cu determination in crude oil distillation products by atomic absorption and inductively coupled plasma mass spectrometry after analyte transfer to aqueous solution

NASA Astrophysics Data System (ADS)

Kowalewska, Zofia; Ruszczyńska, Anna; Bulska, Ewa

2005-03-01

Cu was determined in a wide range of petroleum products from crude oil distillation using flame atomic absorption spectrometry (FAAS), electrothermal atomic absorption spectrometry (ETAAS) and inductively coupled plasma mass spectrometry (ICP-MS). Different procedures of sample preparation were evaluated: (i) mineralization with sulfuric acid in an open system, (ii) mineralization in a closed microwave system, (iii) combustion in hydrogen-oxygen flame in the Wickbold's apparatus, (iv) matrix evaporation followed by acid dissolution, and (v) acidic extraction. All the above procedures led to the transfer of the analyte into an aqueous solution for the analytical measurement step. It was found that application of FAAS was limited to the analysis of the heaviest petroleum products of high Cu content. In ICP-MS, the use of internal reference method (with Rh or In as internal reference element) was required to eliminate the matrix effects in the analysis of extracts and the concentrated solutions of mineralized heavy petroleum products. The detection limits (in original samples) were equal to, respectively, 10, 86, 3.3, 0.9 and 0.4 ng g - 1 in procedures i-v with ETAAS detection and 10, 78, 1.1 and 0.5 ng g - 1 in procedures i-iii and v with ICP-MS detection. The procedures recommended here were validated by recovery experiments, certified reference materials analysis and comparison of results, obtained for a given sample, in different ways. The Cu content in the analyzed samples was: 50-110 ng g - 1 in crude oil, < 0.4-6 ng g - 1 in gasoline, < 0.5-2 ng g - 1 in atmospheric oil, < 6-100 ng g - 1 in heavy vacuum oil and 140-300 ng g - 1 in distillation residue.

Utilization of fish meal and fish oil for production of Cryptococcus sp. MTCC 5455 lipase and hydrolysis of polyurethane thereof.

PubMed

Thirunavukarasu, K; Purushothaman, S; Gowthaman, M K; Nakajima-Kambe, T; Rose, C; Kamini, N R

2015-09-01

Fish meal has been used as an additional nitrogen source and fish oil as inducer for the growth and production of lipase from Cryptococcus sp. MTCC 5455. A response surface design illustrated that the optimum factors influencing lipase production were fish meal, 1.5 %, w/v, Na2HPO4, 0.2 %, w/v, yeast extract, 0.25 %, w/v and sardine oil, 2.0 %, w/v with an activity of 71.23 U/mL at 96 h and 25 °C, which was 48.39 % higher than the conventional one-factor-at-a-time method. The crude concentrated enzyme hydrolyzed polyurethane (PUR) efficiently and hydrolysis was 94 % at 30 °C and 96 h. The products, diethylene glycol and adipic acid were quantified by HPLC and scanning electron microscopic studies of the degraded polymer showed significant increase in size of the holes from 24 to 72 h of incubation. Hydrolysis of PUR within 96 h makes the lipase novel for disposal of PUR and provides an innovative solution to the problems created by plastic wastes.

A Multifunctional Sensor in Ternary Solution Using Canonical Correlations for Variable Links Assessment

PubMed Central

Liu, Dan; Wang, Qisong; Liu, Xin; Niu, Ruixin; Zhang, Yan; Sun, Jinwei

2016-01-01

Accurately measuring the oil content and salt content of crude oil is very important for both estimating oil reserves and predicting the lifetime of an oil well. There are some problems with the current methods such as high cost, low precision, and difficulties in operation. To solve these problems, we present a multifunctional sensor, which applies, respectively, conductivity method and ultrasound method to measure the contents of oil, water, and salt. Based on cross sensitivity theory, these two transducers are ideally integrated for simplifying the structure. A concentration test of ternary solutions is carried out to testify its effectiveness, and then Canonical Correlation Analysis is applied to evaluate the data. From the perspective of statistics, the sensor inputs, for instance, oil concentration, salt concentration, and temperature, are closely related to its outputs including output voltage and time of flight of ultrasound wave, which further identify the correctness of the sensing theory and the feasibility of the integrated design. Combined with reconstruction algorithms, the sensor can realize the content measurement of the solution precisely. The potential development of the proposed sensor and method in the aspect of online test for crude oil is of important reference and practical value. PMID:27775640

Optimization of biosurfactant production in soybean oil by rhodococcus rhodochrous and its utilization in remediation of cadmium-contaminated solution

NASA Astrophysics Data System (ADS)

Suryanti, Venty; Hastuti, Sri; Andriani, Dewi

2016-02-01

Biosurfactant production by Rhodococcus rhodochrous in soybean oil was developed, where the effect of medium composition and fermentation time were evaluated. The optimum condition for biosurfactant production was achieved when a medium containing 30 g/L TSB (tryptic soy broth) and 20% v/v soybean oil was used as media with 7 days of fermentation. Biosurfactant was identified as glycolipids type biosurfactant which had critical micelle concentration (CMC) value of 896 mg/L. The biosurfactant had oil in water emulsion type and was able to reduce the surface tension of palm oil about 52% which could stabilize the emulsion up to 12 days. The batch removal of cadmium metal ion by crude and partially purified biosurfactants have been examined from synthetic aqueous solution at pH 6. The results exhibited that the crude biosurfactant had a much better adsorption ability of Cd(II) than that of partially purified biosurfactant. However, it was found that there was no significant difference in the adsorption of Cd(II) with 5 and 10 minutes of contact time. The results indicated that the biosurfactant could be used in remediation of heavy metals from contaminated aqueous solution.

Production of Mozzarella Cheese Using Rennin Enzyme from Mucor miehei Grown at Rice Bran Molasses Medium

NASA Astrophysics Data System (ADS)

Rusdan, I. H.; Kusnadi, J.

2017-04-01

The research aimed to study the characteristic and yield of Mozzarella cheese produced by using rennin enzyme from Mucor miehei which is grown at rice bran and molasses medium. The popularity of Mozzarella cheese in Indonesia is increased caused by the spreading of western foods in Indonesia such as pizza and spaghetti that use Mozzarella cheese for ingredient. In Italy, Mozzarella and pizza cheeses are dominating 78% of the total Italian Cheese products. In producing Mozzarella cheese, rennin enzyme is always used as milk coagulant. Even now, Indonesia has not produced the rennin enzyme yet. The rennin enzyme from Mucor miehei growing at rice bran and molases medium which have the availability can be managed purposively within short period of time. The completly randomized design methode used to get the best crude extracts of Mucor miehei rennin enzyme, then is employed to produce mozzarella cheese. The result of Mozzarella cheese has various characteristics such as the yield’s weight is 9.1%, which consists of 50% moisture content, 36.64% peotein levels, 0.1 melting ability and 82.72% stretch ability or 0.79/N. With that characteristic it is concluded that rennin enzyme from Mucor miehei grown at rice bran molasses medium has the potential to alternatively subtitute calf rennin to produce Mozzarella cheese, and the characteristics fulfill the standart.

Detergents as selective inhibitors and inactivators of enzymes.

PubMed

Vincenzini, M T; Favilli, F; Stio, M; Vanni, P; Treves, C

1985-01-01

In order to study the detergent-enzyme interaction and to clarify whether such an interaction produces specific or non-specific effects, we investigated the action of natural and synthetic detergents on enzymatic systems of different levels of complexity (crystalline enzymes, crude homogenates, organ preparations, organisms in toto i.e. rats and germinating seeds). The enzyme-detergent interaction was examined both as a time-independent phenomenon (inhibition) and as a time-dependent phenomenon (inactivation). In in vitro experiments a clear inhibition of pyridine-dependent dehydrogenases by long-chain anionic detergents was found. Cationic detergents have their greatest effect on lipase, LDH, MDH and ICDH from rat liver homogenates. At low concentrations SDS inactivates all the dehydrogenase enzymes studied. With high concentrations (10 mM) of SDS and dodecyltrimethylammonium bromide (C12), there was a sharp and non-specific decrease of enzymatic activities. In the in vivo studies, rats were given detergents to drink; the cationic detergent (C12) was far more effective than SDS with enzymes from both intestine and liver homogenates. SDS and C12 do not seem to interfere with enzyme activities at the beginning of the germination of Pinus pinea and Triticum durum seeds. However a marked reduction of activities does occur at the respective maximum germination times of these seeds. The nonionic detergent is ineffective both as inhibitor and as inactivator.

Generic HPLC platform for automated enzyme reaction monitoring: Advancing the assay toolbox for transaminases and other PLP-dependent enzymes.

PubMed

Börner, Tim; Grey, Carl; Adlercreutz, Patrick

2016-08-01

Methods for rapid and direct quantification of enzyme kinetics independent of the substrate stand in high demand for both fundamental research and bioprocess development. This study addresses the need for a generic method by developing an automated, standardizable HPLC platform monitoring reaction progress in near real-time. The method was applied to amine transaminase (ATA) catalyzed reactions intensifying process development for chiral amine synthesis. Autosampler-assisted pipetting facilitates integrated mixing and sampling under controlled temperature. Crude enzyme formulations in high and low substrate concentrations can be employed. Sequential, small (1 µL) sample injections and immediate detection after separation permits fast reaction monitoring with excellent sensitivity, accuracy and reproducibility. Due to its modular design, different chromatographic techniques, e.g. reverse phase and size exclusion chromatography (SEC) can be employed. A novel assay for pyridoxal 5'-phosphate-dependent enzymes is presented using SEC for direct monitoring of enzyme-bound and free reaction intermediates. Time-resolved changes of the different cofactor states, e.g. pyridoxal 5'-phosphate, pyridoxamine 5'-phosphate and the internal aldimine were traced in both half reactions. The combination of the automated HPLC platform with SEC offers a method for substrate-independent screening, which renders a missing piece in the assay and screening toolbox for ATAs and other PLP-dependent enzymes. Copyright © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

The effect of deep eutectic solvents on catalytic function and structure of bovine liver catalase.

PubMed

Harifi-Mood, Ali Reza; Ghobadi, Roohollah; Divsalar, Adeleh

2017-02-01

Aqueous solutions of reline and glyceline, the most common deep eutectic solvents, were used as a medium for Catalase reaction. By some spectroscopic methods such as UV-vis, fluorescence and circular dichroism (CD) function and structure of Catalase were investigated in aqueous solutions of reline and glyceline. These studies showed that the binding affinity of the substrate to the enzyme increased in the presence of 100mM glyceline solution, which contrasts with reline solution that probably relates to instructive changes in secondary structure of protein. Meanwhile, enzyme remained nearly 70% and 80% active in this concentration of glyceline and reline solutions respectively. In the high concentration of DES solutions, enzyme became mainly inactive but surprisingly stayed in nearly 40% active in choline chloride solution, which is the common ion species in reline and glyceline solvents. It is proposed that the chaotropic nature of choline cation might stop the reducing trend of activity in concentrated choline chloride solutions but this instructive effect is lost in aqueous deep eutectic solvents. In this regard, the presence of various concentrations of deep eutectic solvents in the aqueous media of human cells would be an activity adjuster for this important enzyme in its different operation conditions. Copyright © 2016 Elsevier B.V. All rights reserved.

Molecular Identification of a Newly Isolated Bacillus subtilis BI19 and Optimization of Production Conditions for Enhanced Production of Extracellular Amylase

PubMed Central

Dash, Biplab Kumar; Rahman, M. Mizanur; Sarker, Palash Kumar

2015-01-01

A study was carried out with a newly isolated bacterial strain yielding extracellular amylase. The phylogenetic tree constructed on the basis of 16S rDNA gene sequences revealed this strain as clustered with the closest members of Bacillus sp. and identified as Bacillus subtilis BI19. The effect of various fermentation conditions on amylase production through shake-flask culture was investigated. Rice flour (1.25%) as a cheap natural carbon source was found to induce amylase production mostly. A combination of peptone and tryptone as organic and ammonium sulfate as inorganic nitrogen sources gave highest yield. Maximum production was obtained after 24 h of incubation at 37°C with an initial medium pH 8.0. Addition of surfactants like Tween 80 (0.25 g/L) and sodium lauryl sulfate (0.2 g/L) resulted in 28% and 15% increase in enzyme production, respectively. Amylase production was 3.06 times higher when optimized production conditions were used. Optimum reaction temperature and pH for crude amylase activity were 50°C and 6.0, respectively. The crude enzyme showed activity and stability over a fair range of temperature and pH. These results suggest that B. subtilis BI19 could be exploited for production of amylase at relatively low cost and time. PMID:26180814

Anti-angiotensin converting enzyme (ACE) proteins from mycelia of Ganoderma lucidum (Curtis) P. Karst

PubMed Central

2013-01-01

Background Ganoderma lucidum has been purported as a potent remedy in the treatment and prevention of several ailments, including hypertension. This study aimed to explore the anti-ACE potential of protein fractions from the mycelia of G. lucidum. Methods Ganoderma lucidum mycelia were cultivated by submerged fermentation in a liquid medium containing brown sugar and spent brewer’s yeast. Intracellular proteins were fractionated from mycelia crude water extract by ammonium sulphate precipitation, and their angiotensin converting enzyme inhibitory activity was evaluated. The potential anti-ACE protein fractions were further separated by RP-HPLC and characterised using proteomics platforms. Results Preliminary result demonstrated that the mycelia crude water extract inhibited ACE at IC50 value of 1.134 ± 0.036 mg/mL. Following protein fractionation and HPLC purification, the presence of highly potential anti-ACE proteins with the IC50 values less than 200 μg/mL was detected. Characterisation of these proteins demonstrated the presence of four different antihypertensive-related proteins involved in the regulation of blood pressure through different mechanisms. Conclusions This study suggests that the mycelia of G. lucidum has high potential in lowering blood pressure level due to the presence of several antihypertensive-related proteins such as cystathionine beta synthase-like protein, DEAD/DEAH box helicase-like protein, paxillin-like protein, and alpha/beta hydrolase-like protein. PMID:24093919

Evaluation of Enzyme-Linked Immunosorbent Assay for Diagnosis of Post-Kala-Azar Dermal Leishmaniasis with Crude or Recombinant k39 Antigen

PubMed Central

Salotra, P.; Sreenivas, G.; Nasim, A. A.; Subba Raju, B. V.; Ramesh, V.

2002-01-01

The diagnosis of post-kala-azar dermal leishmaniasis (PKDL), a dermatosis that provides the only known reservoir for the parasite Leishmania donovani in India, remains a problem. Timely recognition and treatment of PKDL would contribute significantly to the control of kala-azar. We evaluated here the potential of the enzyme-linked immunosorbent assay (ELISA) as a diagnostic tool for PKDL. Antigen prepared from promastigotes and axenic amastigotes with parasite isolates that were derived from skin lesions of a PKDL patient gave sensitivities of 86.36 and 92%, respectively, in the 88 PKDL cases examined. The specificity of the ELISA test was examined by testing groups of patients with other skin disorders (leprosy and vitiligo) or coendemic infections (malaria and tuberculosis), as well as healthy controls from areas where this disease is endemic or is not endemic. A false-positive reaction was obtained in 14 of 144 (9.8%) of the controls with the promastigote antigen and in 14 of 145 (9.7%) of the controls with the amastigote antigen. Evaluation of the serodiagnostic potential of recombinant k39 by ELISA revealed a higher sensitivity (94.5%) and specificity (93.7%) compared to the other two antigens used. The data demonstrate that ELISA with crude or recombinant antigen k39 provides a relatively simple and less-invasive test for the reliable diagnosis of PKDL. PMID:11874880

Development and application of ab initio QM/MM methods for mechanistic simulation of reactions in solution and in enzymes

PubMed Central

Hu, Hao; Yang, Weitao

2013-01-01

Determining the free energies and mechanisms of chemical reactions in solution and enzymes is a major challenge. For such complex reaction processes, combined quantum mechanics/molecular mechanics (QM/MM) method is the most effective simulation method to provide an accurate and efficient theoretical description of the molecular system. The computational costs of ab initio QM methods, however, have limited the application of ab initio QM/MM methods. Recent advances in ab initio QM/MM methods allowed the accurate simulation of the free energies for reactions in solution and in enzymes and thus paved the way for broader application of the ab initio QM/MM methods. We review here the theoretical developments and applications of the ab initio QM/MM methods, focusing on the determination of reaction path and the free energies of the reaction processes in solution and enzymes. PMID:24146439

Possibility of wax control techniques in Indonesian oil fields

NASA Astrophysics Data System (ADS)

Abdurrahman, M.; Ferizal, F. H.; Husna, U. Z.; Pangaribuan, L.

2018-03-01

Wax is one of the common problem which can reduce the oil production, especially for the reservoir with high paraffin content case. When the temperature of crude oil is lower than pour point, wax molecules can begin rapidly precipitated. The impacts of this problem are the clogging of production equipment, sealing off the pores in the reservoir, and decreasing production flow rate. In order to solve the wax problem, several methods have been applied in some oil fields in the world. For example, chemical methods in Jiangsu field (China) and Mumbai High field (India), hot water in Mangala field (India), magnetic method in Daqing field (China), water-dispersible in Bakken basin (US), and microbial in Jidong field (China). In general, the various crude oils present in the Indonesia contain wax content between 10%-39% and pour point of 22°C-49°C. Hot water and chemical method are commonly used to solve wax problems in Indonesian oil fields. However, the primary solution is magnetic method, and the secondary solution is water dispersible.

Optimization of palm oil extraction from Decanter cake of small crude palm oil mill by aqueous surfactant solution using RSM

NASA Astrophysics Data System (ADS)

Ahmadi Pirshahid, Shewa; Arirob, Wallop; Punsuvon, Vittaya

2018-04-01

The use of hexane to extract vegetable oil from oilseeds or seed cake is of growing concern due to its environmental impact such as its smelling and toxicity. In our method, used Response Surface Methodology (RSM) was applied to study the optimum condition of decanter cake obtained from small crude palm oil with aqueous surfactant solution. For the first time, we provide an optimum condition of preliminary study with decanter cake extraction to obtain the maximum of oil yield. The result from preliminary was further used in RSM study by using Central Composite Design (CCD) that consisted of thirty experiments. The effect of four independent variables: the concentration of Sodium Dodecyl Sulfate (SDS) as surfactant, temperature, the ratio by weight to volume of cake to surfactant solution and the amount of sodium chloride (NaCl) on dependent variables are studied. Data were analyzed using Design-Expert 8 software. The results showed that the optimum condition of decanter cake extraction were 0.016M of SDS solution concentration, 73°C of extraction temperature, 1:10 (g:ml) of the ratio of decanter cake to SDS solution and 2% (w/w) of NaCl amount. This condition gave 77.05% (w/w) oil yield. The chemical properties of the extracted palm oil from this aqueous surfactant extraction are further investigated compared with the hexane extraction. The obtained result showed that all properties of both extractions were nearly the same.

Catalytic activity of enzymes immobilized on AlGaN /GaN solution gate field-effect transistors

NASA Astrophysics Data System (ADS)

Baur, B.; Howgate, J.; von Ribbeck, H.-G.; Gawlina, Y.; Bandalo, V.; Steinhoff, G.; Stutzmann, M.; Eickhoff, M.

2006-10-01

Enzyme-modified field-effect transistors (EnFETs) were prepared by immobilization of penicillinase on AlGaN /GaN solution gate field-effect transistors. The influence of the immobilization process on enzyme functionality was analyzed by comparing covalent immobilization and physisorption. Covalent immobilization by Schiff base formation on GaN surfaces modified with an aminopropyltriethoxysilane monolayer exhibits high reproducibility with respect to the enzyme/substrate affinity. Reductive amination of the Schiff base bonds to secondary amines significantly increases the stability of the enzyme layer. Electronic characterization of the EnFET response to penicillin G indicates that covalent immobilization leads to the formation of an enzyme (sub)monolayer.

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.

Production, purification and characterization of fibrinolytic enzyme from Serratia sp. KG-2-1 using optimized media.

PubMed

Taneja, Kapila; Bajaj, Bijender Kumar; Kumar, Sandeep; Dilbaghi, Neeraj

2017-07-01

Intravascular thrombosis is one of the major causes of variety of cardiovascular disorders leading to high mortality worldwide. Fibrinolytic enzymes from microbial sources possess ability to dissolve these clots and help to circumvent these problems in more efficient and safer way. In the present study, fibrinolytic protease with higher fibrinolytic activity than plasmin was obtained from Serratia sp. KG-2-1 isolated from garbage dump soil. Response surface methodology was used to study the interactive effect of concentration of maltose, yeast extract + peptone (1:1), incubation time, and pH on enzyme production and biomass. Maximum enzyme production was achieved at 33 °C after 24 h at neutral pH in media containing 1.5% Maltose, 4.0% yeast extract + peptone and other trace elements resulting in 1.82 folds increased production. The enzyme was purified from crude extract using ammonium sulfate precipitation and DEAE-Sephadex chromatography resulting in 12.9 fold purification with 14.9% yield. The purified enzyme belongs to metalloprotease class and had optimal activity in conditions similar to physiological environment with temperature optima of 40 °C and pH optima of 8. The enzyme was found to be stable in various solvents and its activity was enhanced in presence of Na + , K + , Ba 2+ , Cu 2+ , Mn 2+ , Hg 2+ but inhibited by Ca 2+ and Fe 3+ . Hence, the obtained enzyme may be used as potential therapeutic agent in combating various thrombolytic disorders.

Effects of exogenous phytase and xylanase, individually or in combination, and pelleting on nutrient digestibility, available energy content of wheat and performance of growing pigs fed wheat-based diets

PubMed Central

Yang, Y. Y.; Fan, Y. F.; Cao, Y. H.; Guo, P. P.; Dong, B.; Ma, Y. X.

2017-01-01

Objective Two experiments were conducted to determine the effects of adding exogenous phytase and xylanase, individually or in combination, as well as pelleting on nutrient digestibility, available energy content of wheat and the performance of growing pigs fed wheat-based diets. Methods In Experiment 1, forty-eight barrows with an initial body weight of 35.9±0.6 kg were randomly assigned to a 2×4 factorial experiment with the main effects being feed form (pellet vs meal) and enzyme supplementation (none, 10,000 U/kg phytase, 4,000 U/kg xylanase or 10,000 U/kg phytase plus 4,000 U/kg xylanase). The basal diet contained 97.8% wheat. Pigs were placed in metabolic cages for a 7-d adaptation period followed by a 5-d total collection of feces and urine. Nutrient digestibility and available energy content were determined. Experiment 2 was conducted to evaluate the effects of pelleting and enzymes on performance of wheat for growing pigs. In this experiment, 180 growing pigs (35.2±9.0 kg BW) were allocated to 1 of 6 treatments according to a 2×3 factorial treatment arrangement with the main effects being feed form (meal vs pellet) and enzyme supplementation (0, 2,500 or 5,000 U/kg xylanase). Results In Experiment 1, there were no interactions between feed form and enzyme supplementation. Pelleting reduced the digestibility of acid detergent fiber (ADF) by 6.4 percentage units (p<0.01), increased the digestibility of energy by 0.6 percentage units (p<0.05), and tended to improve the digestibility of crude protein by 0.5 percentage units (p = 0.07) compared with diets in mash form. The addition of phytase improved the digestibility of phosphorus (p<0.01) and calcium (p<0.01) by 6.9 and 7.6 percentage units respectively compared with control group. Adding xylanase tended to increase the digestibility of crude protein by 1.0 percentage units (p = 0.09) and increased the digestibility of neutral detergent fiber (NDF) (p<0.01) compared with control group. Supplementation of the xylanase-phytase combination improved the digestibility of phosphorus (p<0.01) but impaired NDF digestibility (p<0.05) compared with adding xylanase alone. In Experiment 2, adding xylanase increased average daily gain (p<0.01) and linearly improved the feed:gain ratio (p<0.01) compared with control group. Conclusion Pelleting improved energy digestibility but decreased ADF digestibility. Adding xylanase increased crude protein digestibility and pig performance. Phytase increased the apparent total tract digestibility of phosphorus and calcium. The combination of phytase-xylanase supplementation impaired the effects of xylanase on NDF digestibility. PMID:27004820

Effects of exogenous phytase and xylanase, individually or in combination, and pelleting on nutrient digestibility, available energy content of wheat and performance of growing pigs fed wheat-based diets.

PubMed

Yang, Y Y; Fan, Y F; Cao, Y H; Guo, P P; Dong, B; Ma, Y X

2017-01-01

Two experiments were conducted to determine the effects of adding exogenous phytase and xylanase, individually or in combination, as well as pelleting on nutrient digestibility, available energy content of wheat and the performance of growing pigs fed wheat-based diets. In Experiment 1, forty-eight barrows with an initial body weight of 35.9±0.6 kg were randomly assigned to a 2×4 factorial experiment with the main effects being feed form (pellet vs meal) and enzyme supplementation (none, 10,000 U/kg phytase, 4,000 U/kg xylanase or 10,000 U/kg phytase plus 4,000 U/kg xylanase). The basal diet contained 97.8% wheat. Pigs were placed in metabolic cages for a 7-d adaptation period followed by a 5-d total collection of feces and urine. Nutrient digestibility and available energy content were determined. Experiment 2 was conducted to evaluate the effects of pelleting and enzymes on performance of wheat for growing pigs. In this experiment, 180 growing pigs (35.2±9.0 kg BW) were allocated to 1 of 6 treatments according to a 2×3 factorial treatment arrangement with the main effects being feed form (meal vs pellet) and enzyme supplementation (0, 2,500 or 5,000 U/kg xylanase). In Experiment 1, there were no interactions between feed form and enzyme supplementation. Pelleting reduced the digestibility of acid detergent fiber (ADF) by 6.4 percentage units (p<0.01), increased the digestibility of energy by 0.6 percentage units (p<0.05), and tended to improve the digestibility of crude protein by 0.5 percentage units (p = 0.07) compared with diets in mash form. The addition of phytase improved the digestibility of phosphorus (p<0.01) and calcium (p<0.01) by 6.9 and 7.6 percentage units respectively compared with control group. Adding xylanase tended to increase the digestibility of crude protein by 1.0 percentage units (p = 0.09) and increased the digestibility of neutral detergent fiber (NDF) (p<0.01) compared with control group. Supplementation of the xylanase-phytase combination improved the digestibility of phosphorus (p<0.01) but impaired NDF digestibility (p<0.05) compared with adding xylanase alone. In Experiment 2, adding xylanase increased average daily gain (p<0.01) and linearly improved the feed:gain ratio (p<0.01) compared with control group. Pelleting improved energy digestibility but decreased ADF digestibility. Adding xylanase increased crude protein digestibility and pig performance. Phytase increased the apparent total tract digestibility of phosphorus and calcium. The combination of phytase-xylanase supplementation impaired the effects of xylanase on NDF digestibility.

Chemiluminescence-based pesticide biosensor utilizing the intelligent evolved properties of the enzyme alkaline phosphatase

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

Ayyagari, M.; Kamtekar, S.; Pande, R.

A methodology is described for immobilizing the enzyme alkaline phosphatase onto a glass surface using a novel biotinylated copolymer, poly(3-undecylthiophene-co-3- methanoithiophene). A streptavidin conjugate of alkaline phosphatase is used in this study. The biotinylated polymer is attached to the silanized glass surface via hydrophobic interactions and the enzyme is interfaced with the polymer through the classical biotin- streptavidin interaction. Alkaline phosphatase catalyzes the dephosphorylation of a macrocyclic compound, chloro-3-(4-methoxy spiro) (1,2 dioxetane-3-2`-tricyclo-) (3.3.1.1 )-(decani-4-yl) phenyl phosphate, to a species which emits energy by chemiluminescence. This chemiluminescence signal can be detected with a photomultiplier tube for enzymatic catalysis with the biocatalystmore » both in solution and immobilized on a glass surface. The signal generation is inhibited by the organophosphorus based insecticides such as paraoxon as well as nerve agents. We demonstrate in this study that a number of organophosphorus based insecticides inhibit the enzyme-mediated generation of chemiluminescence signal. This is true for the enzyme conjugate both free in solution and immobilized on a glass surface. In solution, the inhibition resembles the case of a partially uncompetitive system. By this type of inhibition we are able to detect pesticides down to about 50 ppb for the enzyme in solution. The pesticide detection limit of immobilized enzyme is currently being investigated. The enzyme is capable of a number of measurement cycles without significant loss of signal level.« less

Homogeneous solutions of hydrophilic enzymes in nonpolar organic solvents. New systems for fundamental studies and biocatalytic transformations.

PubMed

Mozhaev, V V; Poltevsky, K G; Slepnev, V I; Badun, G A; Levashov, A V

1991-11-04

A typical hydrophilic enzyme, CT, can be dissolved in nonpolar organic solvents (n-octane, cyclohexane and toluene) up to microM concentrations. In the homogeneous solution obtained, the enzyme possesses catalytic activity and enormously high thermostability. It does not lose this activity even after several hours refluxing in octane (126 degrees C) or cyclohexane (81 degrees C).

Contamination by oil crude extraction - Refinement and their effects on human health.

PubMed

Ramirez, Maria Isabel; Arevalo, Ana Paulina; Sotomayor, Santiago; Bailon-Moscoso, Natalia

2017-12-01

The harmful effects of oil on various species of flora and fauna have been studied extensively; however, few studies have studied the effects of oil exposure on human health. The objective of this research was to collect information on the acute health effects and serious psychological symptoms of the possible consequences of such exposure to crude oil. Some studies focused on the composition of different chemicals used in the extraction process, and wastes generated proved to be highly harmful to human health. Thus, studies have shown that individuals who live near oil fields or wells - or who take part in activities of cleaning oil spills - have presented health conditions, such as irritation to the skin, eyes, mucous membranes, kidney damage, liver, reproductive, among others. In Ecuador, this reality is not different from other countries, and some studies have shown increased diseases related with oil crude and oil spills, like skin irritation, throat, liver, lung, infertility, and abortions, and it has been linked to childhood leukemia. Other studies suggest a direct relationship between DNA damage because of oil resulting in a genetic instability of the main enzymes of cellular metabolism as well as a relationship with some cancers, such as leukemia. Copyright © 2017 Elsevier Ltd. All rights reserved.

[Analysis of variation of monoterpene glycosides and polyhydroxy compounds in paeoniae radix alba during preliminary processing].

PubMed

Xu, Yuan; Liu, Pei; Yan, Hui; Qian, Da-Wei; Duan, Jin-Ao

2014-05-01

To investigate variation of monoterpene glycosides and polyhydroxy compounds in Paeoniae Radix Alba dried by different processing methods. The crude drugs were processed sequentially as washed, removed the head, tail, fine roots and dried. The samples were divided into eight groups by whether peeled and decocted or not. Each group was dried by 35, 45, 60, 80,100, 120 degrees C, sun-dried and shade-dried. HPLC-PDA method was adopted to determine the content of monoterpene glycosides compounds (paeoniflorin alibiflorin, oxypaeoniflorin and benzoylpaeoniflorin), polyhydroxy compounds (catechin and gallic acid) and benzoic acid. Chromatographic conditions: Phecad C18 column (250 mm x 4.6 mm, 5 microm). A principal component analysis (PCA) method was used subsequently to get data processed. The retained content of seven constituents decreased in those peeled crude drug, and after cooked, monoterpene glycosides and polyhydroxy compounds increased while the benzoic acid decreased. It was believed that rele- vant enzymes were inactivated while being cooked so that drying temperature showed little influence on the biotransformation. Contents of effective ingredients in Paeoniae Radix Alba are influenced by drying processing. The preferable method shows to be that crude drug should be cooked before being peeled and dried. As a matter of processing convtence, it is suggested to be peeled and sliced before being dried.

Isolation of quercetin and mandelic acid from Aesculus indica fruit and their biological activities.

PubMed

Zahoor, Muhammad; Shafiq, Sadaf; Ullah, Habib; Sadiq, Abdul; Ullah, Farhat

2018-06-26

In this study Aesculus indica fruit was subjected to isolation of phytochemicals. Two antioxidants quercetin and Mandelic acid were isolated in pure state. The free radical scavenging and acetyl choline esterase inhibitory potential of the crude extract and sub fractions were also determined. The antioxidant capacity of crude extract, fractions and isolated compounds were determined by DPPH and ABTS methods. Folin-Ciocalteu reagent method was used to estimate the total phenolic contents and were found to be 78.34 ± 0.96, 44.16 ± 1.05, 65.45 ± 1.29, 37.85 ± 1.44 and 50.23 ± 2.431 (mg/g of gallic acid) in crude extract, ethyl acetate, chloroform, n-hexane and aqueous fractions respectively. The flavonoid concentration in crude extract, ethyl acetate, chloroform, n-hexane and aqueous fraction were; 85.30 ± 1.20, 53.80 ± 1.07, 77.50 ± 1.12, 26.30 ± 1.35 and 37.78 ± 1.25 (mg/g of quercetin) respectively. The chloroform fraction was more potent against enzymes, acetyl choline esterase and butyryl choline esterase (IC 50  = 85 and 160 μg/ml respectively). The phenolic compounds in the crude extract and fractions were determined using HPLC standard method. Chlorogenic acid, quercetin, phloroglucinol, rutin, mandelic acid and hydroxy benzoic acid were detected at retention times 6.005, 10.062, 22.623, 30.597, 35.490 and 36.211 in crude extract and different fractions. The ethyl acetate fraction was rich in the targeted compounds and was therefore subjected to column isolation. The HPLC chromatogram of isolated compounds showed single peak at specified retention times which confirms their isolation in pure state. The isolated compounds were then characterized by FTIR and NMR spectrophotometric techniques. The Aesculus indica fruit extracts showed antioxidant and anticholine esterase inhibitory potentials. Two bioactive compounds were isolated in the pure form ethyl acetate fraction. From results it was concluded that the fruit of this plant could be used to minimize oxidative stress caused by reactive oxygen species.

[Analyzing crude/processed root of Polygonum multiflorum from different habitats by UPLC fingerprint and mode identification methods].

PubMed

Xiao, Rong; Lin, Yan; Lei, Si-Min; Zhang, Ying; Huang, Jie; Xia, Bo-Hou; Li, Chun; Liao, Duan-Fang; Wu, Ping; Lin, Li-Mei

2017-06-01

To establish a content determination method for 2,3,5,4'-tetrahydroxy stilbene-2-O-β-D-glucoside (TSG) of the crude/processed root of Polygonum multiflorum from different habitats in China and set up the fingerprint by using UPLC. Various samples were pretreated by macro-porous resin. Then UPLC analysis was performed on Waters ACQUITY UPLC@BEH C18 chromatographic column (2.1 mm×50 mm, 1.7 μm) at (25±5) ℃. A binary gradient elution system was composed of acetonitrile (phase A) and 0.5% acetic acid solution (phase B). Detection was performed at the wavelength of 254 nm, and the mobile flow rate was set at 0.3 mL•min⁻¹. Results showed that the yield of extraction of the 2,3,5,4'-tetrahydroxy stilbene-2-O-β-D-glucoside from root of P. multiflorum was all over 25.0% after macro-porous resin separation; an exclusive UPLC fingerprint method of the crude/processed root of P. multiflorum from different habitats was successfully set up and 17 chromatographic peaks were calibrated. Cluster analysis can not entirely distinguish the crude one from the processed one, while principal component analysis absolutely can. 2,3,5,4'-tetrahydroxy stilbene-2-O-β-D-glucoside is the composition that has largest differences in variable importance in projection (VIP) between crude and processed root of P. multiflorum. The separating method can gain high-purity 2,3,5,4'-tetrahydroxy stilbene-2-O-β-D-glucoside, and the determination method is simple, sensitive, reliable and can be used in fast identifying the crude/processed root of P. multiflorum or as a method for overall quality control of root of P. multiflorum. Copyright© by the Chinese Pharmaceutical Association.

Enhanced stability and chemical resistance of a new nanoscale biocatalyst for accelerating CO2 absorption into a carbonate solution.

PubMed

Zhang, Shihan; Lu, Hong; Lu, Yongqi

2013-12-03

A novel potassium-carbonate-based absorption process is currently being developed to reduce the energy consumption when capturing CO2 from coal combustion flue gas. The process employs the enzyme carbonic anhydrase (CA) as a catalyst to accelerate the rate of CO2 absorption. This study focused on the immobilization of a new variant of the CA enzyme onto a new group of nonporous nanoparticles to improve the enzyme's thermal stability and its chemical resistance to major impurities from the flue gas. The CA enzyme was manufactured at the pilot scale by a leading enzyme company. As carrier materials, two different batches of SiO2-ZrO2 composite nanoparticles and one batch of silica nanoparticle were synthesized using a flame spray pyrolysis method. Classic Danckwerts absorption theory with reaction was applied to determine the kinetics of the immobilized enzymes for CO2 absorption. The immobilized enzymes retained 56-88% of their original activity in a K2CO3/KHCO3 solution over a 60-day test period at 50 °C, compared with a 30% activity retention for their free CA enzyme counterpart. The immobilized CA enzymes also revealed improved chemical stability. The inactivation kinetics of the free and immobilized CA enzymes in the K2CO3/KHCO3 solution were experimentally quantified.

Alkaline thermostable pectinase enzyme from Aspergillus niger strain MCAS2 isolated from Manaslu Conservation Area, Gorkha, Nepal.

PubMed

Khatri, Bhim Prakash; Bhattarai, Tribikram; Shrestha, Sangita; Maharjan, Jyoti

2015-01-01

Pectinase enzymes are one of the commercially important enzymes having great potential in various industries especially in food industry. Pectinases accounts for 25 % of global food enzymes produced and their market is increasing day by day. Therefore, the exploration of microorganism with novel characteristics has always been the focus of the research. Microorganism dwelling in unique habitat may possess unique characteristics. As such, a pectinase producing fungus Aspergillus niger strain MCAS2 was isolated from soil of Manaslu Conservation Area (MCA), Gorkha, Nepal. The optimum production of pectinase enzyme was observed at 48 h of fermentation. The pectinase enzyme was partially purified by cold acetone treatment followed by Sephadex G-75 gel filtration chromatography. The partially purified enzyme exhibited maximum activity 60 U/mg which was almost 8.5-fold higher than the crude pectinase. The approximate molecular weight of the enzyme was found to be 66 kDa as observed from SDS-PAGE. The pectinase enzyme was active at broad range of temperature (30-70 °C) and pH (6.2-9.2). Optimum temperature and pH of the pectinase enzyme were 50 °C and 8.2 respectively. The enzyme was stable up to 70 °C and about 82 % of pectinase activity was still observed at 100 °C. The thermostable and alkaline nature of this pectinase can meet the demand of various industrial processes like paper and pulp industry, in textile industry, fruit juice industry, plant tissue maceration and wastewater treatment. In addition, the effect of different metal ions on pectinase activity was also studied.

Optimization of parameters for enhanced oil recovery from enzyme treated wild apricot kernels.

PubMed

Rajaram, Mahatre R; Kumbhar, Baburao K; Singh, Anupama; Lohani, Umesh Chandra; Shahi, Navin C

2012-08-01

Present investigation was undertaken with the overall objective of optimizing the enzymatic parameters i.e. moisture content during hydrolysis, enzyme concentration, enzyme ratio and incubation period on wild apricot kernel processing for better oil extractability and increased oil recovery. Response surface methodology was adopted in the experimental design. A central composite rotatable design of four variables at five levels was chosen. The parameters and their range for the experiments were moisture content during hydrolysis (20-32%, w.b.), enzyme concentration (12-16% v/w of sample), combination of pectolytic and cellulolytic enzyme i.e. enzyme ratio (30:70-70:30) and incubation period (12-16 h). Aspergillus foetidus and Trichoderma viride was used for production of crude enzyme i.e. pectolytic and cellulolytic enzyme respectively. A complete second order model for increased oil recovery as the function of enzymatic parameters fitted the data well. The best fit model for oil recovery was also developed. The effect of various parameters on increased oil recovery was determined at linear, quadric and interaction level. The increased oil recovery ranged from 0.14 to 2.53%. The corresponding conditions for maximum oil recovery were 23% (w.b.), 15 v/w of the sample, 60:40 (pectolytic:cellulolytic), 13 h. Results of the study indicated that incubation period during enzymatic hydrolysis is the most important factor affecting oil yield followed by enzyme ratio, moisture content and enzyme concentration in the decreasing order. Enzyme ratio, incubation period and moisture content had insignificant effect on oil recovery. Second order model for increased oil recovery as a function of enzymatic hydrolysis parameters predicted the data adequately.

The planning and establishment of a sample preparation laboratory for drug discovery

PubMed Central

Dufresne, Claude

2000-01-01

Nature has always been a productive source of new drugs. With the advent of high-throughput screening, it has now become possible to rapidly screen large sample collections. In addition to seeking greater diversity from natural product sources (micro-organisms, plants, etc.), fractionation of the crude extracts prior to screening is becoming a more important part of our efforts. As sample preparation protocols become more involved, automation can help to achieve and maintain a desired sample throughput. To address the needs of our screening program, two robotic systems were designed. The first system processes crude extracts all the way to 96-well plates, containing solutions suitable for screening in biological and biochemical assays. The system can dissolve crude extracts, fractionate them on solid-phase extraction cartridges, dry and weigh each fraction, re-dissolve them to a known concentration, and prepare mother plates. The second system replicates mother plates into a number of daughter plates. PMID:18924691

Bioaccessibility of Ca, Cu, Fe, Mg, Zn, and crude protein in beef, pork and chicken after thermal processing.

PubMed

Menezes, Eveline A; Oliveira, Aline F; França, Celia J; Souza, Gilberto B; Nogueira, Ana Rita A

2018-02-01

The bioaccessibility of Ca, Cu, Fe, Mg, Zn, and crude protein was evaluated after submitting beef, pork, and chicken to five different thermal treatments. The bioaccessibility of crude protein and metals were simulated by using in vitro enzymatic digestion with a gastric fluid solution and dialysability approach. Inductively coupled plasma optical spectrometry was used to quantify the dialyzable fraction and the total mineral content after microwave-assisted digestion. Graphite furnace atomic absorption spectrometry quantified Cu in chicken dialyzable fraction. The increase of temperature and heat exposure period decreased the protein bioaccessibility. Considering the total and dialyzable fraction, beef is an important source of Cu, Fe, Mg, and Zn to the human diet. The results of Fourier-transform infrared spectroscopy indicated physical changes in the treated samples related to protein denaturation, which was probably responsible for the decreased bioaccessibility of minerals and protein, mainly at higher temperatures. Copyright © 2017 Elsevier Ltd. All rights reserved.

Central carbon metabolism in marine bacteria examined with a simplified assay for dehydrogenases.

PubMed

Wen, Weiwei; Wang, Shizhen; Zhou, Xiaofen; Fang, Baishan

2013-06-01

A simplified assay platform was developed to measure the activities of the key oxidoreductases in central carbon metabolism of various marine bacteria. Based on microplate assay, the platform was low-cost and simplified by unifying the reaction conditions of enzymes including temperature, buffers, and ionic strength. The central carbon metabolism of 16 marine bacteria, involving Pseudomonas, Exiguobacterium, Marinobacter, Citreicella, and Novosphingobium were studied. Six key oxidoreductases of central carbon metabolism, glucose-6-phosphate dehydrogenase, pyruvate dehydrogenase, 2-ketoglutarate dehydrogenase, malate dehydrogenase, malic enzyme, and isocitrate dehydrogenase were investigated by testing their activities in the pathway. High activity of malate dehydrogenase was found in Citreicella marina, and the specific activity achieved 22 U/mg in cell crude extract. The results also suggested that there was a considerable variability on key enzymes' activities of central carbon metabolism in some strains which have close evolutionary relationship while they adapted to the requirements of the niche they (try to) occupy.

Inhibition of partially purified K+/H+-ATPase from guinea-pig isolated and enriched parietal cells by substituted benzimidazoles.

PubMed Central

Beil, W.; Sewing, K. F.

1984-01-01

The cellular and subcellular distributions of adenosinetriphosphatases (ATPases) were examined in guinea-pig gastric mucosal cells. All cell types displayed Mg2+-ATPase and bicarbonate (HCO3-)-stimulated ATPase activity. K+-ATPase was located only in fractions derived from parietal cells. Differential and density-gradient centrifugation of material prepared from parietal cells revealed that K+-ATPase activity was located in a tubulo-vesicular membrane fraction. Enzyme activity was ten fold greater in this fraction than in a crude parietal cell homogenate. The substituted benzimidazoles, omeprazole and picoprazole, inhibited K+-ATPase (IC50 1.8 +/- 0.5 mumol l-1 and 3.1 +/- 0.4 mumol l-1, respectively). Detailed kinetic analysis indicated that these compounds were non-competitive and reversible inhibitors of the enzyme. In contrast cimetidine and verapamil were without effect on the enzyme. The relevance of the inhibition of K+-ATPase to the antisecretory activity of the benzimidazoles, in experimental animals and man, is discussed. PMID:6146367

Biological pretreatment of corn stover with ligninolytic enzyme for high efficient enzymatic hydrolysis.

PubMed

Wang, Feng-Qin; Xie, Hui; Chen, Wei; Wang, En-Tao; Du, Feng-Guang; Song, An-Dong

2013-09-01

Aiming at increasing the efficiency of transferring corn stover into sugars, a biological pretreatment was developed and investigated in this study. The protocol was characterized by the pretreatment with crude ligninolytic enzymes from Phanerochete chrysosporium and Coridus versicolor to break the lignin structure in corn stover, followed by a washing procedure to eliminate the inhibition of ligninolytic enzyme on cellulase. By a 2 d-pretreatment, sugar yield from corn stover hydrolysis could be increased by 50.2% (up to 323 mg/g) compared with that of the control. X-ray diffractometry and FT-IR analysis revealed that biological pretreatment could partially remove the lignin of corn stover, and consequently enhance the enzymatic hydrolysis efficiency of cellulose and hemeicellulose. In addition, the amount of microbial inhibitors, such as acetic acid and furfural, were much lower in biological pretreatment than that in acid pretreatment. This study provided a promising pretreatment method for biotransformation of corn stovers. Copyright © 2013 Elsevier Ltd. All rights reserved.

Effects of aeration on formation and localization of the acetyl coenzyme A synthetases of Saccharomyces cerevisiae

NASA Technical Reports Server (NTRS)

Klein, H. P.; Jahnke, L.

1979-01-01

Previous studies on the yeast Saccharomyces cerevisiae have shown that two different forms of the enzyme acetyl coenzyme A synthetase (ACS) are present, depending on the conditions under which the cells are grown. The paper evaluates the usefulness of a method designed to assay both synthetases simultaneously in yeast homogenates. The data presented confirm the possibility of simultaneous detection and estimation of the amount of both ACSs of S. cerevisiae in crude homogenates of this strain, making possible the study of physiological factors involved in the formation of these isoenzymes. One important factor for specifying which of the two enzymes is found in these yeast cells is the presence or absence of oxygen in their environment. Aeration not only affects the ratio of the two ACSs but also appears to affect the cellular distribution of these enzymes. Most of the data presented suggest the possibility that the nonaerobic ACS may serve as a precursor to the aerobic form.

The first characterized asparaginase from a basidiomycete, Flammulina velutipes.

PubMed

Eisele, Nadine; Linke, Diana; Bitzer, Katrin; Na'amnieh, Shukry; Nimtz, Manfred; Berger, Ralf G

2011-02-01

Flammulina velutipes enjoys high popularity as an edible mushroom in Asian cuisines. Investigating the secretion of peptidases in nutrient media enriched with gluten, an enzyme was noticed that catalyzed the deamidation of L-asparagine and L-glutamine. The enzyme was purified to electrophoretic homogeneity by foaming and SEC. PAGE analysis revealed a protein of about 85 kDa with 13 kDa subunits indicating a hexameric protein. Degenerated primers were deduced from peptide fragments and the complete coding sequence of 372 bp was determined. The gene of Flammulina velutipes asparaginase (FvNase) over expressed in E. coli achieved an L-asparagine-hydrolyzing activity of 16 U/mL in crude extract, which was five times higher than its L-glutamine-hydrolyzing ability. The enzyme showed a pH-optimum at pH 7, remarkable tolerance towards elevated temperature and sodium chloride concentration in both the native and recombinant form, and no significant homology to any conserved domains of published asparaginases or glutaminases. Copyright © 2010 Elsevier Ltd. All rights reserved.

Purification and characterisation of a novel iso-propanol dehydrogenase from Phytomonas sp.

PubMed

Uttaro, A D; Opperdoes, F R

1997-04-01

An alcohol dehydrogenase with two identical subunits and a subunit molecular mass of 40,000 was purified from Phytomonas sp. isolated from the lactiferous tubes of Euphorbia characias. Digitonin titration and subcellular fractionation suggest that the enzyme is present in the mitochondrion. It utilises as substrates, primary and secondary alcohols, is specific for NAD+ as coenzyme and is inhibited by HgCl(2). The pH optimum for the oxidation of ethanol is 9.5, and for the reverse reaction 8.5. The apparent Km values for iso-propanol and ethanol are 40 and 34 microM, respectively and for the reverse reaction, with acetone as substrate, 14 microM. The respective specific activities with iso-propanol and ethanol as substrate, as measured in crude extracts are 300 and 16 mU (milligram of protein)-1. In isoelectric focusing the enzyme showed three major bands with slightly differing isoelectric points that ranged from 6.4 to 6.8. The name, iso-propanol dehydrogenase is proposed for this enzyme.

Applicability of Yeast Extracellular Proteinases in Brewing: Physiological and Biochemical Aspects

PubMed Central

Bilinski, Carl A.; Russell, Inge; Stewart, Graham G.

1987-01-01

A general screening survey for expression of extracellular acid proteinase production was performed on over 100 cultures belonging to the genus Saccharomyces. Although two strains of Saccharomyces cerevisiae showed positive extracellular proteinase phenotypes in plate tests, it was not possible to demonstrate proteolytic activities in cell-free culture supernatants in assays performed at beer pH values. Of several yeasts from other genera examined, Saccharomycopsis fibuligera and Torulopsis magnoliae produced extracellular proteinases with desirable properties. Proteolytic activities were detected in assays performed at beer pH values and at lower temperature. Brewer's wort served as a highly inducing medium for extracellular proteinase production, with T. magnoliae yielding enzyme of highest specific activity. In fact, commencement of enzyme production was detected shortly after the onset of exponential growth in brewer's wort. Inclusion of crude enzyme preparations in brewer's wort inoculated simultaneously with brewer's yeast reduced final ethanol yields slightly and was found to be effective in reducing chill haze formation in bottled beer. PMID:16347298

Regiospecific Ester Hydrolysis by Orange Peel Esterase - An Undergraduate Experiment.

NASA Astrophysics Data System (ADS)

Bugg, Timothy D. H.; Lewin, Andrew M.; Catlin, Eric R.

1997-01-01

A simple but effective experiment has been developed to demonstrate the regiospecificity of enzyme catalysis using an esterase activity easily isolated from orange peel. The experiment involves the preparation of diester derivatives of para-, meta- and ortho-hydroxybenzoic acid (e.g. methyl 4-acetoxy-benzoic acid). The derivatives are incubated with orange peel esterase, as a crude extract, and with commercially available pig liver esterase and porcine pancreatic lipase. The enzymatic hydrolysis reactions are monitored by thin layer chromatography, revealing which of the two ester groups is hydrolysed, and the rate of the enzyme-catalysed reaction. The results of a group experiment revealed that in all cases hydrolysis was observed with at least one enzyme, and in most cases the enzymatic hydrolysis was specific for production of either the hydroxy-ester or acyl-acid product. Specificity towards the ortho-substituted series was markedly different to that of the para-substituted series, which could be rationalised in the case of pig liver esterase by a published active site model.

Antibacterial and Hypoglycemic Diterpenoids from Salvia chamaedryoides.

PubMed

Bisio, Angela; De Mieri, Maria; Milella, Luigi; Schito, Anna M; Parricchi, Anita; Russo, Daniela; Alfei, Silvana; Lapillo, Margherita; Tuccinardi, Tiziano; Hamburger, Matthias; De Tommasi, Nunziatina

2017-02-24

A surface extract of the aerial parts of Salvia chamaedryoides afforded 13 diterpenes (1-13), with seven compounds (1, 3, 4, 7-9, 12) described for the first time. The structures of the new compounds were established using 1D and 2D NMR spectroscopic methods, HRESIMS, and ECD data. The potential hypoglycemic effects of the crude extract, fractions, and pure compounds from S. chamaedryoides were investigated by inhibition of α-glucosidase and α-amylase enzymes. The extract and its fractions showed a moderate dose-dependent inhibition; the pure compounds exhibited differential inhibitory activity against these two enzymes. Molecular modeling studies were also performed to suggest the interaction mode of compound 3 in the α-glucosidase enzyme active site. The antimicrobial activity of the purified compounds was investigated against 26 clinical pathogens. No activity was detected for the Gram-negative species tested nor on Candida albicans and C. glabrata, while variable susceptibilities were observed using Gram-positive staphylococcal and enterococcal species.

From by-product to valuable components: Efficient enzymatic conversion of lactose in whey using β-galactosidase from Streptococcus thermophilus.

PubMed

Geiger, Barbara; Nguyen, Hoang-Minh; Wenig, Stefanie; Nguyen, Hoang Anh; Lorenz, Cindy; Kittl, Roman; Mathiesen, Geir; Eijsink, Vincent G H; Haltrich, Dietmar; Nguyen, Thu-Ha

2016-12-15

β-Galactosidase from Streptococcus thermophilus was overexpressed in a food-grade organism, Lactobacillus plantarum WCFS1. Laboratory cultivations yielded 11,000 U of β-galactosidase activity per liter of culture corresponding to approximately 170 mg of enzyme. Crude cell-free enzyme extracts obtained by cell disruption and subsequent removal of cell debris showed high stability and were used for conversion of lactose in whey permeate. The enzyme showed high transgalactosylation activity. When using an initial concentration of whey permeate corresponding to 205 g L -1 lactose, the maximum yield of galacto-oligosaccharides (GOS) obtained at 50°C reached approximately 50% of total sugar at 90% lactose conversion, meaning that efficient valorization of the whey lactose was obtained. GOS are of great interest for both human and animal nutrition; thus, efficient conversion of lactose in whey into GOS using an enzymatic approach will not only decrease the environmental impact of whey disposal, but also create additional value.

Simultaneous saccharification and fermentation of fungal pretreated cornstalk for hydrogen production using Thermoanaerobacterium thermosaccharolyticum W16.

PubMed

Zhao, Lei; Cao, Guang-Li; Wang, Ai-Jie; Guo, Wan-Qian; Ren, Hong-Yu; Ren, Nan-Qi

2013-10-01

In this research, environmentally friendly fungal pretreatment was first adopted for deconstruction of cornstalk. Then the fungal-pretreated cornstalk was employed to produce hydrogen in simultaneous saccharification and fermentation (SSF) using crude enzyme from Trichoderma viride and Thermoanaerobacterium thermosaccharolyticum W16. The influence of various factors including substrate concentration, initial pH, and enzyme loading on hydrogen production were evaluated. The highest hydrogen yield of 89.3 ml/g-cornstalk was obtained with an initial pH 6.5, 0.75% substrate concentration, and 34 FPU/g cellulose. Compared the result with SSF of physical or chemical pretreated lignocellulosic materials, this research suggested an economic and efficient way for hydrogen production from lignocellulosic biomass. Copyright © 2013 Elsevier Ltd. All rights reserved.

[Specific immunotherapy with depigmented allergoids].

PubMed

Klimek, L; Thorn, C; Pfaar, O

2010-01-01

Specific immunotherapy is the only available causative treatment for IgE-mediated allergic conditions. The state of the art is treatment via the subcutaneous route with crude extracts in a water solution, with physically linked (semidepot) extracts or chemically modified semidepot extracts (allergoids). A relatively new purification method combines depigmentation followed by polymerization with glutaraldehyde. This modification results in increased tolerance with a reduction in both local and systemic adverse effects. As controlled clinical trials have shown, the effectiveness is comparable to that of specific immunotherapy with crude allergen extracts. Recent data suggest that the modified polymerized allergoids allow a safe rush titration in a few days or even in 1 day (ultra-rush titration).

In situ hydrogen, acetone, butanol, ethanol and microdiesel production by Clostridium acetobutylicum ATCC 824 from oleaginous fungal biomass.

PubMed

Hassan, Elhagag Ahmed; Abd-Alla, Mohamed Hemida; Bagy, Magdy Mohamed Khalil; Morsy, Fatthy Mohamed

2015-08-01

An in situ batch fermentation technique was employed for biohydrogen, acetone, butanol, ethanol and microdiesel production from oleaginous fungal biomass using the anaerobic fermentative bacterium Clostridium acetobutylicum ATCC 824. Oleaginous fungal Cunninghamella echinulata biomass which has ability to accumulate up to 71% cellular lipid was used as the substrate carbon source. The maximum cumulative hydrogen by C. acetobutylicum ATCC 824 from crude C. echinulata biomass was 260 ml H2 l(-1), hydrogen production efficiency was 0.32 mol H2 mole(-1) glucose and the hydrogen production rate was 5.2 ml H2 h(-1). Subsequently, the produced acids (acetic and butyric acids) during acidogenesis phase are re-utilized by ABE-producing clostridia and converted into acetone, butanol, and ethanol. The total ABE produced by C. acetobutylicum ATCC 824 during batch fermentation was 3.6 g l(-1) from crude fungal biomass including acetone (1.05 g l(-1)), butanol (2.19 g l(-1)) and ethanol (0.36 g l(-1)). C. acetobutylicum ATCC 824 has ability to produce lipolytic enzymes with a specific activity 5.59 U/mg protein to hydrolyze ester containing substrates. The lipolytic potential of C. acetobutylicum ATCC 824 was used as a biocatalyst for a lipase transesterification process using the produced ethanol from ABE fermentation for microdiesel production. The fatty acid ethyl esters (microdiesel) generated from the lipase transesterification of crude C. echinulata dry mass was analyzed by GC/MS as 15.4% of total FAEEs. The gross energy content of biohydrogen, acetone, butanol, ethanol and biodiesel generated through C. acetobutylicum fermentation from crude C. echinulata dry mass was 3113.14 kJ mol(-1). These results suggest a possibility of integrating biohydrogen, acetone, butanol and ethanol production technology by C. acetobutylicum with microdiesel production from crude C. echinulata dry mass and therefore improve the feasibility and commercialization of bioenergy production. Copyright © 2015 Elsevier Ltd. All rights reserved.

40 CFR 417.41 - Specialized definitions.

Code of Federal Regulations, 2010 CFR

2010-07-01

... this subpart. (b) The term anhydrous product shall mean the theoretical product that would result if all water were removed from the actual product. (c) The term sweet water shall mean the solution of 8-10 percent crude glycerine and 90-22 percent water that is a by-product of saponification or fat...

Stability of thermostable alkaline protease from Bacillus licheniformis RP1 in commercial solid laundry detergent formulations.

PubMed

Sellami-Kamoun, Alya; Haddar, Anissa; Ali, Nedra El-Hadj; Ghorbel-Frikha, Basma; Kanoun, Safia; Nasri, Moncef

2008-01-01

The stability of crude extracellular protease produced by Bacillus licheniformis RP1, isolated from polluted water, in various solid laundry detergents was investigated. The enzyme had an optimum pH and temperature at pH 10.0-11.0 and 65-70 degrees C. Enzyme activity was inhibited by PMSF, suggesting that the preparation contains a serine-protease. The alkaline protease showed extreme stability towards non-ionic (5% Tween 20% and 5% Triton X-100) and anionic (0.5% SDS) surfactants, which retained 100% and above 73%, respectively, of its initial activity after preincubation 60 min at 40 degrees C. The RP1 protease showed excellent stability and compatibility with a wide range of commercial solid detergents at temperatures from 40 to 50 degrees C, suggesting its further application in detergent industry. The enzyme retained 95% of its initial activity with Ariel followed by Axion (94%) then Dixan (93.5%) after preincubation 60 min at 40 degrees C in the presence of 7 mg/ml of detergents. In the presence of Nadhif and New Det, the enzyme retained about 83.5% of the original activity. The effects of additives such as maltodextrin, sucrose and PEG 4000 on the stability of the enzyme during spray-drying and during subsequent storage in New Det detergent were also examined. All additives tested enhanced stability of the enzyme.

Production of Recombinant Trichoderma reesei Cellobiohydrolase II in a New Expression System Based on Wickerhamomyces anomalus

PubMed Central

Díaz-Rincón, Dennis J.; Duque, Ivonne; Osorio, Erika; Rodríguez-López, Alexander; Espejo-Mojica, Angela; Parra-Giraldo, Claudia M.

2017-01-01

Cellulase is a family of at least three groups of enzymes that participate in the sequential hydrolysis of cellulose. Recombinant expression of cellulases might allow reducing their production times and increasing the low proteins concentrations obtained with filamentous fungi. In this study, we describe the production of Trichoderma reesei cellobiohydrolase II (CBHII) in a native strain of Wickerhamomyces anomalus. Recombinant CBHII was expressed in W. anomalus 54-A reaching enzyme activity values of up to 14.5 U L−1. The enzyme extract showed optimum pH and temperature of 5.0–6.0 and 40°C, respectively. Enzyme kinetic parameters (KM of 2.73 mM and Vmax of 23.1 µM min−1) were between the ranges of values reported for other CBHII enzymes. Finally, the results showed that an enzymatic extract of W. anomalus 54-A carrying the recombinant T. reesei CBHII allows production of reducing sugars similar to that of a crude extract from cellulolytic fungi. These results show the first report on the use of W. anomalus as a host to produce recombinant proteins. In addition, recombinant T. reesei CBHII enzyme could potentially be used in the degradation of lignocellulosic residues to produce bioethanol, based on its pH and temperature activity profile. PMID:28951785

Application of ionic liquids based enzyme-assisted extraction of chlorogenic acid from Eucommia ulmoides leaves.

PubMed

Liu, Tingting; Sui, Xiaoyu; Li, Li; Zhang, Jie; Liang, Xin; Li, Wenjing; Zhang, Honglian; Fu, Shuang

2016-01-15

A new approach for ionic liquid based enzyme-assisted extraction (ILEAE) of chlorogenic acid (CGA) from Eucommia ulmoides is presented in which enzyme pretreatment was used in ionic liquids aqueous media to enhance extraction yield. For this purpose, the solubility of CGA and the activity of cellulase were investigated in eight 1-alkyl-3-methylimidazolium ionic liquids. Cellulase in 0.5 M [C6mim]Br aqueous solution was found to provide better performance in extraction. The factors of ILEAE procedures including extraction time, extraction phase pH, extraction temperatures and enzyme concentrations were investigated. Moreover, the novel developed approach offered advantages in term of yield and efficiency compared with other conventional extraction techniques. Scanning electronic microscopy of plant samples indicated that cellulase treated cell wall in ionic liquid solution was subjected to extract, which led to more efficient extraction by reducing mass transfer barrier. The proposed ILEAE method would develope a continuous process for enzyme-assisted extraction including enzyme incubation and solvent extraction process. In this research, we propose a novel view for enzyme-assisted extraction of plant active component, besides concentrating on enzyme facilitated cell wall degradation, focusing on improvement of bad permeability of ionic liquids solutions. Copyright © 2015 Elsevier B.V. All rights reserved.

Saccharification of rice straw by cellulase from a local Trichoderma harzianum SNRS3 for biobutanol production.

PubMed

Rahnama, Nooshin; Foo, Hooi Ling; Abdul Rahman, Nor Aini; Ariff, Arbakariya; Md Shah, Umi Kalsom

2014-12-12

Rice straw has shown to be a promising agricultural by-product in the bioconversion of biomass to value-added products. Hydrolysis of cellulose, a main constituent of lignocellulosic biomass, is a requirement for fermentable sugar production and its subsequent bioconversion to biofuels such as biobutanol. The high cost of commercial enzymes is a major impediment to the industrial application of cellulases. Therefore, the use of local microbial enzymes has been suggested. Trichoderma harzianum strains are potential CMCase and β-glucosidase producers. However, few researches have been reported on cellulase production by T. harzianum and the subsequent use of the crude cellulase for cellulose enzymatic hydrolysis. For cellulose hydrolysis to be efficiently performed, the presence of the whole set of cellulase components including exoglucanase, endoglucanase, and β-glucosidase at a considerable concentration is required. Biomass recalcitrance is also a bottleneck in the bioconversion of agricultural residues to value-added products. An effective pretreatment could be of central significance in the bioconversion of biomass to biofuels. Rice straw pretreated using various concentrations of NaOH was subjected to enzymatic hydrolysis. The saccharification of rice straw pretreated with 2% (w/v) NaOH using crude cellulase from local T. harzianum SNRS3 resulted in the production of 29.87 g/L reducing sugar and a yield of 0.6 g/g substrate. The use of rice straw hydrolysate as carbon source for biobutanol fermentation by Clostridium acetobutylicum ATCC 824 resulted in an ABE yield, ABE productivity, and biobutanol yield of 0.27 g/g glucose, 0.04 g/L/h and 0.16 g/g glucose, respectively. As a potential β-glucosidase producer, T. harzianum SNRS3 used in this study was able to produce β-glucosidase at the activity of 173.71 U/g substrate. However, for cellulose hydrolysis to be efficient, Filter Paper Activity at a considerable concentration is also required to initiate the hydrolytic reaction. According to the results of our study, FPase is a major component of cellulose hydrolytic enzyme complex system and the reducing sugar rate-limiting enzyme. Our study revealed that rice straw hydrolysate served as a potential substrate for biobutanol production and FPase is a rate-limiting enzyme in saccharification.

A comparative study of extracellular glucanhydrolase and glucosyltransferase enzyme activities of five different serotypes of oral Streptococcus mutans.

PubMed

Felgenhauer, B; Trautner, K

1982-01-01

The activities of glucanhydrolase (EC 3.2.1.11) and glucosyltransferase (EC 2.4.1.5) in crude enzyme preparations of 44 strains of Streptococcus mutans of five serotypes were investigated. The strains were grown in a laboratory fermentor for 16 h and the enzymes were isolated by adding solid ammonium sulphate to the culture supernatant, resulting in a 12-fold enrichment of the enzymes. For glucanhydrolase, strains of serotype a showed the lowest total activity (0.768 U, approx. 120 ml), whereas strains of serotype d had an activity 39 times higher (29.9 U). The total activities of strains of serotypes b, c and e were 5.56, 6.30 and 7.06 U, respectively. For glucosyltransferase, strains of type e showed the highest total activity (293 U), whereas differences between strains of the other four types were insignificant (type a: 158 U; type b: 175 U; type c: 191 U; type d: 225 U; approx. 120 ml). A strong correlation was found between the glucanhydrolase activity and the percentage of insoluble glucan synthesized in vitro by the respective strains. This correlation was not substantially changed if the enzyme activities were expressed as specific activities, or as total activities against bacterial weight.

Optimization, purification, and characterization of L-asparaginase from Actinomycetales bacterium BkSoiiA.

PubMed

Dash, Chitrangada; Mohapatra, Sukanti Bala; Maiti, Prasanta Kumar

2016-01-01

Actinobacteria are promising source of a wide range of important enzymes, some of which are produced in industrial scale, with others yet to be harnessed. L-Asparaginase is used as an antineoplastic agent. The present work deals with the production and optimization of L-asparaginase from Actinomycetales bacterium BkSoiiA using submerged fermentation in M9 medium. Production optimization resulted in a modified M9 medium with yeast extract and fructose as carbon and nitrogen sources, respectively, at pH 8.0, incubated for 120 hr at 30 ± 2 °C. The crude enzyme was purified to near homogeneity by ammonium sulfate precipitation following dialysis, ion-exchange column chromatography, and finally gel filtration. The sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) revealed an apparent molecular weight of 57 kD. The enzyme was purified 95.06-fold and showed a final specific activity of 204.37 U/mg with 3.49% yield. The purified enzyme showed maximum activity at a pH 10.0 and was stable at pH 7.0 to 9.0. The enzyme was activated by Mn(2+) and strongly inhibited by Ba(2+). All these preliminary characterization suggests that the L-asparaginase from the source may be a tool useful to pharmaceutical industries after further research.

Ion-exchange chromatography: basic principles and application to the partial purification of soluble mammalian prolyl oligopeptidase.

PubMed

Cummins, Philip M; Dowling, Oonagh; O'Connor, Brendan F

2011-01-01

Ion-exchange chromatography (IEC) allows for the separation of ionizable molecules on the basis of differences in charge properties. Its large sample-handling capacity, broad applicability (particularly to proteins and enzymes), moderate cost, powerful resolving ability, and ease of scale-up and automation have led to it becoming one of the most versatile and widely used of all liquid chromatography (LC) techniques. In this chapter, we review the basic principles of IEC, as well as the broader criteria for selecting IEC conditions. By way of further illustration, we outline protocols necessary to partially purify a serine peptidase from bovine whole brain cytosolic fraction, covering crude tissue extract preparation through to partial purification of the target enzyme using anion-exchange chromatography. Protocols for assaying total protein and enzyme activity in both pre- and post-IEC fractions are also described. The target serine peptidase, prolyl oligopeptidase (POP, EC3.4.21.26), is an 80-kDa enzyme with endopeptidase activity towards peptide substrates of ≤30 amino acids. POP is a ubiquitous post-proline cleaving enzyme with particularly high expression levels in the mammalian brain, where it participates in the metabolism of neuroactive peptides and peptide-like hormones (e.g. thyroliberin, gonadotropin-releasing hormone).

Canola Oil Fuel Cell Demonstration: Volume 3 - Technical, Commercialization, and Application Issues Associated with Harvested Biomass

DTIC Science & Technology

2006-08-17

Hydratable and non-hydratable phosphatides are removed from the oil using a degumming solution (0.1 percent of 0.85 wt percent phosphoric acid aqueous...solution or 2500 ppm citric acid may be used) followed by the addition of soft water equal to 75 percent of the phosphatide content in the crude oil...converted to water-soluble phosphatidic acid through the addition of phosphoric acid , and hydratable phosphatides are formed from the addition of soft

Polymer-Coated Nanoparticles for Reversible Emulsification and Recovery of Heavy Oil.

PubMed

Qi, Luqing; Song, Chen; Wang, Tianxiao; Li, Qilin; Hirasaki, George J; Verduzco, Rafael

2018-06-05

Heavy crude oil has poor solubility and a high density, making recovery and transport much more difficult and expensive than for light crude oil. Emulsifiers can be used to produce low viscosity oil-in-water emulsions for recovery and transport, but subsequent demulsification can be challenging. Here, we develop and implement interfacially active, pH-responsive polymer-coated nanoparticles (PNPs) to reversibly stabilize, recover, and break oil/water emulsions through variation of solution pH. Silica particles with poly(2-(dimethylamino)ethyl methacrylate) (DMAEMA) chains covalently grafted to the surface are prepared although a reversible addition fragmentation chain transfer grafting-through technique. The resulting DMAEMA PNPs can stabilize emulsions of high viscosity Canadian heavy oil at PNP concentrations as low as 0.1 wt % and at neutral pH. The performance of the DMAEMA PNPs exceeds that of DMAEMA homopolymer additives, which we attribute to the larger size and irreversible adsorption of DMAEMA PNPs to the oil/water interface. After recovery, the emulsion can be destabilized by the addition of acid to reduce pH, resulting in separation and settling of the heavy oil from the aqueous phase. Recovery of more than 10 wt % of the crude heavy oil-in-place is achieved by flooding with aqueous solution of 0.1 wt % DMAEMA PNPs without any additional surfactant or chemical. This work demonstrates the applicability of PNPs as surface active materials for enhanced oil recovery processes and for heavy oil transport.

Enzyme electrochemical sensor electrode and method of making it

DOEpatents

Rishpon, Judith; Zawodzinski, Thomas A.; Gottesfeld, Shimshon

1992-01-01

An electrochemical sensor electrode is formed from an electronic conductor coated with a casting solution containing a perfluorosulfonic acid ionomer and a selected enzyme. The selected enzyme catalyzes a reaction between a predetermined substance in a solution and oxygen to form an electrochemically active compound that is detected at the electronic conductor. The resulting perfluorosulfonic acid polymer provides a stable matrix for the enzyme for long lived enzyme activity, wherein only thin coatings are required on the metal conductor. The polymer also advantageously repels interfering substances from contacting the enzyme and contains quantities of oxygen to maintain a sensing capability during conditions of oxygen depletion in the sample. In one particular embodiment, glucose oxidase is mixed with the perfluorosulfonic acid ionomer to form an electrode for glucose detection.

Multipoint attachment to a support protects enzyme from inactivation by organic solvents: alpha-Chymotrypsin in aqueous solutions of alcohols and diols.

PubMed

Mozhaev, V V; Sergeeva, M V; Belova, A B; Khmelnitsky, Y L

1990-03-25

Inactivation of alpha-chymotrypsin in aqueous solutions of alcohols and diols proceeds both reversibly and irreversibly. Reversible loss of the specific enzyme activity results from conformational changes (unfolding) of the enzyme detected by fluorescence spectroscopy. Multipoint covalent attachment to the matrix of polyacryl-amide gel by copolymerization method stabilizes alpha-chymotrypsin from denaturation by alcohols, the stabilizing effect increasing with the number of bonds between the protein and the support. Immobilization protects the enzyme also from irreversible inactivation by organic solvents resulting from bimolecular aggregation and autolysis.

Purification and characterization of a thermostable α-galactosidase from Thielavia terrestris NRRL 8126 in solid state fermentation.

PubMed

Saad, Rawia R; Fawzi, Eman M

2012-03-01

Several seeds and husks of some plants belonging to leguminosae, Graminae, Compositae and Palmae were evaluated as carbon substrates to produce α-galactosidase (α-Gal) by the thermophilic fungus, Thielavia terrestris NRRL 8126 in solid substrate fermentation. The results showed that Cicer arietinum (chick pea seed) was the best substrate for α-Gal production. The crude enzyme was precipitated by ammonium sulphate (60%) and purified by gel filtration on sephadex G-100 followed by ion exchange chromatography on DEAE-Cellulose. The final purification fold of the enzyme was 30.42. The temperature and pH optima of purified α-Gal from Thielavia terrestris were 70 °C and 6.5, respectively. The enzyme showed high thermal stability at 70 °C and 75 °C and the half-life of the α-Gal at 90 °C was 45 min. Km of the purified enzyme was 1.31 mM. The purified enzyme was inhibited by Ag2+, Hg2+, Zn2+, Ba2+, Mg2+, Mn2+ and Fe2+ at 5 mM and 10 mM. Also, EDTA, sodium arsenate, L-cysteine and iodoacetate inhibited the enzyme activity. On the other hand, Ca2+, Cu2+, K+ and Na+ slightly enhanced the enzyme activity at 5 mM while at 10 mM they caused inhibition. The molecular weight of the α-Gal was estimated to be 82 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This enzyme displays a number of biochemical properties that make it a potentially strong candidate for biotechnological and medicinal applications.

Acetylcholinesterase immobilization and characterization, and comparison of the activity of the porous silicon-immobilized enzyme with its free counterpart.

PubMed

Saleem, Muhammad; Rafiq, Muhammad; Seo, Sung-Yum; Lee, Ki Hwan

2016-02-02

A successful prescription is presented for acetylcholinesterase physically adsorbed on to a mesoporous silicon surface, with a promising hydrolytic response towards acetylthiocholine iodide. The catalytic behaviour of the immobilized enzyme was assessed by spectrophotometric bioassay using neostigmine methyl sulfate as a standard acetycholinesterase inhibitor. The surface modification was studied through field emission SEM, Fourier transform IR spectroscopy, energy-dispersive X-ray spectroscopy, cathode luminescence and X-ray photoelectron spectroscopy analysis, photoluminescence measurement and spectrophotometric bioassay. The porous silicon-immobilized enzyme not only yielded greater enzyme stability, but also significantly improved the native photoluminescence at room temperature of the bare porous silicon architecture. The results indicated the promising catalytic behaviour of immobilized enzyme compared with that of its free counterpart, with a greater stability, and that it aided reusability and easy separation from the reaction mixture. The porous silicon-immobilized enzyme was found to retain 50% of its activity, promising thermal stability up to 90°C, reusability for up to three cycles, pH stability over a broad pH of 4-9 and a shelf-life of 44 days, with an optimal hydrolytic response towards acetylthiocholine iodide at variable drug concentrations. On the basis of these findings, it was believed that the porous silicon-immobilized enzyme could be exploited as a reusable biocatalyst and for screening of acetylcholinesterase inhibitors from crude plant extracts and synthesized organic compounds. Moreover, the immobilized enzyme could offer a great deal as a viable biocatalyst in bioprocessing for the chemical and pharmaceutical industries, and bioremediation to enhance productivity and robustness. © 2016 Authors.

Acetylcholinesterase immobilization and characterization, and comparison of the activity of the porous silicon-immobilized enzyme with its free counterpart

PubMed Central

Saleem, Muhammad; Rafiq, Muhammad; Seo, Sung-Yum; Lee, Ki Hwan

2016-01-01

A successful prescription is presented for acetylcholinesterase physically adsorbed on to a mesoporous silicon surface, with a promising hydrolytic response towards acetylthiocholine iodide. The catalytic behaviour of the immobilized enzyme was assessed by spectrophotometric bioassay using neostigmine methyl sulfate as a standard acetycholinesterase inhibitor. The surface modification was studied through field emission SEM, Fourier transform IR spectroscopy, energy-dispersive X-ray spectroscopy, cathode luminescence and X-ray photoelectron spectroscopy analysis, photoluminescence measurement and spectrophotometric bioassay. The porous silicon-immobilized enzyme not only yielded greater enzyme stability, but also significantly improved the native photoluminescence at room temperature of the bare porous silicon architecture. The results indicated the promising catalytic behaviour of immobilized enzyme compared with that of its free counterpart, with a greater stability, and that it aided reusability and easy separation from the reaction mixture. The porous silicon-immobilized enzyme was found to retain 50% of its activity, promising thermal stability up to 90°C, reusability for up to three cycles, pH stability over a broad pH of 4–9 and a shelf-life of 44 days, with an optimal hydrolytic response towards acetylthiocholine iodide at variable drug concentrations. On the basis of these findings, it was believed that the porous silicon-immobilized enzyme could be exploited as a reusable biocatalyst and for screening of acetylcholinesterase inhibitors from crude plant extracts and synthesized organic compounds. Moreover, the immobilized enzyme could offer a great deal as a viable biocatalyst in bioprocessing for the chemical and pharmaceutical industries, and bioremediation to enhance productivity and robustness. PMID:26839417

Determination of lytic enzyme activities of indigenous Trichoderma isolates from Pakistan.

PubMed

Asad, Saeed Ahmad; Tabassum, Ayesha; Hameed, Abdul; Hassan, Fayyaz Ul; Afzal, Aftab; Khan, Sabaz Ali; Ahmed, Rafiq; Shahzad, Muhammad

2015-01-01

This study investigated lytic enzyme activities in three indigenous Trichoderma strains namely, Trichoderma asperellum, Trichoderma harzianum and Trichoderma sp. Native Trichoderma strains and a virulent strain of Rhizoctonia solani isolated from infected bean plants were also included in the study. Enzyme activities were determined by measuring sugar reduction by dinitrosalicylic acid (DNS) method using suitable substrates. The antagonists were cultured in minimal salt medium with the following modifications: medium A (1 g of glucose), medium B (0.5 g of glucose + 0.5 g of deactivated R. solani mycelia), medium C (1.0 g of deactivated respective antagonist mycelium) and medium D (1 g of deactivated R. solani mycelia). T asperellum showed presence of higher amounts of chitinases, β-1, 3-glucanases and xylanases in extracellular protein extracts from medium D as compared to medium A. While, the higher activities of glucosidases and endoglucanses were shown in medium D extracts by T. harzianum. β-glucosidase activities were lower compared with other enzymes; however, activities of the extracts of medium D were significantly different. T. asperellum exhibited maximum inhibition (97.7%). On the other hand, Trichoderma sp. did not show any effect on mycelia growth of R. solani on crude extract.

Optimization of the Production of 1-Phenylethanol Using Enzymes from Flowers of Tea (Camellia sinensis) Plants.

PubMed

Dong, Fang; Zhou, Ying; Zeng, Lanting; Watanabe, Naoharu; Su, Xinguo; Yang, Ziyin

2017-01-13

1-Phenylethanol (1PE) can be used as a fragrance in food flavoring and cosmetic industries and as an intermediate in the pharmaceutical industry. 1PE can be synthesized from acetophenone, and the cost of 1PE is higher than the cost of acetophenone. Therefore, it is important to establish an effective and low-cost approach for producing 1PE. Our previous studies found that tea ( Camellia sinensis ) flowers, which are an abundant and waste resource, contained enzymes that could transform acetophenone to 1PE. In the present study, we extracted crude enzymes from tea flowers and optimized the production conditions of 1PE using response surface methodology. The optimized conditions were an extraction pH of 7.0, a reaction pH of 5.3, a reaction temperature of 55 °C, a reaction time of 100 min, a coenzyme NADPH concentration of 3.75 μmol/mL in the reaction assay, and a substrate acetophenone concentration of 1.25 μmol/mL in the reaction assay. The results provide essential information for future industrial 1PE production using plant-derived enzymes.

Nylon bead enzyme-linked immunosorbent assay for detection of sub-picogram quantities of Brucella antigens.

PubMed Central

Perera, V Y; Creasy, M T; Winter, A J

1983-01-01

An indirect sandwich enzyme-linked immunosorbent assay, using antibody covalently coupled to nylon beads, has been adapted for the detection of Brucella antigens. Optimum conditions were achieved by incubation of 1 ml of reaction mixture with a single bead, and by minimizing nonspecific interactions through the use of beads coated with purified bovine antibodies, preabsorption of third layer rabbit antibodies with normal bovine serum, and treatment of beads with normal goat serum before addition of the goat anti-rabbit enzyme conjugate. Beta-galactosidase was selected for use with clinical samples primarily because of low levels of endogenous enzyme in bovine leukocytes. Use of a fluorogenic substrate enhanced sensitivity 20-fold. Under these conditions, 100 fg of solubilized crude lipopolysaccharide or 8 to 10 Brucella cells was detectable in a fixed volume of 1 ml. A system was also devised for concentrating antigen which permitted ready detection of 2 pg of lipopolysaccharide in a volume of 50 ml (40 fg/ml). Attempts to detect lipopolysaccharide in the presence of concentrated serum or plasma were unsuccessful, but 10 brucellae added to a suspension of leukocytes from 100 ml of normal bovine blood were easily measured. PMID:6415094

Determination of lytic enzyme activities of indigenous Trichoderma isolates from Pakistan

PubMed Central

Asad, Saeed Ahmad; Tabassum, Ayesha; Hameed, Abdul; Hassan, Fayyaz ul; Afzal, Aftab; Khan, Sabaz Ali; Ahmed, Rafiq; Shahzad, Muhammad

2015-01-01

Abstract This study investigated lytic enzyme activities in three indigenous Trichoderma strains namely, Trichoderma asperellum, Trichoderma harzianum and Trichoderma sp. Native Trichoderma strains and a virulent strain of Rhizoctonia solani isolated from infected bean plants were also included in the study. Enzyme activities were determined by measuring sugar reduction by dinitrosalicylic acid (DNS) method using suitable substrates. The antagonists were cultured in minimal salt medium with the following modifications: medium A (1 g of glucose), medium B (0.5 g of glucose + 0.5 g of deactivated R. solani mycelia), medium C (1.0 g of deactivated respective antagonist mycelium) and medium D (1 g of deactivated R. solani mycelia). T asperellum showed presence of higher amounts of chitinases, β-1, 3-glucanases and xylanases in extracellular protein extracts from medium D as compared to medium A. While, the higher activities of glucosidases and endoglucanses were shown in medium D extracts by T. harzianum. β-glucosidase activities were lower compared with other enzymes; however, activities of the extracts of medium D were significantly different. T. asperellum exhibited maximum inhibition (97.7%). On the other hand, Trichoderma sp. did not show any effect on mycelia growth of R. solani on crude extract. PMID:26691463

Photometric Characterization of the Reductive Amination Scope of the Imine Reductases from Streptomyces tsukubaensis and Streptomyces ipomoeae.

PubMed

Matzel, Philipp; Krautschick, Lukas; Höhne, Matthias

2017-10-18

Imine reductases (IREDs) have emerged as promising enzymes for the asymmetric synthesis of secondary and tertiary amines starting from carbonyl substrates. Screening the substrate specificity of the reductive amination reaction is usually performed by time-consuming GC analytics. We found two highly active IREDs in our enzyme collection, IR-20 from Streptomyces tsukubaensis and IR-Sip from Streptomyces ipomoeae, that allowed a comprehensive substrate screening with a photometric NADPH assay. We screened 39 carbonyl substrates combined with 17 amines as nucleophiles. Activity data from 663 combinations provided a clear picture about substrate specificity and capabilities in the reductive amination of these enzymes. Besides aliphatic aldehydes, the IREDs accepted various cyclic (C 4 -C 8 ) and acyclic ketones, preferentially with methylamine. IR-Sip also accepted a range of primary and secondary amines as nucleophiles. In biocatalytic reactions, IR-Sip converted (R)-3-methylcyclohexanone with dimethylamine or pyrrolidine with high diastereoselectivity (>94-96 % de). The nucleophile acceptor spectrum depended on the carbonyl substrate employed. The conversion of well-accepted substrates could also be detected if crude lysates were employed as the enzyme source. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Medium pH in submerged cultivation modulates differences in the intracellular protein profile of Fusarium oxysporum.

PubMed

da Rosa-Garzon, Nathália Gonsales; Laure, Hélen Julie; Souza-Motta, Cristina Maria de; Rosa, José César; Cabral, Hamilton

2017-08-09

Fusarium oxysporum is a filamentous fungus that damages a wide range of plants and thus causes severe crop losses. In fungal pathogens, the genes and proteins involved in virulence are known to be controlled by environmental pH. Here, we report the influence of culture-medium pH (5, 6, 7, and 8) on the production of degradative enzymes involved in the pathogenesis of F. oxysporum URM 7401 and on the 2D-electrophoresis profile of intracellular proteins in this fungus. F. oxysporum URM 7401 was grown in acidic, neutral, and alkaline culture media in a submerged bioprocess. After 96 hr, the crude extract was processed to enzyme activity assays, while the intracellular proteins were obtained from mycelium and analyzed using 2D electrophoresis and mass spectrometry. We note that the diversity of secreted enzymes was changed quantitatively in different culture-medium pH. Also, the highest accumulated biomass and the intracellular protein profile of F. oxysporum URM 7401 indicate an increase in metabolism in neutral-alkaline conditions. The differential profiles of secreted enzymes and intracellular proteins under the evaluated conditions indicate that the global protein content in F. oxysporum URM 7401 is modulated by extracellular pH.

Semi-continuous production of high-activity pectinases by immobilized Rhizopus oryzae using tobacco wastewater as substrate and their utilization in the hydrolysis of pectin-containing lignocellulosic biomass at high solid content.

PubMed

Zheng, Yu-Xi; Wang, Yuan-Liang; Pan, Jun; Zhang, Jian-Rong; Dai, Ya; Chen, Kun-Yan

2017-10-01

In this study, highly reactive endo- and exo-polygalacturonases (PGs) were produced from the tobacco industry wastewater using immobilized Rhizopus oryzae. Compared with free cells, immobilized cells increased enzyme activity 2.8-fold and reduced production time to 24h by shake-flask production. Moreover, the immobilized cells enabled the semi-continuous production of enzymes through repeated-batch mode for seven consecutive cycles in a scale-up bioreactor. During the first five cycles, the average endo-PG and exo-PG activities reached 307.5 and 242.6U/ml, respectively. The addition of crude enzyme for the hydrolysis of pectin-containing lignocellulosic biomass under high-gravity conditions increased glucose release 4.2-fold (115.4 vs. 29.0g/L), compared with hydrolysis using cellulase alone. This process achieves the efficient production of pectin-degrading enzymes, provides a cost-effective method for tobacco wastewater treatment, and offers the possibility to obtain fermentable sugars with high-titer from pectin-containing lignocellulosic biomass, which has important potential for the commercial production of bio-fuels. Copyright © 2017 Elsevier Ltd. All rights reserved.

(Evaluation of neutralizing ability of four commercially available antivenoms against the venom of Bothrops asper from Costa Rica)

PubMed

Bogarín, G; Segura, E; Durán, G; Lomonte, B; Rojas, G; Gutiérrez, J M

1995-09-01

We studied the ability of four commercially available antivenoms to neutralize several toxic and enzymatic activities of Bothrops asper (terciopelo) venom from Costa Rica. Experiments with preincubation of venom and antivenom were carried out to test the neutralization of lethal, hemorrhagic, coagulant and indirect hemolytic activities. In addition, antibody titers against crude venom and myotoxin II purified from this venom were determined by enzyme immunoassay (ELISA). Results indicate that polyvalent antivenom from Instituto Clodomiro Picado (Costa Rica) has the highest neutralizing ability against lethal, coagulant and indirect hemolytic activities, whereas MYN polyvalent antivenom (México) has the highest neutralization against hemorrhagic activity. Antivenom from Instituto Clodomiro Picado also has the highest antibody titers against crude B. asper venom and against myotoxin II. Antivenoms from Universidad Central de Venezuela (Venezuela), Vencofarma (Brazil) and MYN (México) failed to neutralize the lethal effect of this venom. These results stress the need for rigorous quality control systems to evaluate the neutralizing ability of antivenoms in Central America.

Enzymatic dehalogenation of pentachlorophenol by extracts from Arthrobacter sp. strain ATCC 33790.

PubMed Central

Schenk, T; Müller, R; Mörsberger, F; Otto, M K; Lingens, F

1989-01-01

Arthrobacter sp. strain ATCC 33790 was grown with pentachlorophenol (PCP) as the sole source of carbon and energy. Crude extracts, which were prepared by disruption of the bacteria with a French pressure cell, showed no dehalogenating activity with PCP as the substrate. After sucrose density ultracentrifugation of the crude extract at 145,000 x g, various layers were found in the gradient. One yellow layer showed enzymatic conversion of PCP. One chloride ion was released per molecule of PCP. The product of the enzymatic conversion was tetrachlorohydroquinone. NADPH and oxygen were essential for this reaction. EDTA stimulated the enzymatic activity by 67%. The optimum pH for the enzyme activity was 7.5, and the temperature optimum was 25 degrees C. Enzymatic activity was also detected with 2,4,5-trichlorophenol, 2,3,4-trichlorophenol, 2,4,6-trichlorophenol, and 2,3,4,5-tetrachlorophenol as substrates, whereas 3,4,5-trichlorophenol, 2,4-dichlorophenol, 3,4-dichlorophenol, and 4-chlorophenol did not serve as substrates. PMID:2793827

Development of a monoclonal-based enzyme-linked immunoassay for saxitoxin-induced protein.

PubMed

Smith, D S; Kitts, D D

1994-03-01

A monoclonal antibody was generated against saxitoxin-induced protein (SIP) from the small shore crab Hemigrapsus oregenesis. SIP was induced by saxitoxin injection and could be detected in the crude crab extracts with both polyclonal and monoclonal antibody preparations. On Western blots, the polyclonal serum reacted against several bands which were induced by saxitoxin in the crude extracts. These bands represented proteins related to SIP. The monoclonal (4G5), however, was specific for the 79,000 mol. wt subunit of SIP. A triple antibody sandwich ELISA was developed in which polyclonal anti-SIP IgG was used as a trapping layer and monoclonal 4G5 was used as the detection layer. This assay was shown to be more specific and more accurate than a direct bind assay which employed the polyclonal antiserum alone. Although the polyclonal serum was more sensitive than the monoclonal on Western blots, the triple antibody sandwich and direct bind ELISAs were of comparable sensitivity.

Formulation of fermentation media from flour-rich waste streams for microbial lipid production by Lipomyces starkeyi.

PubMed

Tsakona, Sofia; Kopsahelis, Nikolaos; Chatzifragkou, Afroditi; Papanikolaou, Seraphim; Kookos, Ioannis K; Koutinas, Apostolis A

2014-11-10

Flour-rich waste (FRW) and by-product streams generated by bakery, confectionery and wheat milling plants could be employed as the sole raw materials for generic fermentation media production, suitable for microbial oil synthesis. Wheat milling by-products were used in solid state fermentations (SSF) of Aspergillus awamori for the production of crude enzymes, mainly glucoamylase and protease. Enzyme-rich SSF solids were subsequently employed for hydrolysis of FRW streams into nutrient-rich fermentation media. Batch hydrolytic experiments using FRW concentrations up to 205 g/L resulted in higher than 90% (w/w) starch to glucose conversion yields and 40% (w/w) total Kjeldahl nitrogen to free amino nitrogen conversion yields. Starch to glucose conversion yields of 98.2, 86.1 and 73.4% (w/w) were achieved when initial FRW concentrations of 235, 300 and 350 g/L were employed in fed-batch hydrolytic experiments, respectively. Crude hydrolysates were used as fermentation media in shake flask cultures with the oleaginous yeast Lipomyces starkeyi DSM 70296 reaching a total dry weight of 30.5 g/L with a microbial oil content of 40.4% (w/w), higher than that achieved in synthetic media. Fed-batch bioreactor cultures led to a total dry weight of 109.8 g/L with a microbial oil content of 57.8% (w/w) and productivity of 0.4 g/L/h. Copyright © 2014 Elsevier B.V. All rights reserved.

Metabolic characterization of anaerobic fungi provides a path forward for bioprocessing of crude lignocellulose

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

Henske, John K.; Wilken, St. Elmo; Solomon, Kevin V.

The conversion of lignocellulose-rich biomass to bio-based chemicals and higher order fuels remains a grand challenge, as single-microbe approaches often cannot drive both deconstruction and chemical production steps. In contrast, consortia based bioprocessing leverages the strengths of different microbes to distribute metabolic loads and achieve process synergy, product diversity, and bolster yields. Here, we describe a biphasic fermentation scheme that combines the lignocellulolytic action of anaerobic fungi isolated from large herbivores with domesticated microbes for bioproduction. When grown in batch culture, anaerobic fungi release excess sugars from both cellulose and crude biomass due to a wealth of highly expressed carbohydratemore » active enzymes (CAZymes), converting as much as 49% of cellulose to free glucose. This sugar-rich hydrolysate readily supports growth of Saccharomyces cerevisiae, which can be engineered to produce a range of value-added chemicals. Further, construction of metabolic pathways from transcriptomic data reveals that anaerobic fungi do not catabolize all sugars that their enzymes hydrolyze from biomass, leaving other carbohydrates such as galactose, arabinose, and mannose available as nutritional links to other microbes in their consortium. Although basal expression of CAZymes in anaerobic fungi is high, it is drastically amplified by cellobiose breakout products encountered during biomass hydrolysis. Overall, these results suggest that anaerobic fungi provide a nutritional benefit to the rumen microbiome, which can be harnessed to design synthetic microbial communities that compartmentalize biomass degradation and bioproduct formation.« less

Integrated biomarker response index used in laboratory exposure of the mussel Mytilus edulis to water accommodated fractions of crude oil

NASA Astrophysics Data System (ADS)

Yuan, Mengqi; Wang, You; Zhang, Xinxin; Hu, Shunxin; Tang, Xuexi

2017-03-01

Oil pollution is a serious environmental problem in coastal areas and to set up a quantitative evaluation method in monitoring the environmental pollution is of great importance. Individuals of Mytilus edulis, collected from the coastal area of Qingdao, were thus exposed to the water accommodated fractions (WAFs) of crude oil under controlled lab conditions. Mussels were exposed to different concentrations of WAFs for 96h acute toxicity experiment and 15d chronic toxicity experiment, a 7d restoration experiment was conducted after toxicity experiment as well. The activities of the antioxidant enzymes (CAT, GST, GPx and GR) were measured in gills of mussels. The purpose of the study was to screen out a series of sensitive biomarkers and set up an Integrated Biomarker Response index (IBR) for marine oil pollution monitoring. Results showed: (1) WAFs exposure induced the increased activities of CAT, GST, GPx and GR in gills, revealing a stimulation effect on the antioxidant system. Activities of enzymes were more significantly altered in response to higher concentrations of WAFs (2.3 and 5.0 mg.L-1) than lower concentrations (0.5 and 1.1 mg.L-1). After restoration experiment, the activities decreased to the initial levels. (2) IBR was set up base on the laboratory experiment, which showed a positive relationship with WAFs exposure concentrations. Therefore, IBR was suggested to be a potentially useful tool in assessing the oil pollution.

Valorization of By-Products from Palm Oil Mills for the Production of Generic Fermentation Media for Microbial Oil Synthesis.

PubMed

Tsouko, Erminda; Kachrimanidou, Vasiliki; Dos Santos, Anderson Fragoso; do Nascimento Vitorino Lima, Maria Eduarda; Papanikolaou, Seraphim; de Castro, Aline Machado; Freire, Denise Maria Guimarães; Koutinas, Apostolis A

2017-04-01

This study demonstrates the production of a generic nutrient-rich feedstock using by-product streams from palm oil production that could be used as a substitute for commercial fermentation supplements. Solid-state fermentations of palm kernel cake (PKC) and palm-pressed fiber (PPF) were conducted in tray bioreactors and a rotating drum bioreactor by the fungal strain Aspergillus oryzae for the production of crude enzymes. The production of protease was optimized (319.3 U/g) at an initial moisture content of 55 %, when PKC was used as the sole substrate. The highest free amino nitrogen (FAN) production (5.6 mg/g) obtained via PKC hydrolysis using the crude enzymes produced via solid-state fermentation was achieved at 50 °C. Three initial PKC concentrations (48.7, 73.7, and 98.7 g/L) were tested in hydrolysis experiments, leading to total Kjeldahl nitrogen to FAN conversion yields up to 27.9 %. Sequential solid-state fermentation followed by hydrolysis was carried out in the same rotating drum bioreactor, leading to the production of 136.7 U/g of protease activity during fermentation and 196.5 mg/L of FAN during hydrolysis. Microbial oil production was successfully achieved with the oleaginous yeast strain Lipomyces starkeyi DSM 70296 cultivated on the produced PKC hydrolysate mixed with commercial carbon sources, including glucose, xylose, mannose, galactose, and arabinose.

Heterologous expression of Aspergillus aculeatus endo-polygalacturonase in Pichia pastoris by high cell density fermentation and its application in textile scouring.

PubMed

Abdulrachman, Dede; Thongkred, Paweena; Kocharin, Kanokarn; Nakpathom, Monthon; Somboon, Buppha; Narumol, Nootsara; Champreda, Verawat; Eurwilaichitr, Lily; Suwanto, Antonius; Nimchua, Thidarat; Chantasingh, Duriya

2017-02-16

Removal of non-cellulosic impurities from cotton fabric, known as scouring, by conventional alkaline treatment causes environmental problems and reduces physical strength of fabrics. In this study, an endo-polygalacturonase (EndoPG) from Aspergillus aculeatus produced in Pichia pastoris was evaluated for its efficiency as a bioscouring agent while most current bioscouring process has been performed using crude pectinase preparation. The recombinant EndoPG exhibited a specific activity of 1892.08 U/mg on citrus pectin under the optimal condition at 50 °C, pH 5.0 with a V max and K m of 65,451.35 μmol/min/mL and 15.14 mg/mL, respectively. A maximal activity of 2408.70 ± 26.50 U/mL in the culture supernatant was obtained by high cell density batch fermentation, equivalent to a 4.8 times greater yield than that from shake-flask culture. The recombinant enzyme was shown to be suitable for application as a bioscouring agent, in which the wettability of cotton fabric was increased by treatment with enzyme at 300 U/mL scouring solution at 40 °C, pH 5.0 for 1 h. The bio-scoured fabric has comparable wettability to that obtained by conventional chemical scouring, but has higher tensile strength. The work has demonstrated for the first time functions of A. aculeatus EndoPG on bioscouring in eco-textile processing. EndoPG alone was shown to possess effective scouring activity. High expression level and homogeneity could be achieved in bench-scale bioreactor.

A deadenylase assay by size-exclusion chromatography.

PubMed

He, Guang-Jun; Yan, Yong-Bin

2012-01-01

The shortening of the 3'-end poly(A) tail, also called deadenylation, is crucial to the regulation of mRNA processing, transportation, translation and degradation. The deadenylation process is achieved by deadenylases, which specifically catalyze the removal of the poly(A) tail at the 3'-end of eukaryotic mRNAs and release 5'-AMP as the product. To achieve their physiological functions, all deadenylases have numerous binding partners that may regulate their catalytic properties or recruit them into various protein complexes. To study the effects of various partners, it is important to develop new deadenylase assay that can be applied either in vivo or in vitro. In this research, we developed the deadenylase assay by the size-exclusion chromatography (SEC) method. The SEC analysis indicated that the poly(A) or oligo(A) substrate and the product AMP could be successfully separated and quantified. The enzymatic parameters of deadenylase could be obtained by quantifying the AMP generation. When using the commercial poly(A) as the substrate, a biphasic catalytic process was observed, which might correlate to the two distinct states of poly(A) in the commercial samples. Different lots of commercial poly(A) had dissimilar size distributions and were dissimilar in response to the degradation of deadenylase. The deadenylation pattern, processive or distributive, could also be investigated using the SEC assay by monitoring the status of the substrate and the generation kinetics of AMP and A2. The SEC assay was applicable to both simple samples using the purified enzyme and complex enzyme reaction conditions such as using protein mixtures or crude cell extracts as samples. The influence of solutes with absorption at 254 nm could be successfully eliminated by constructing the different SEC profiles.

Asphaltene Aggregation and Fouling Behavior

NASA Astrophysics Data System (ADS)

Derakhshesh, Marzie

This thesis explored the properties of asphaltene nano-aggregates in crude oil and toluene based solutions and fouling at process furnace temperatures, and the links between these two phenomena. The link between stability of asphaltenes at ambient conditions and fouling at the conditions of a delayed coker furnace, at over 450 °C, was examined by blending crude oil with an aliphatic diluent in different ratios. The stability of the blends were measured using a S-value analyzer, then fouling rates were measured on electrically heated stainless steel 316 wires in an autoclave reactor. The less stable the blend, the greater the rate and extent of fouling. The most severe fouling occurred with the unstable asphaltenes. SEM imaging of the foulant illustrates very different textures, with the structure becoming more porous with lower stability. Under cross-polarized light, the coke shows the presence of mesophase in the foulant layer. These data suggest a correlation between the fouling rate at high temperature furnace conditions and the stability index of the crude oil. Three organic polysulfides were introduced to the crude oil to examine their effect on fouling. The polysulfides are able to reduce coking and carbon monoxide generation in steam crackers. The fouling results demonstrated that polysulfide with more sulfur content increased the amount of corrosion-fouling of the wire. Various additives, solvents, ultrasound, and heat were employed to attempt to completely disaggregate the asphaltene nano-aggregates in solution at room temperature. The primary analytical technique used to monitor the nano-aggregation state of the asphaltenes in solution was the UV-visible spectroscopy. The results indicate that stronger solvents, such as pyridine and quinoline, combined with ionic liquids yield a slight reduction in the apparent absorbance at longer wavelengths, indicative of a decrease in the nano-aggregate size although the magnitude of the decrease is not significant. Analysis of the spectra of the whole asphaltene samples in toluene indicates that the absorbance of visible light with wavelengths > 600 nm follows a lambda--4 dependence. This functional dependence is consistent with Rayleigh scattering. Rayleigh scattering provides strong evidence that the apparent absorption of visible light by asphaltenes from 600-800 nm is not a molecular absorption phenomenon but rather a scattering mechanism. Rayleigh scattering equations were combined with experimental visible spectra to estimate the average nanoaggregate sizes, which were in a very good agreement with the sizes reported in the literature. The occlusion of two polynuclear aromatic hydrocarbons (PAHs) (pyrene and phenanthrene) in asphaltene precipitates was tested by adding PAHs to the asphaltene in toluene solutions, precipitating by n-pentane and determining the amount of PAHs in precipitates using simulated distillation instrument. Pyrene and phenanthrene, which are normally soluble in the toluene-n-pentane solutions, were detected in the asphaltene precipitates at up to 6 wt% concentration. Trapping of PAHs outside of the nanoaggregates during precipitation gave 7-14 times less of the PAHs in the solid precipitate. This study shows that asphaltene aggregates can interact significantly with PAHs. The results are consistent with the presence open porous asphaltene nanoaggregates in solutions such as toluene.

Stability of the anti-oxidative enzymes in aqueous and detergent solution.

PubMed

Mailer, K; Del Maestro, R F

1991-09-18

Activities of the anti-oxidative enzymes, superoxide dismutase (SOD), glutathione peroxidase (GPx) and catalase were studied in rat tissues to determine the ability of detergents both to solubilize the enzymes and also to stabilize enzyme activity. Rat brain, heart and liver were homogenized in 0.1M KCl, 0.1% sodium dodecyl sulfate, 0.1% lubrol, or 0.1% cetyl-trimethylammonium bromide. In general lubrol was more effective than the other solutions in solubilizing GPx and catalase. Lubrol and 0.1M KCl were equally effective in solubilizing SOD. The highest enzyme activities were (1) SOD: 2484 ng/mg (brain), 2501 ng/mg (heart), and 5586 ng/mg (liver); (2) GPx: 224 mU/mg (brain), 1870 mU/mg (heart), and 7332 mU/mg (liver); (3) catalase: 2.8 mU/mg (brain), 10.6 mU/mg (heart), and 309 mU/mg (liver). While cetyl trimethylammonium bromide is marginally better than sodium dodecyl sulfate in solubilizing active enzyme, neither ionic detergent has any advantage over lubrol or 0.1M KCl. For catalase and GPx, enzyme activity loss with time is biphasic. After initial, rapid activity loss (1-5 days for GPx and 7-10 days for catalase) the differences noted among the homogenizing solutions disappear and very little if any activity loss is noted over the next 2-3 weeks. For catalase and GPx, only baseline enzyme activity from t = 0-3 weeks is found in the most chaotropic solution, 0.1% sodium dodecyl sulfate while biphasic activity loss is most pronounced in 0.1% lubrol. These results may indicate active GPx and catalase species stabilized by a lipid-like environment. Correlating in vitro catalase or GPx measurements with in vivo anti-oxidative protection may underestimate tissue defences.

The effects of xylanase supplementation on growth, digestion, circulating hormone and metabolite levels, immunity and gut microflora in cockerels fed on wheat-based diets.

PubMed

Gao, F; Jiang, Y; Zhou, G H; Han, Z K

2007-08-01

1. The xylanase product used in this study was derived from a genetically modified isolate of Aspergillus niger. Two trials were conducted to investigate the effects of xylanase supplementation on growth, digestion, circulating hormone and metabolite levels, immune parameters and composition of the gut microflora in cockerels fed on wheat-based diets. 2. The experimental diets consisted of a wheat-based control diet supplemented with 0 or 0.1% enzyme preparation. The diets were fed between 7 and 21 d of age. 3. Enzyme supplementation improved growth and feed conversion efficiency. The addition of enzyme to wheat-based diet increased the apparent total digestibility of dry matter (DM), crude protein and fat. 4. Enzyme supplementation reduced the relative weight of digestive organs to a certain extent, but there was no significant difference. Enzyme supplementation reduced digesta viscosity in the jejunum. There was no significant difference between the two experimental groups in counts of lactobacillus and coliform bacteria in the caeca. 5. Enzyme supplementation increased the concentration of blood thyroxine (T(4)), insulin-like growth factor I (IGF-I) and insulin, reduced the concentrations of blood uric acid, but had no significant effect on the concentrations of blood glucose and triiodothyronine (T(3)). 6. Enzyme supplementation increased the relative weight of spleen of cockerels, serum antibody titres to Newcastle disease virus (NDV), lymphocyte proliferation in response to phytohaemagglutinin (PHA) and the natural killer (NK) cell activity. 7. It is concluded that supplementation with an enzyme preparation (xylanase), which hydrolyses non-starch polysaccharides can improve growth in cockerels fed on wheat-based diets. This improvement is achieved through enzyme effects on digestion, absorption, metabolism and immunity of cockerels.

Production and characterization of multi-polysaccharide degrading enzymes from Aspergillus aculeatus BCC199 for saccharification of agricultural residues.

PubMed

Suwannarangsee, Surisa; Arnthong, Jantima; Eurwilaichitr, Lily; Champreda, Verawat

2014-10-01

Enzymatic hydrolysis of lignocellulosic biomass into fermentable sugars is a key step in the conversion of agricultural by-products to biofuels and value-added chemicals. Utilization of a robust microorganism for on-site production of biomass-degrading enzymes has gained increasing interest as an economical approach for supplying enzymes to biorefinery processes. In this study, production of multi-polysaccharide-degrading enzymes from Aspergillus aculeatus BCC199 by solid-state fermentation was improved through the statistical design approach. Among the operational parameters, yeast extract and soybean meal as well as the nonionic surfactant Tween 20 and initial pH were found as key parameters for maximizing production of cellulolytic and hemicellulolytic enzymes. Under the optimized condition, the production of FPase, endoglucanase, β-glucosidase, xylanase, and β-xylosidase was achieved at 23, 663, 88, 1,633, and 90 units/g of dry substrate, respectively. The multi-enzyme extract was highly efficient in the saccharification of alkaline-pretreated rice straw, corn cob, and corn stover. In comparison with commercial cellulase preparations, the BCC199 enzyme mixture was able to produce remarkable yields of glucose and xylose, as it contained higher relative activities of β-glucosidase and core hemicellulases (xylanase and β-xylosidase). These results suggested that the crude enzyme extract from A. aculeatus BCC199 possesses balanced cellulolytic and xylanolytic activities required for the efficient saccharification of lignocellulosic biomass feedstocks, and supplementation of external β-glucosidase or xylanase was dispensable. The work thus demonstrates the high potential of A. aculeatus BCC199 as a promising producer of lignocellulose-degrading enzymes for the biomass conversion industry.

Protective Effect of Fragaria ananassa Crude Extract on Cadmium-Induced Lipid Peroxidation, Antioxidant Enzymes Suppression, and Apoptosis in Rat Testes

PubMed Central

Elmallah, Mohammed I. Y.; Elkhadragy, Manal F.; Al-Olayan, Ebtesam M.; Abdel Moneim, Ahmed E.

2017-01-01

Cadmium is a deleterious environmental pollutant that threats both animals and human health. Oxidative stress and elevated levels of reactive oxygen species (ROS) have recently been reported to be the main cause of cellular damage as a result of cadmium exposure. We investigate, here, the protective effect of strawberry crude extracts on cadmium-induced oxidative damage of testes in rats. Four groups (n = 8) of 32 adult male Wistar rats weighing 160–180 g were used. The control group received 0.9% saline solution all over the experimental period (5 days). Group 2 was intraperitoneally injected with 6.5 mg/kg CdCl2. Group 3 was provided only with an oral administration of strawberry methanolic extract (SME) at a dose of 250 mg/kg. Group 4 was treated with SME before cadmium injection with the same mentioned doses. It was shown that cadmium exposure results in a significant decrease in both relative testicular weight and serum testosterone level. Analyzing the oxidative damaging effect of cadmium on the testicular tissue revealed the induction of oxidative stress markers represented in the elevated level of lipid peroxidation (LPO), nitric oxide (NO), and a decrease in the reduced glutathione (GSH) content. Considering cadmium toxicity, the level of the antioxidant enzyme activities including catalase (CAT), superoxide dismutase (SOD2), glutathione peroxidase (GPx1), and glutathione reductase (GR) were markedly decreased. Moreover, gene expression analysis indicated significant upregulation of the pro-apoptotic proteins, bcl-2-associated-X-protein (BAX), and tumor necrosis factor-α (TNFA) in response to cadmium intoxication, while significant downregulation of the anti-apoptotic, B-cell lymphoma 2 (BCL2) gene was detected. Immunohistochemistry of the testicular tissue possessed positive immunostaining for the increased level of TNF-α, but decreased number of proliferating cell nuclear antigen (PCNA) stained cells. Administration of SME debilitated the deleterious effect of cadmium via reduction of both LPO and NO levels followed by a significant enhancement in the gene expression level of CAT, SOD2, GPX1, GR, nuclear factor-erythroid 2-related factor 2 (NFE2L2), heme oxygenase-1 (HMOX1), Bcl-2, and PCNA. In addition, the SME treated group revealed a significant increase in the level of testosterone and GSH accompanied by a marked decrease in the gene expression level of Bax and TNF-α. In terms of the summarized results, the SME of Fragaria ananassa has a protective effect against cadmium-induced oxidative damage of testes. PMID:28475120

Simulation of aerobic and anaerobic biodegradation processes at a crude oil spill site

USGS Publications Warehouse

Essaid, Hedeff I.; Bekins, Barbara A.; Godsy, E. Michael; Warren, Ean; Baedecker, Mary Jo; Cozzarelli, Isabelle M.

1995-01-01

A two-dimensional, multispecies reactive solute transport model with sequential aerobic and anaerobic degradation processes was developed and tested. The model was used to study the field-scale solute transport and degradation processes at the Bemidji, Minnesota, crude oil spill site. The simulations included the biodegradation of volatile and nonvolatile fractions of dissolved organic carbon by aerobic processes, manganese and iron reduction, and methanogenesis. Model parameter estimates were constrained by published Monod kinetic parameters, theoretical yield estimates, and field biomass measurements. Despite the considerable uncertainty in the model parameter estimates, results of simulations reproduced the general features of the observed groundwater plume and the measured bacterial concentrations. In the simulation, 46% of the total dissolved organic carbon (TDOC) introduced into the aquifer was degraded. Aerobic degradation accounted for 40% of the TDOC degraded. Anaerobic processes accounted for the remaining 60% of degradation of TDOC: 5% by Mn reduction, 19% by Fe reduction, and 36% by methanogenesis. Thus anaerobic processes account for more than half of the removal of DOC at this site.

Pilot-scale resin adsorption as a means to recover and fractionate apple polyphenols.

PubMed

Kammerer, Dietmar R; Carle, Reinhold; Stanley, Roger A; Saleh, Zaid S

2010-06-09

The purification and fractionation of phenolic compounds from crude plant extracts using a food-grade acrylic adsorbent were studied at pilot-plant scale. A diluted apple juice concentrate served as a model phenolic solution for column adsorption and desorption trials. Phenolic concentrations were evaluated photometrically using the Folin-Ciocalteu assay and by HPLC-DAD. Recovery rates were significantly affected by increasing phenolic concentrations of the feed solutions applied to the column. In contrast, the flow rate during column loading hardly influenced adsorption efficiency, whereas the temperature and pH value were shown to be crucial parameters determining both total phenolic recovery rates and the adsorption behavior of individual polyphenols. As expected, the eluent composition had the greatest impact on the desorption characteristics of both total and individual phenolic compounds. HPLC analyses revealed significantly different elution profiles of individual polyphenols depending on lipophilicity. This technique allows fractionation of crude plant phenolic extracts, thus providing the opportunity to design the functional properties of the resulting phenolic fractions selectively, and the present study delivers valuable information with regard to the adjustment of individual process parameters.

SOLVENT EXTRACTION PROCESS FOR THE RECOVERY OF METALS FROM PHOSPHORIC ACID

DOEpatents

Bailes, R.H.; Long, R.S.

1958-11-01

> A solvent extraction process is presented for recovering metal values including uranium, thorium, and other lanthanide and actinide elements from crude industrial phosphoric acid solutions. The process conslsts of contacting said solution with an immisclble organic solvent extractant containing a diluent and a material selected from the group consisting of mono and di alkyl phosphates, alkyl phosphonates and alkyl phosphites. The uranlum enters the extractant phase and is subsequently recovered by any of the methods known to the art. Recovery is improved if the phosphate solution is treated with a reducing agent such as iron or aluminum powder prior to the extraction step.

Plant Molecular Farming: Much More than Medicines.

PubMed

Tschofen, Marc; Knopp, Dietmar; Hood, Elizabeth; Stöger, Eva

2016-06-12

Plants have emerged as commercially relevant production systems for pharmaceutical and nonpharmaceutical products. Currently, the commercially available nonpharmaceutical products outnumber the medical products of plant molecular farming, reflecting the shorter development times and lower regulatory burden of the former. Nonpharmaceutical products benefit more from the low costs and greater scalability of plant production systems without incurring the high costs associated with downstream processing and purification of pharmaceuticals. In this review, we explore the areas where plant-based manufacturing can make the greatest impact, focusing on commercialized products such as antibodies, enzymes, and growth factors that are used as research-grade or diagnostic reagents, cosmetic ingredients, and biosensors or biocatalysts. An outlook is provided on high-volume, low-margin proteins such as industrial enzymes that can be applied as crude extracts or unprocessed plant tissues in the feed, biofuel, and papermaking industries.

Characterization of two acyl-acyl carrier protein thioesterases from developing Cuphea seeds specific for medium-chain- and oleoyl-acyl carrier protein.

PubMed

Dörmann, P; Spener, F; Ohlrogge, J B

1993-03-01

Two acyl-acyl carrier protein (ACP) thioesterases were partially purified from developing seeds of Cuphea lanceolata Ait., a plant with decanoic acid-rich triacylglycerols. The two enzymes differ markedly in their substrate specificity. One is specific for medium-chain acyl-ACPs, the other one for oleoyl-ACP. In addition, these enzymes are distinct with regard to molecular weight, pH optimum and sensitivity to salt. The thioesterases could be separated by Mono Q chromatography or gel filtration. The medium-chain acyl-ACP thioesterase and oleoyl-ACP thioesterase were purified from a crude extract 29- and 180-fold, respectively. In Cuphea wrightii A. Gray, which predominantly contains decanoic a nd lauric acid in the seeds, two different thioesterases were also found with a similar substrate specificity as in Cuphea lanceolata.

Cell-Wall-Bound Proteinase of Lactobacillus delbrueckii subsp. lactis ACA-DC 178: Characterization and Specificity for β-Casein

PubMed Central

Tsakalidou, E.; Anastasiou, R.; Vandenberghe, I.; van Beeumen, J.; Kalantzopoulos, G.

1999-01-01

Lactobacillus delbrueckii subsp. lactis ACA-DC 178, which was isolated from Greek Kasseri cheese, produces a cell-wall-bound proteinase. The proteinase was removed from the cell envelope by washing the cells with a Ca2+-free buffer. The crude proteinase extract shows its highest activity at pH 6.0 and 40°C. It is inhibited by phenylmethylsulfonyl fluoride, showing that the enzyme is a serine-type proteinase. Considering the substrate specificity, the enzyme is similar to the lactococcal PI-type proteinases, since it hydrolyzes β-casein mainly and α- and κ-caseins to a much lesser extent. The cell-wall-bound proteinase from L. delbrueckii subsp. lactis ACA-DC 178 liberates four main peptides from β-casein, which have been identified. PMID:10223997

Proteolysin, a Novel Highly Thermostable and Cosolvent-Compatible Protease from the Thermophilic Bacterium Coprothermobacter proteolyticus

PubMed Central

Toplak, Ana; Wu, Bian; Fusetti, Fabrizia; Quaedflieg, Peter J. L. M.

2013-01-01

Through genome mining, we identified a gene encoding a putative serine protease of the thermitase subgroup of subtilases (EC 3.4.21.66) in the thermophilic bacterium Coprothermobacter proteolyticus. The gene was functionally expressed in Escherichia coli, and the enzyme, which we called proteolysin, was purified to near homogeneity from crude cell lysate by a single heat treatment step. Proteolysin has a broad pH tolerance and is active at temperatures of up to 80°C. In addition, the enzyme shows good activity and stability in the presence of organic solvents, detergents, and dithiothreitol, and it remains active in 6 M guanidinium hydrochloride. Based on its stability and activity profile, proteolysin can be an excellent candidate for applications where resistance to harsh process conditions is required. PMID:23851086

Effects of method of detachment on electrophoretic mobility of mammalian cells grown in monolayer culture

NASA Technical Reports Server (NTRS)

Plank, L. D.; Kunze, M. E.; Todd, P. W.

1985-01-01

A variety of proteolytic and micolytic enzumes, mechanical procedures, and changes in the ionic environment, especially Ca chelation, are used for dispersal of monolayer grown cells. If either chelating agents or mechanical dispersion are used alone, the cell yield is often low and suspensions of single cells are difficult to obtain. Confluent monolayers treated with EDTA tend to be released from their surfaces in sheets, and clumps of cells remain even after further incubation in EDTA. Crude trypsin is the most popular dispersal agent and is known to contain a variety of contaminating enzymes which contribute to the dispersal of cells. A variety of cell injuries resulting from the activity of proteolytic enzymes are reported. It is shown that crystalline trypsin is least harmful to cell integrity as judged by trypan blue uptake.

Plant Molecular Farming: Much More than Medicines

NASA Astrophysics Data System (ADS)

Tschofen, Marc; Knopp, Dietmar; Hood, Elizabeth; Stöger, Eva

2016-06-01

Plants have emerged as commercially relevant production systems for pharmaceutical and nonpharmaceutical products. Currently, the commercially available nonpharmaceutical products outnumber the medical products of plant molecular farming, reflecting the shorter development times and lower regulatory burden of the former. Nonpharmaceutical products benefit more from the low costs and greater scalability of plant production systems without incurring the high costs associated with downstream processing and purification of pharmaceuticals. In this review, we explore the areas where plant-based manufacturing can make the greatest impact, focusing on commercialized products such as antibodies, enzymes, and growth factors that are used as research-grade or diagnostic reagents, cosmetic ingredients, and biosensors or biocatalysts. An outlook is provided on high-volume, low-margin proteins such as industrial enzymes that can be applied as crude extracts or unprocessed plant tissues in the feed, biofuel, and papermaking industries.

Small-Angle X-Ray Scattering Analysis of the Bifunctional Antibiotic Resistance Enzyme Aminoglycoside (6′) Acetyltransferase-Ie/Aminoglycoside (2″) Phosphotransferase-Ia Reveals a Rigid Solution Structure

PubMed Central

Caldwell, Shane J.

2012-01-01

Aminoglycoside (6′) acetyltransferase-Ie/aminoglycoside (2″) phosphotransferase-Ia [AAC(6′)-Ie/APH(2″)-Ia] is one of the most problematic aminoglycoside resistance factors in clinical pathogens, conferring resistance to almost every aminoglycoside antibiotic available to modern medicine. Despite 3 decades of research, our understanding of the structure of this bifunctional enzyme remains limited. We used small-angle X-ray scattering (SAXS) to model the structure of this bifunctional enzyme in solution and to study the impact of substrate binding on the enzyme. It was observed that the enzyme adopts a rigid conformation in solution, where the N-terminal AAC domain is fixed to the C-terminal APH domain and not loosely tethered. The addition of acetyl-coenzyme A, coenzyme A, GDP, guanosine 5′-[β,γ-imido]triphosphate (GMPPNP), and combinations thereof to the protein resulted in only modest changes to the radius of gyration (RG) of the enzyme, which were not consistent with any large changes in enzyme structure upon binding. These results imply some selective advantage to the bifunctional enzyme beyond coexpression as a single polypeptide, likely linked to an improvement in enzymatic properties. We propose that the rigid structure contributes to improved electrostatic steering of aminoglycoside substrates toward the two active sites, which may provide such an advantage. PMID:22290965

Immobilization of NTPDase-1 from Trypanosoma cruzi and Development of an Online Label-Free Assay.

PubMed

Calil, Felipe Antunes; Lima, Juliana Maria; de Oliveira, Arthur Henrique Cavalcante; Mariotini-Moura, Christiane; Fietto, Juliana Lopes Rangel; Cardoso, Carmen Lucia

2016-01-01

The use of IMERs (Immobilized Enzyme Reactors) as a stationary phase coupled to high performance chromatographic systems is an interesting approach in the screening of new ligands. In addition, IMERs offer many advantages over techniques that employ enzymes in solution. The enzyme nucleoside triphosphate diphosphohydrolase (NTPDase-1) from Trypanosoma cruzi acts as a pathogen infection facilitator, so it is a good target in the search for inhibitors. In this paper, immobilization of NTPDase-1 afforded ICERs (Immobilized Capillary Enzyme Reactors). A liquid chromatography method was developed and validated to monitor the ICER activity. The conditions for the application of these bioreactors were investigated, and excellent results were obtained. The enzyme was successfully immobilized, as attested by the catalytic activity detected in the Tc NTPDase-1-ICER chromatographic system. Kinetic studies on the substrate ATP gave K M of 0.317 ± 0.044 mmol·L -1 , which still presented high affinity compared to in solution. Besides that, the ICER was stable for 32 days, enough time to investigate samples of possible inhibitors, including especially the compound Suramin, that inhibited 51% the enzyme activity at 100  µ mol·L -1 , which is in accordance with the data for the enzyme in solution.

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.

Immobilization of NTPDase-1 from Trypanosoma cruzi and Development of an Online Label-Free Assay

PubMed Central

Lima, Juliana Maria; de Oliveira, Arthur Henrique Cavalcante

2016-01-01

The use of IMERs (Immobilized Enzyme Reactors) as a stationary phase coupled to high performance chromatographic systems is an interesting approach in the screening of new ligands. In addition, IMERs offer many advantages over techniques that employ enzymes in solution. The enzyme nucleoside triphosphate diphosphohydrolase (NTPDase-1) from Trypanosoma cruzi acts as a pathogen infection facilitator, so it is a good target in the search for inhibitors. In this paper, immobilization of NTPDase-1 afforded ICERs (Immobilized Capillary Enzyme Reactors). A liquid chromatography method was developed and validated to monitor the ICER activity. The conditions for the application of these bioreactors were investigated, and excellent results were obtained. The enzyme was successfully immobilized, as attested by the catalytic activity detected in the TcNTPDase-1-ICER chromatographic system. Kinetic studies on the substrate ATP gave K M of 0.317 ± 0.044 mmol·L−1, which still presented high affinity compared to in solution. Besides that, the ICER was stable for 32 days, enough time to investigate samples of possible inhibitors, including especially the compound Suramin, that inhibited 51% the enzyme activity at 100 µmol·L−1, which is in accordance with the data for the enzyme in solution. PMID:28070446

Bioconjugation of silk fibroin nanoparticles with enzyme and Peptide and their characterization.

PubMed

Wang, Fei; Zhang, Yu-Qing

2015-01-01

Bombyx mori silk fibroin is a type of protein-based polymer with unique characteristics that is widely used in the research and development of medical biomaterials. The degummed filament of silk fibroin can be dissolved in a highly concentrated salt solution. After desalination, the regenerated liquid silk fibroin (LSF) solution could be made into various forms of silk biomaterials, such as powder, fiber, film, porous matrix, 3D scaffold, and hydrogel, depending on its application. In this study, we mixed the liquid silk solution with enzymes, including oxidase and hydrolase, and rapidly injected the mixture into an excess of acetone. The enzyme retained most of its enzymatic activity and was also captured in silk fibroin nanoparticles (SFNs), which instantly formed via a configuration transition of the regenerated silk protein from a random coil and α-helix to a β-sheet. The resulting enzyme-captured SFNs displayed a fine crystal structure with a high activity recovery and good thermal stability. Moreover, the affinities of these modified enzymes to their substrate did not evidently suffer from the capture. When only the liquid silk solution was rapidly injected into acetone, the resulting globular SFNs with the same crystallinity were also a good carrier that was covalently conjugated to enzymes and insulin. Thus, silk protein nanoparticles are of potential value as an enzyme or peptide delivery system for the research and development of medical biomaterials. In this report, the bioconjugation of SFNs with glucose oxidase, superoxidase, β-glucosidase, L-asparaginase, neutral protease, and insulin and their characterization are described in detail. © 2015 Elsevier Inc. All rights reserved.

Comparison of the anti-ulcer activity between the crude and bran-processed Atractylodes lancea in the rat model of gastric ulcer induced by acetic acid.

PubMed

Yu, Yan; Jia, Tian-Zhu; Cai, Qian; Jiang, Ning; Ma, Ming-Yue; Min, Dong-Yu; Yuan, Yuan

2015-02-03

The rhizome of Atractylodes lancea (AL, Compositae, Chinese name: Cangzhu; Japanese name: Sou-ju-tsu) has been used traditionally for the treatment of various diseases such as digestive disorders, rheumatic diseases, and influenza in China, Korea and Japan. The crude AL and AL bran-processed are both listed in the Chinese Pharmacopoeia. However, the differences between the effects of the crude and AL bran-processed on gastric ulcer were poorly understood, and the mechanisms for the treatment of gastric ulcer were not clear. This study aimed at comparing the anti-ulcer effects between the crude AL and AL processed in acetic acid induced model in rats and evaluating the mechanisms of action involved in the anti-ulcer properties of AL. The model of gastric ulcer was imitated by acetic acid in rats, and AL was gavaged. The serum and gastric tissues were collected. The levels of epidermal growth factor (EGF), trefoil factor2 (TFF2), tumor necrosis factor-α (TNF-α), interleukin 6, 8 (IL-6, 8) and prostaglandin E2 (PGE2) in serum and gastric tissues were determined by the double-antibody sandwich enzyme-linked immunosorbent assay (ELISA), and the mRNA expressions of EGF, TFF2, TNF-α, and IL-8 in stomach were analyzed by quantitative real-time reverse transcription polymerase chain reaction (RT-PCR). Meanwhile, histopathological changes were evaluated by hematoxylin and eosin (HE) stain. The protein expressions of EGF, TFF2, TNF-α, and IL-8 were examined by immunohistochemistry in stomach. The results demonstrated that the damage of gastric tissue was obviously alleviated and the productions of TNF-α, IL-8, IL-6, and PGE2 and the mRNA expressions of TNF-α, and IL-8 were notably inhibited. Furthermore, the productions of EGF and TFF2 and the mRNA expressions of EGF and TFF2 were significantly stimulated by both crude AL and AL processed in a dose-dependent manner. Compared with the crude AL, the processed AL was more effective. The AL processed had more satisfactory effects in treatment of gastric-ulcer than the crude AL. The anti-ulcer effects of AL could be attributed to the anti-inflammatory properties via down-regulating TNF-α, IL-8, IL-6 and PGE2 and to the gastroprotective effects via up-regulating EGF and TFF2. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Large-scale isotype-specific quantification of Serum amyloid A 1/2 by multiple reaction monitoring in crude sera.

PubMed

Sung, Hye-Jin; Jeon, Seon-Ae; Ahn, Jung-Mo; Seul, Kyung-Jo; Kim, Jin Young; Lee, Ju Yeon; Yoo, Jong Shin; Lee, Soo-Youn; Kim, Hojoong; Cho, Je-Yoel

2012-04-03

Quantification is an essential step in biomarker development. Multiple reaction monitoring (MRM) is a new modified mass spectrometry-based quantification technology that does not require antibody development. Serum amyloid A (SAA) is a positive acute-phase protein identified as a lung cancer biomarker in our previous study. Acute SAA exists in two isoforms with highly similar (92%) amino acid sequences. Until now, studies of SAA have been unable to distinguish between SAA1 and SAA2. To overcome the unavailability of a SAA2-specific antibody, we developed MRM methodology for the verification of SAA1 and SAA2 in clinical crude serum samples from 99 healthy controls and 100 lung adenocarcinoma patients. Differential measurement of SAA1 and SAA2 was made possible for the first time with the developed isotype-specific MRM method. Most healthy control samples had small or no MS/MS peaks of the targeted peptides otherwise, higher peak areas with 10- to 34-fold increase over controls were detected in lung cancer samples. In addition, our SAA1 MRM data demonstrated good agreement with the SAA1 enzyme-linked immunosorbent assay (ELISA) data. Finally, successful quantification of SAA2 in crude serum by MRM, for the first time, shows that SAA2 can be a good biomarker for the detection of lung cancers. Copyright © 2012 Elsevier B.V. All rights reserved.

Effect of Saccharomyces Boulardii Cell Wall Extracts on Colon Cancer Prevention in Male F344 Rats Treated with 1,2-Dimethylhydrazine.

PubMed

Fortin, Olivier; Aguilar-Uscanga, Blanca R; Vu, Khanh D; Salmieri, Stephane; Lacroix, Monique

2018-01-01

The effect of Saccharomyces boulardii cell wall extracts on colon cancer prevention in rats treated with 1,2-dimethylhydrazine was investigated. A crude insoluble glucan (0.5 and 1.0 mg/kg/day) and a crude mannoprotein extract (0.3 and 3.0 mg/kg/day) were administered in rats by gavage for 12 weeks along with a high fat low fiber diet whereupon rats were sacrificed and aberrant crypt foci (ACF) were counted in the colon. Moreover, NAD(P)H: quinone reductase (QR) and harmful fecal enzymes (β-glucosidase and β-glucuronidase) were quantified in the liver and in the caecum, respectively. Results showed a reduction in ACF counts, a decreased β-glucuronidase activity and an increased QR activity when rats were treated only with insoluble glucan. While these enzymatic modulations may be constituted one of the mechanisms that is responsible for the reduction of ACF counts observed, the reduction of ACF counts caused by insoluble glucan should be addressed, at least, as a biomarker of their cancer-prevention properties. To our knowledge, this is the first study demonstrated that crude cell wall extract obtained from S. boulardii could have a potential role in colon cancer prevention in vivo by revealing the potential implication of QR and β-glucuronidase modulation.

Production of Trametes pubescens laccase under submerged and semi-solid culture conditions on agro-industrial wastes.

PubMed

Gonzalez, Juan C; Medina, Sandra C; Rodriguez, Alexander; Osma, Johann F; Alméciga-Díaz, Carlos J; Sánchez, Oscar F

2013-01-01

Laccases are copper-containing enzymes involved in the degradation of lignocellulosic materials and used in the treatment of phenol-containing wastewater. In this study we investigated the effect of culture conditions, i.e. submerged or semi-solid, and copper supplementation on laccase production by Trametespubescens grown on coffee husk, soybean pod husk, or cedar sawdust. The highest specific laccase activity was achieved when the culture was conducted under submerged conditions supplemented with copper (5 mM), and using coffee husk as substrate. The crude extracts presented two laccase isoforms with molecular mass of 120 (Lac1) and 60 kDa (Lac2). Regardless of the substrate, enzymatic crude extract and purified fractions behaved similarly at different temperatures and pHs, most of them presented the maximum activity at 55 °C and a pH range between 2 and 3. In addition, they showed similar stability and electro-chemical properties. At optimal culture conditions laccase activity was 7.69 ± 0.28 U mg(-1) of protein for the crude extract, and 0.08 ± 0.001 and 2.86 ± 0.05 U mg(-1) of protein for Lac1 and Lac2, respectively. In summary, these results show the potential of coffee husk as an important and economical growth medium to produce laccase, offering a new alternative use for this common agro-industrial byproduct.

Serodiagnosis of sporotrichosis infection in cats by enzyme-linked immunosorbent assay using a specific antigen, SsCBF, and crude exoantigens.

PubMed

Fernandes, Geisa Ferreira; Lopes-Bezerra, Leila Maria; Bernardes-Engemann, Andréa Reis; Schubach, Tânia Maria Pacheco; Dias, Maria Adelaide Galvão; Pereira, Sandro Antonio; de Camargo, Zoilo Pires

2011-01-27

The main objective of this study is to standardize an ELISA for the diagnosis of feline sporotrichosis. Sporothrix schenckii is the etiological agent of human and animal sporotrichosis. Cats may act as reservoirs for S. schenckii and can transmit the infection to humans by a bite or scratch. There are few methods for the serological diagnosis of fungal diseases in animals. In this paper, an ELISA test for the diagnosis of cat sporotrichosis is proposed, which detects S. schenckii-specific antibodies in feline sera. Two different kinds of antigens were used: "SsCBF", a specific molecule from S. schenckii that consists of a Con A-binding fraction derived from a peptido-rhamnomannan component of the cell wall, and a S. schenckii crude exoantigen preparation. The ELISA was developed, optimized, and evaluated using sera from 30 cats with proven sporotrichosis (by culture isolation); 22 sera from healthy feral cats from a zoonosis center were used as negative controls. SsCBF showed 90% sensitivity and 96% specificity in ELISA; while crude exoantigens demonstrated 96% sensitivity and 98% specificity. The ELISA assay described here would be a valuable screening tool for the detection of specific S. schenckii antibodies in cats with sporotrichosis. The assay is inexpensive, quick to perform, easy to interpret, and permits the diagnosis of feline sporotrichosis. Copyright © 2010 Elsevier B.V. All rights reserved.

Production of Trametes pubescens Laccase under Submerged and Semi-Solid Culture Conditions on Agro-Industrial Wastes

PubMed Central

Rodriguez, Alexander; Osma, Johann F.; Alméciga-Díaz, Carlos J.; Sánchez, Oscar F.

2013-01-01

Laccases are copper-containing enzymes involved in the degradation of lignocellulosic materials and used in the treatment of phenol-containing wastewater. In this study we investigated the effect of culture conditions, i.e. submerged or semi-solid, and copper supplementation on laccase production by Trametes pubescens grown on coffee husk, soybean pod husk, or cedar sawdust. The highest specific laccase activity was achieved when the culture was conducted under submerged conditions supplemented with copper (5 mM), and using coffee husk as substrate. The crude extracts presented two laccase isoforms with molecular mass of 120 (Lac1) and 60 kDa (Lac2). Regardless of the substrate, enzymatic crude extract and purified fractions behaved similarly at different temperatures and pHs, most of them presented the maximum activity at 55 °C and a pH range between 2 and 3. In addition, they showed similar stability and electro-chemical properties. At optimal culture conditions laccase activity was 7.69±0.28 U mg-1 of protein for the crude extract, and 0.08±0.001 and 2.86±0.05 U mg-1 of protein for Lac1 and Lac2, respectively. In summary, these results show the potential of coffee husk as an important and economical growth medium to produce laccase, offering a new alternative use for this common agro-industrial byproduct. PMID:24019936

Analysis of gas membrane ultra-high purification of small quantities of mono-isotopic silane

DOE PAGES

de Almeida, Valmor F.; Hart, Kevin J.

2017-01-03

A small quantity of high-value, crude, mono-isotopic silane is a prospective gas for a small-scale, high-recovery, ultra-high membrane purification process. This is an unusual application of gas membrane separation for which we provide a comprehensive analysis of a simple purification model. The goal is to develop direct analytic expressions for estimating the feasibility and efficiency of the method, and guide process design; this is only possible for binary mixtures of silane in the dilute limit which is a somewhat realistic case. In addition, analytic solutions are invaluable to verify numerical solutions obtained from computer-aided methods. Hence, in this paper wemore » provide new analytic solutions for the purification loops proposed. Among the common impurities in crude silane, methane poses a special membrane separation challenge since it is chemically similar to silane. Other potential problematic compounds are: ethylene, diborane and ethane (in this order). Nevertheless, we demonstrate, theoretically, that a carefully designed membrane system may be able to purify mono-isotopic, crude silane to electronics-grade level in a reasonable amount of time and expenses. We advocate a combination of membrane materials that preferentially reject heavy impurities based on mobility selectivity, and light impurities based on solubility selectivity. We provide estimates for the purification of significant contaminants of interest. In this study, we suggest cellulose acetate and polydimethylsiloxane as examples of membrane materials on the basis of limited permeability data found in the open literature. We provide estimates on the membrane area needed and priming volume of the cell enclosure for fabrication purposes when using the suggested membrane materials. These estimates are largely theoretical in view of the absence of reliable experimental data for the permeability of silane. And finally, future extension of this work to the non-dilute limit may apply to the recovery of silane from rejected streams of natural silicon semi-conductor processes.« less

Determination of osmium concentrations and (187)Os/(188)Os of crude oils and source rocks by coupling high-pressure, high-temperature digestion with sparging OsO(4) into a multicollector inductively coupled plasma mass spectrometer.

PubMed

Sen, Indra S; Peucker-Ehrenbrink, Bernhard

2014-03-18

The (187)Os/(188)Os ratio that is based on the β(-)-decay of (187)Re to (187)Os (t1/2 = 41.6 billion years) is widely used to investigate petroleum system processes. Despite its broad applicability to studies of hydrocarbon deposits worldwide, a suitable matrix-matched reference material for Os analysis does not exist. In this study, a method that enables Os isotope measurement of crude oil with in-line Os separation and purification from the sample matrix is proposed. The method to analyze Os concentration and (187)Os/(187)Os involves sample digestion under high pressure and high temperature using a high pressure asher (HPA-S, Anton Paar), sparging of volatile osmium tetroxide from the sample solution, and measurements using multicollector inductively coupled plasma mass spectrometry (MC-ICPMS). This methods significantly reduced the total procedural time compared to conventional Carius tube digestion followed by Os separation and purification using solvent extraction, microdistillation and N-TIMS analysis. The method yields Os concentration (28 ± 4 pg g(-1)) and (187)Os/(188)Os (1.62 ± 0.15) of commercially available crude oil reference material NIST 8505 (1 S.D., n = 6). The reference material NIST 8505 is homogeneous with respect to Os concentration at a test portion size of 0.2 g. Therefore, (187)Os/(188)Os composition and Os concentration of NIST 8505 can serve as a matrix-matched reference material for Os analysis. Data quality was assessed by repeated measurements of the USGS shale reference material SCo-1 (sample matrix similar to petroleum source rock) and the widely used Liquid Os Standard solution (LOsSt). The within-laboratory reproducibility of (187)Os/(188)Os for a 5 pg of LOsSt solution, analyzed with this method over a period of 12 months was ∼1.4% (1 S.D., n = 26), respectively.

Analysis of gas membrane ultra-high purification of small quantities of mono-isotopic silane

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

de Almeida, Valmor F.; Hart, Kevin J.

A small quantity of high-value, crude, mono-isotopic silane is a prospective gas for a small-scale, high-recovery, ultra-high membrane purification process. This is an unusual application of gas membrane separation for which we provide a comprehensive analysis of a simple purification model. The goal is to develop direct analytic expressions for estimating the feasibility and efficiency of the method, and guide process design; this is only possible for binary mixtures of silane in the dilute limit which is a somewhat realistic case. In addition, analytic solutions are invaluable to verify numerical solutions obtained from computer-aided methods. Hence, in this paper wemore » provide new analytic solutions for the purification loops proposed. Among the common impurities in crude silane, methane poses a special membrane separation challenge since it is chemically similar to silane. Other potential problematic compounds are: ethylene, diborane and ethane (in this order). Nevertheless, we demonstrate, theoretically, that a carefully designed membrane system may be able to purify mono-isotopic, crude silane to electronics-grade level in a reasonable amount of time and expenses. We advocate a combination of membrane materials that preferentially reject heavy impurities based on mobility selectivity, and light impurities based on solubility selectivity. We provide estimates for the purification of significant contaminants of interest. In this study, we suggest cellulose acetate and polydimethylsiloxane as examples of membrane materials on the basis of limited permeability data found in the open literature. We provide estimates on the membrane area needed and priming volume of the cell enclosure for fabrication purposes when using the suggested membrane materials. These estimates are largely theoretical in view of the absence of reliable experimental data for the permeability of silane. And finally, future extension of this work to the non-dilute limit may apply to the recovery of silane from rejected streams of natural silicon semi-conductor processes.« less

Three RNases in Senescent and Nonsenescent Wheat Leaves 1

PubMed Central

Blank, A.; McKeon, Thomas A.

1991-01-01

We have described three RNases in wheat leaves (Triticum aestivum L. cv Chinese Spring) and developed assays for measuring each RNase individually in crude leaf extracts. We initially used activity staining in sodium dodecyl sulfate-polyacrylamide gels to characterize RNases in extracts of primary and flag leaves. We thus identified acid RNase (EC 3.1.27.1, here designated RNase WLA), and two apparently novel enzymes, designated RNases WLB and WLC. RNase WLB activity displays a distinctive isozyme pattern, a molecular mass of 26 kilodaltons (major species), a broad pH range with an optimum near neutrality, insensitivity to EDTA, and stimulation by moderate concentrations of KCl and by MgCl2. RNase WLC activity exhibits a molecular mass of 27 kilodaltons, a neutral pH optimum, insensitivity to EDTA, and inhibition by KCl, MgCl2, and tri-(hydroxymethyl)aminomethane. Based on distinctive catalytic properties established in gels, we designed conventional solution assays for selective quantitation of each RNase activity. We used the assays to monitor the individual RNases after gel filtration chromatography and native gel electrophoresis of extracts. In accompanying work, we used the assays to monitor RNases WLA, WLB, and WLC, which are present in senescent and nonsenescent leaves, during the course of leaf senescence. ImagesFigure 1Figure 3Figure 4 PMID:16668563

Optimising the operational parameters of a spherical steriliser for the treatment of oil palm fresh fruit bunch

NASA Astrophysics Data System (ADS)

Kumaradevan, D.; Chuah, K. H.; Moey, L. K.; Mohan, V.; Wan, W. T.

2015-09-01

The extraction of crude palm oil (CPO) begins with the sterilization of oil palm fresh fruit bunch (FFB) in a pressurized, saturated-steam chamber. Sterilization loosens the palm fruits from the stalks and deactivates the free fatty acid (FFA)-producing enzymes. Operational parameters affecting the quality and yield of CPO from an industrial spherical sterilizer are studied at a palm oil mill. The factors are the ripeness of FFB, the number of days before treatment of FFB, and the number of pressure peaks applied in the sterilization process. The results indicate that the degree of ripeness of FFB is the most important parameter affecting the quality and yield of CPO. Ripeness is graded based on the fruits’ colour and the presence of loose fruits. Over ripe FFB that goes for the sterilization process has higher FFA content in CPO and more oil loss to the condensate chamber. The spontaneous reaction on FFB due to accumulation at the loading ramp also gives rise to higher FFA content. Oil loss to condensate chamber is reduced using a two-peak sterilization technique for over ripe FFB; the peak refers to the pressure level of stream after a flushing and refilling cycle. Overall, the generated solution improves the quality and yield of the palm oil mill.

Biomarker responses in the bivalve Chlamys farreri to the water-soluble fraction of crude oil

NASA Astrophysics Data System (ADS)

Jiang, Fenghua; Zhang, Li; Yang, Baijuan; Zheng, Li; Sun, Chengjun

2015-07-01

To investigate the effect of the water soluble fraction of crude oil (WSF) on marine bivalves, the scallop Chlamys farreri was exposed to three WSF concentrations (0.18 mg/L, 0.32 mg/L, and 0.51 mg/L, respectively) in seawater. Petroleum hydrocarbon contents in scallops and a suite of enzymes [7-Ethoxyresorufin-O-deethylase (EROD), aryl hydrocarbon hydroxylase (AHH), glutathione S-transferase (GST), and glutathione peroxidase (GPx)] in gills and digestive glands were monitored over 10 days. The results revealed that WSF affected the activity of the four enzymes in the gills and digestive glands. EROD activity in the gills was significantly induced in most individuals of the three test groups, while in the digestive gland it was significantly induced in the low-concentration group within 4 days but was inhibited in the middle- and high-concentration groups on days 1, 4, and 10. AHH activity in the gills of all treatment groups was significantly induced on day 1. In the digestive gland, AHH activity was induced in most individuals from the treatment groups. In all treatment groups, GST activity was significantly inhibited from days 2 to 10 in the gills and was induced after day 4 in the digestive gland. GPx activity in the gills was significantly inhibited throughout the exposure period in all treatment groups. There was no overall significant difference in GPx activity in the digestive gland between the control and treatment groups. Our results also revealed that petroleum hydrocarbon concentrations in the tissues increased linearly with exposure time. EROD activity in the digestive gland and GST and GPx activity in the gill tissue were negatively correlated with petroleum hydrocarbon body burden. These enzymes play important roles in detoxification and can act as potential biomarkers for monitoring petroleum hydrocarbon contaminants in the marine environment.

A Novel Aqueous Two Phase System Composed of Surfactant and Xylitol for the Purification of Lipase from Pumpkin (Cucurbita moschata) Seeds and Recycling of Phase Components.

PubMed

Amid, Mehrnoush; Manap, Mohd Yazid; Hussin, Muhaini; Mustafa, Shuhaimi

2015-06-17

Lipase is one of the more important enzymes used in various industries such as the food, detergent, pharmaceutical, textile, and pulp and paper sectors. A novel aqueous two-phase system composed of surfactant and xylitol was employed for the first time to purify lipase from Cucurbita moschata. The influence of different parameters such as type and concentration of surfactants, and the composition of the surfactant/xylitol mixtures on the partitioning behavior and recovery of lipase was investigated. Moreover, the effect of system pH and crude load on the degree of purification and yield of the purified lipase were studied. The results indicated that the lipase was partitioned into the top surfactant rich phase while the impurities partitioned into the bottom xylitol-rich phase using an aqueous two phase system composed of 24% (w/w) Triton X-100 and 20% (w/w) xylitol, at 56.2% of tie line length (TLL), (TTL is one of the important parameters in this study and it is determined from a bimodal curve in which the tie-line connects two nodes on the bimodal, that represent concentration of phase components in the top and bottom phases) and a crude load of 25% (w/w) at pH 8.0. Recovery and recycling of components was also measured in each successive step process. The enzyme was successfully recovered by the proposed method with a high purification factor of 16.4 and yield of 97.4% while over 97% of the phase components were also recovered and recycled. This study demonstrated that the proposed novel aqueous two phase system method is more efficient and economical than the traditional aqueous two phase system method for the purification and recovery of the valuable enzyme lipase.

Casein Fermentate of Lactobacillus animalis DPC6134 Contains a Range of Novel Propeptide Angiotensin-Converting Enzyme Inhibitors▿

PubMed Central

Hayes, M.; Stanton, C.; Slattery, H.; O'Sullivan, O.; Hill, C.; Fitzgerald, G. F.; Ross, R. P.

2007-01-01

This work evaluated the angiotensin-converting-enzyme (ACE)-inhibitory activities of a bovine sodium caseinate fermentate generated using the proteolytic capabilities of the porcine small intestinal isolate Lactobacillus animalis DPC6134 (NCIMB deposit 41355). The crude 10-kDa L. animalis DPC6134 fermentate exhibited ACE-inhibitory activity of 85.51% (±15%) and had a 50% inhibitory concentration (IC50) of 0.8 mg protein/ml compared to captopril, which had an IC50 value of 0.005 mg/ml. Fractionation of the crude L. animalis DPC6134 fermentate by membrane filtration and reversed-phase high-performance liquid chromatography (HPLC) generated three bioactive fractions from a total of 72 fractions. Fractions 10, 19, and 43 displayed ACE-inhibitory activity percentages of 67.53 (±15), 83.71 (±19), and 42.36 (±11), respectively, where ACE inhibition was determined with 80 μl of the fractions with protein concentrations of 0.5 mg/ml. HPLC and mass spectrometry analysis identified 25 distinct peptide sequences derived from α-, β-, and κ-caseins. In silico predictions, based on the C-terminal tetrapeptide sequences, suggested that peptide NIPPLTQTPVVVPPFIQ, corresponding to β-casein f(73-89); peptide IGSENSEKTTMP, corresponding to αs1-casein f(201212); peptide SQSKVLPVPQ, corresponding to β-casein f(166-175); peptide MPFPKYPVEP, corresponding to β-casein f(124133); and peptide EPVLGPVRGPFP, corresponding to β-casein f(210-221), contained ACE-inhibitory activities. These peptides were chosen for chemical synthesis to confirm the ACE-inhibitory activity of the fractions. Chemically synthesized peptides displayed IC50 values in the range of 92 μM to 790 μM. Additionally, a simulated gastrointestinal digestion confirmed that the ACE-inhibitory 10-kDa L. animalis DPC6134 fermentation was resistant to a cocktail of digestive enzymes found in the gastrointestinal tract. PMID:17483275

Antimicrobial activity of sodium hypochlorite-based irrigating solutions.

PubMed

Poggio, Claudio; Arciola, Carla Renata; Dagna, Alberto; Chiesa, Marco; Sforza, Dario; Visai, Livia

2010-09-01

The objective of the present study was the in vitro evaluation of the antimicrobial activity of three different NaOCl-based endodontic irrigating solutions: a 5.25% conventional sodium hypochlorite solution; and two new irrigating solutions, a 5.25% sodium hypochlorite solution with the addition of a proteolytic enzyme and a surfactant; and a 5.25% sodium hypochlorite gel with inorganic silicate. Enterococcus faecalis, Staphylococcus aureus and Streptococcus mutans strains were selected to evaluate the antimicrobial activity of the endodontic irrigating solutions by the agar disc diffusion test. Paper disks were saturated with each one of the tested solutions (at room temperature and pre-warmed at 45°C) and placed onto culture agar-plates pre-adsorbed with bacterial cells and further incubated for 24 h at 37°C. The growth inhibition zones around each irrigating solution were recorded and compared for each bacterial strain. The results were significantly different among the tested irrigating solutions: 5.25% sodium hypochlorite solution produced the highest inhibition areas; 5.25% sodium hypochlorite solution with a proteolytic enzyme and a surfactant, and 5.25% sodium hypochlorite gel with inorganic silicate showed the lowest zones of inhibition. Even if all tested irrigating solution possessed antibacterial activity versus all tested bacterial strains, 5.25% sodium hypochlorite solution with a proteolytic enzyme and a surfactant, and 5.25% sodium hypochlorite gel with inorganic silicate showed lower in vitro efficacy than 5.25% conventional sodium hypochlorite solution.

Glycogen-bound polyphosphate kinase from the archaebacterium Sulfolobus acidocaldarius.

PubMed

Skórko, R; Osipiuk, J; Stetter, K O

1989-09-01

Glycogen-bound polyphosphate kinase has been isolated from a crude extract of Sulfolobus acidocaldarius by isopycnic centrifugation in CsCl. Divalent cations (Mn2+ greater than Mg2+) stimulated the reaction. The enzyme does not require the presence of histones for its activity; it is inhibited strongly by phosphate and slightly by fluoride. The protein from the glycogen complex migrated in a sodium dodecyl sulfate-polyacrylamide gel as a 57-kilodalton protein band; after isoelectric focusing it separated into several spots in the pH range of 5.6 to 6.7.

Enhanced production of raw starch degrading enzyme using agro-industrial waste mixtures by thermotolerant Rhizopus microsporus for raw cassava chip saccharification in ethanol production.

PubMed

Trakarnpaiboon, Srisakul; Srisuk, Nantana; Piyachomkwan, Kuakoon; Sakai, Kenji; Kitpreechavanich, Vichien

2017-09-14

In the present study, solid-state fermentation for the production of raw starch degrading enzyme was investigated by thermotolerant Rhizopus microsporus TISTR 3531 using a combination of agro-industrial wastes as substrates. The obtained crude enzyme was applied for hydrolysis of raw cassava starch and chips at low temperature and subjected to nonsterile ethanol production using raw cassava chips. The agro-industrial waste ratio was optimized using a simplex axial mixture design. The results showed that the substrate mixture consisting of rice bran:corncob:cassava bagasse at 8 g:10 g:2 g yielded the highest enzyme production of 201.6 U/g dry solid. The optimized condition for solid-state fermentation was found as 65% initial moisture content, 35°C, initial pH of 6.0, and 5 × 10 6 spores/mL inoculum, which gave the highest enzyme activity of 389.5 U/g dry solid. The enzyme showed high efficiency on saccharification of raw cassava starch and chips with synergistic activities of commercial α-amylase at 50°C, which promotes low-temperature bioethanol production. A high ethanol concentration of 102.2 g/L with 78% fermentation efficiency was achieved from modified simultaneous saccharification and fermentation using cofermentation of the enzymatic hydrolysate of 300 g raw cassava chips/L with cane molasses.

An isolated Amycolatopsis sp. GDS for cellulase and xylanase production using agricultural waste biomass.

PubMed

Kshirsagar, S D; Saratale, G D; Saratale, R G; Govindwar, S P; Oh, M K

2016-01-01

The aim of this study was to evaluate an isolate of Amycolatopsis sp. GDS for cellulase and xylanase production, their characterization, and its application to the preparation of biomass feedstock for ethanol production. A novel potent cellulolytic bacterial strain was isolated and identified as Amycolatopsis sp. GDS. The strain secreted high levels of cellulase and xylanase in the presence of agricultural waste biomass. The enzymes were thermostable and active up to 70°C. Interestingly, the enzymes were expressed well at higher NaCl (up to 2·5 mol l(-1) ) and ionic liquid (10%) concentrations, so that they could be used during the pretreatment of biomass. Enzyme stability in the presence of organic solvents, surfactants and oxidizing agents was also noted. Crude enzymes from Amycolatopsis sp. GDS resulted in comparable saccharification (60%) of wheat straw to commercial enzymes (64%). The cellulolytic enzymes from Amycolatopsis sp. GDS were stable, expressed well under conditions with various chemicals, and yielded significant amounts of hydrolysates from the biomass. The high bioethanol production using yeast co-cultures with enzymatic hydrolysates highlights the significance of selecting the strain and substrate for biofuel production. This study demonstrates the importance of the isolate Amycolatopsis sp. GDS that secretes high levels of cellulase and hemicellulase by utilizing agricultural waste biomass and its application in the preparation of biomass feedstock and sequential ethanol fermentation. © 2015 The Society for Applied Microbiology.

The antimicrobial, antioxidative, anti-inflammatory activity and cytotoxicity of different fractions of four South African Bauhinia species used traditionally to treat diarrhoea.

PubMed

Ahmed, Aroke S; Elgorashi, Esameldin E; Moodley, Nivan; McGaw, Lyndy J; Naidoo, Vinasan; Eloff, Jacobus N

2012-10-11

Many Bauhinia species, including those indigenous to South Africa, are used in traditional medicine across the world for treating ailments such as gastrointestinal tract (GIT) disorders, diabetes, infectious diseases and inflammation. Several relevant aspects of different fractions of leaf extracts of Bauhinia bowkeri (BAB), Bauhinia galpinii (BAG), Bauhinia petersiana (BAP), and Bauhinia variegata (BAV) used in South African traditional medicine to alleviate diarrhoea related symptoms were evaluated. The antioxidative activities of the extracts were determined using the 2, 2-diphenyl-1-picrylhydrazyl (DPPH), 2, 2'-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid (ABTS(+)) radical scavenging and ferric reducing antioxidant power (FRAP) methods. In vitro antimicrobial activities of the extracts were determined against bacterial strains (Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli and Enterococcus faecalis) and clinical isolates of the opportunistic fungal strains (Aspergillus fumigatus, Candida albicans, and Cryptococcus neoformans) using a serial dilution microplate method. The polyphenolic contents were quantified using standard methods, and anti-inflammatory activities of the crude extracts were determined using the cyclooxygenase and soybean 15-lipoxygenase enzyme inhibitory assays. The safety of the extracts was evaluated by determining the cytotoxicity against Vero cell lines. The acidified 70% acetone crude extract and their fractions had good antiradical potency against the DPPH and ABTS radicals. The methanol soluble portions of the butanol fractions were more potent (EC(50) ranges from 0.64 ± 0.05 to 1.51 ± 0.07 and 0.88 ± 0.18 to 1.49 ± 0.09 μg/ml against DPPH and ABTS radical respectively) compared to the standard, trolox and ascorbic acid (EC(50) ranges from 1.47 ± 0.24 to 1.70 ± 0.27 μg/ml) for both DPPH and ABTS. The crude extracts contained variable quantities of phenolic content. The crude extracts and their fractions had weak to good antimicrobial activities, inhibiting the growth of the organisms at concentrations ranging from 39 to 2500 μg/ml. The BAG crude extract and its fractions were the most active against the fungi (MICs ranging from 39 to 625 μg/ml) while the BAB extract and its fractions were the least active with the MICs ranging between 39 and 2500 μg/ml. Aspergillus fumigatus was the least susceptible fungus while Cryptococcus neoformans was the most susceptible. The phenolic-rich crude extracts of BAB, BAG, and BAP had moderate to good dose-dependent cyclooxygenase-1 enzyme inhibitory activity with inhibitions between 22.8% and 71.4%. The extracts were however, inactive against cyclooxygenase-2. The extracts had some level of cytotoxicity towards Vero cell lines, reducing cell viability to less than 10% at concentrations more than 50 μg/ml. The biological activities observed in Bauhinia species provide a scientific basis for the use of the plants in traditional medicines to treat diseases with multi-factorial pathogenesis such as diarrhoea, with each aspect of activity contributing to the ultimate therapeutic benefit of the plants. However, the use of the phenolic-rich extracts of these plants to treat diarrhoea or any other ailments in traditional medicine needs to be monitored closely because of potential toxic effects and selective inhibition of COX-1 with the associated GIT injury. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

Functionalized three-dimensional graphene sponges for highly efficient crude and diesel oil adsorption.

PubMed

Bagoole, Oscar; Rahman, Md Mahfuzur; Shah, Sohail; Hong, Haiping; Chen, Hang; Al Ghaferi, Amal; Younes, Hammad

2018-06-02

Modified Hummer's method has been used in this study to synthesize graphene oxide (GO) solution that was utilized for the fabrication of three-dimensional (3D) graphene sponges and their subsequent functionalization through a low-cost and facile vapor-based surface enhancement approach. The functionalized 3D-graphene sponge is an excellent absorbent, which can remove more than 3300 wt.% of crude oil (calculated with respect to the original sorbent mass). The functionalization of the obtained graphene sponges with trichloro (1H,1H,2H,2H-perfluorooctyl)silane enhanced their wettability properties due to the super-hydrophobic nature of the resulting materials characterized by the contact angles in water greater than 150°. Furthermore, their elastic compression modulus (estimated by conducting a series of compression tests) was about 22.3 kPa. The equilibrium modeling of the oil removal process, which was performed by plotting Langmuir, Freundlich, Temkin, and Dubinin-Radushkevich isotherms, confirmed the properties of the fabricated 3D graphene sponges as exceptional absorbents for crude and diesel oil, which could be attributed to the oleophilic nature of graphene. Moreover, the obtained 3D graphene sponges could be regenerated via heat treatment, which was conducted to release the adsorbed species. After five adsorption-desorption cycles, the sorption capacity of the produced 3D graphene sponges towards crude oil reached 95% of the initial value.

Method for the quantification of vanadyl porphyrins in fractions of crude oils by High Performance Liquid Chromatography-Flow Injection-Inductively Coupled Plasma Mass Spectrometry

NASA Astrophysics Data System (ADS)

Wandekoken, Flávia G.; Duyck, Christiane B.; Fonseca, Teresa C. O.; Saint'Pierre, Tatiana D.

2016-05-01

High performance liquid chromatography hyphenated by flow injection to inductively coupled plasma mass spectrometry (HPLC-FI-ICP-MS) was used to investigate V linked to porphyrins present in fractions of crude oil. First, the crude oil sample was submitted to fractionation by preparative liquid chromatography with UV detection, at the porphyrin Soret band wavelength (400 nm). The obtained porphyrin fractions were then separated in a 250 mm single column, in the HPLC, and eluted with different mobile phases (methanol or methanol:toluene (80:20; v:v)). The quantification of V-porphyrins in the fractions eluted from HPLC was carried out by online measuring the 51V isotope in the ICP-MS, against vanadyl octaethylporphine standard solutions (VO-OEP), prepared in the same solvent as the mobile phase, and injected post-column directly into the plasma. A 20 μg L- 1 Ge in methanol was used as internal standard for minimizing non-spectral interference, such as short-term variations due to injection. The mathematical treatment of the signal based on Fast Fourier Transform smoothing algorithm was employed to improve the precision. The concentrations of V as V-porphyrins were between 2.7 and 11 mg kg- 1 in the fractions, which were close to the total concentration of V in the porphyrin fractions of the studied crude oil.

[Studies on the intradermal reactions with the fractions of Ascaris lumbricoides

PubMed

Lee, Chan Wook

1967-06-01

INTRODUCTION : The studies on allergic reaction with the substances of Ascaris lumbricoides have long been studied by various worers; Conventry(1929), Campbell(1936), Sakei(1949), Miyakawa(1950), Ikeda(1952), Matsumoto and Imawari(1952), Morishita and Kobayashi(1953, 1954), Komiyayama(1954) and Yammoto(1956). Campbell(1936) and some other workers reported that the polysaccharides from ascaris produced the stronger intrademal reactions than protein fraction, though Yamamoto(1954) and others found the reverse results. On the other hand, Hosotani(1954) reported that the crude antigen or mixed antigen with polysaccharide and protein fraction of the ascaris produced the strongest skin reaction than with the other single fraction. As are shown in above reports, the intensity of the allergic reaction with the substances from ascaris is still remained under dipute. The reason might be due to the difference of the method of preparation, technique and evaluation. The aim of the present study is to elucidate the intensity of allergic reactivity fo protein and polysaccharide fration and mixed substance of two fractions and crude antigen of Ascaris lumbricoides. MATERIALS AND METHODS : A Intrdermal Test 1. Human Experiment. The intradermal test was performed on several groups of people. A: Ascaris lumbricoides egg positive cases among adult ages. B: Ascaris lumbricoides egg negative cases but who bad doubtful symptom. C: Ascaris lumbricoides egg negative cases but who had past history of ascaris infection. D: Ascaris lumbricoides egg negative cases and aged 3-8 months old. 2. Animal Experiment: Same breeds of 6 dogs were raised i cages of laboratory for 6 months, During the period, special attention was paid to keep them in parasite free conditon. The body weight was 10kg in average. B. Antigens. The adults worms of Ascaris lumbricoides, which were obtained during laparotomy, were first washed with sterilized saline solution. Each ascaris was placed in 50ml of saline solution and kept in 30 degrees C incubator for 24 hours. Among of them, the active one was seleted and put to sudden freeze at -70 degrees C for 20 hours. the whole body was powdered in a dried condition and kept it in ampoule at -4 degrees C. a) Crude antigen: The ether extract of powdered Ascaris lumbrucoides were motared by adding veronal buffer solution (1:100) and kept in icebox for 48 hours. The suspension was diluted with veronal buffer solution in the ratio of 1:10,000. b) Protein antigen : This antigen was prepared by Chaffee's modified methods and ammonium sulfate extraction mehtod. c) Polysaccharide antigen: Chaffee's modified method and ethanol extration method were applied. d) Mixed antigen : The same amount of preparation of protein and polysaccharide antigen were mixed. C. Intrdermal test 1. The intradermal test : 0.02ml of natigen was injected on the anterior surface of the frearm in human and on the back in aninmal, with tuberculin syringe. The criteria of the skin reaction were determined as follows; wheal: -; 0-4mm, +/-; 5-7mm, +;8-9mm, +; 21-32mm, ++; 33-44mm, +++; 45-56mm, ++++; over 57mm, in diameter. D. Stool examination : All the stool examination was done by formalin-ether concectration(M.G.L) method. E.P.G (egg per gram) was also determined by Stoll's egg counting method. RESULT : The intradermal reaction after the injection of each antigen was observed at 15, 30, 60 minutes and 3, 24 hours. In 58 ascariasis cases, the peak of wheal was appeared at 30 minute; 93.0% with the crude antigen, 15.5% with the mixed antigen, 10.3% with the Protein antigen, but all were negative in the polysaccharide antigen. The erythema reaction paralledled, in general, to the wheal; 75.8% at 15 minutes, 72.5% at 30 minutes and 48.3% at 60 mintues, with the crude antigen. Only 3.4% showed erythema at 15,30, and 60 minutes int the case of mixed antigen, and 1.7% fo positive was appeared at 30 minutes in the case of protein antigen, but none was observed in the polysaccharide antigen. The wheal and erythema revealed correlationship each other. Both of them showed 65.5% positive boundary in the case of crude antigen. Generally, the crude antigen resulted the highest and strongest reaction than the other fractions; the mixed, protein and polysaccharide in decreasing order. In adult age group, who showed egg negative at the time of injection, 81.5% were positive in skin reaction with the crude antigen, and 88.6% in the group who complained doubtful symptom but egg negative. In the group who had past history of ascaris infection showed 66.6% positive and the egg negative infant group with the same crude antigen. The sheal size was not always paralleled with the worm burden. The cross reaction with the antigen from Ascaris lumbricoides and Toxocara canis was examined by the intradermal test. There was no cross reaction between the two antigen. The infected dog with Toxocara canis showed positive reaction by the crude antigen of same species, but mot by the human species. Experimentally, the positive skin reaction was appeared only by the crude antigen at four weeks after the infection of Toxocara canis. CONCLUSION: The intradermal studies with the fraction of Ascaris lumbricoides and Toxocara canis were performed to human and dog, and the following results were observed. 1) Wheal and erythema were appeared in the cases of ascaris infection or who had past history, but not in the ascaris free before. 2) The size of wheal reached to peak 30 minutes after the injection. 3) The crude antigen had specificity and showed no cross reaction. 4) The crude antigen cause the strongest and largest reaction than the other substances; protein, polysaccharide and the mixed antigen. No cutaneous reaction was observed with the fraction of polysaccharides. 5) The size of wheal did not parallel with the worm burden. 6) The skin reaction was appeared four weeks after the infection.

Biodegradation of phenolic compounds by Basidiomycota and its phenol oxidases: A review.

PubMed

Martínková, L; Kotik, M; Marková, E; Homolka, L

2016-04-01

The phylum Basidiomycota include organisms with enormous bioremediation potential. A variety of processes were proposed at the lab scale for using these fungi and their phenol oxidases in the degradation of phenolics. Here we present a survey of this topic using literature published mostly over the last 10 years. First, the sources of the enzymes are summarized. The laccase and tyrosinase were mainly from Trametes versicolor and Agaricus bisporus, respectively. Recently, however, new promising wild-type producers of the enzymes have emerged and a number of recombinant strains were also constructed, based mainly on yeasts or Aspergillus strains as hosts. The next part of the study summarizes the enzyme and whole-cell applications for the degradation of phenols, polyphenols, cresols, alkylphenols, naphthols, bisphenols and halogenated (bis)phenols in model mixtures or real wastewaters from the food, paper and coal industries, or municipal and hospital sewage. The enzymes were applied as free (crude or purified) enzymes or as enzymes immobilized in various supports or CLEAs, and optionally recycled or used in continuous mode. Alternatively, growing cultures or harvested mycelia were used instead. The products, which were characterized as quinones and their polymers in some cases, could be eliminated by filtration, flocculation or adsorption onto chitosan. The purity of a treated wastewater was monitored using a sensitive aquatic organism. It is concluded that low-cost sources of these enzymes should be searched for and the benefits of enzymatic, biological and physico-chemical methods could be combined to make the processes fit for industrial use. Copyright © 2016 Elsevier Ltd. All rights reserved.

Characterization of NADP-dependent 7 beta-hydroxysteroid dehydrogenases from Peptostreptococcus productus and Eubacterium aerofaciens.

PubMed Central

Hirano, S; Masuda, N

1982-01-01

Peptostreptococcus productus strain b-52 (a human fecal isolate) and Eubacterium aerofaciens ATCC 25986 were found to contain NADP-dependent 7 beta-hydroxysteriod dehydrogenase activity. The enzyme was synthesized constitutively by both organisms, and the enzyme yields were suppressed by the addition of 0.5 mM 7 beta-hydroxy bile acid to the growth medium. Purification of the enzyme by chromatography resulted in preparations with 3.5 (P. productus b-52, on Sephadex G-200) and 1.8 (E. aerofaciens, on Bio-Gel A-1.5 M) times the activity of the crude cell extracts. A pH optimum of 9.8 and a molecular weight of approximately 53,000 were shown for the enzyme of strain b-52, and an optimum pH at 10.5 and a molecular weight of 45,000 was shown for that from strain ATCC 25986. Kinetic studies revealed that both enzyme preparations oxidized the 7 beta-hydroxy group in unconjugated and conjugated bile acids, a lower Km value being demonstrated with free bile acid than with glycine and taurine conjugates. No measureable activity against 3 alpha-, 7 alpha-, or 12 alpha-hydroxy groups was detected in either enzyme preparation. When tested with strain ATCC 25986, little 7 beta-hydroxy-steroid dehydrogenase activity was detected in cells grown in the presence of glucose in excess. The enzyme from strain b-52 was found to be heat labile (90% inactivation at 50 degrees C for 3 min) and highly sensitive to sulfhydryl inhibitors. PMID:6954878

Evolution of cyclohexadienyl dehydratase from an ancestral solute-binding protein.

PubMed

Clifton, Ben E; Kaczmarski, Joe A; Carr, Paul D; Gerth, Monica L; Tokuriki, Nobuhiko; Jackson, Colin J

2018-04-23

The emergence of enzymes through the neofunctionalization of noncatalytic proteins is ultimately responsible for the extraordinary range of biological catalysts observed in nature. Although the evolution of some enzymes from binding proteins can be inferred by homology, we have a limited understanding of the nature of the biochemical and biophysical adaptations along these evolutionary trajectories and the sequence in which they occurred. Here we reconstructed and characterized evolutionary intermediate states linking an ancestral solute-binding protein to the extant enzyme cyclohexadienyl dehydratase. We show how the intrinsic reactivity of a desolvated general acid was harnessed by a series of mutations radiating from the active site, which optimized enzyme-substrate complementarity and transition-state stabilization and minimized sampling of noncatalytic conformations. Our work reveals the molecular evolutionary processes that underlie the emergence of enzymes de novo, which are notably mirrored by recent examples of computational enzyme design and directed evolution.

Curious Cases of the Enzymes.

PubMed

Ulusu, Nuriye Nuray

2015-07-01

Life as we know it heavily relies on biological catalysis, in fact, in a very nonromantic version of it, life could be considered as a series of chemical reactions, regulated by the guarding principles of thermodynamics. In ancient times, a beating heart was a good sign of vitality, however, to me, it is actually the presence of active enzymes that counts… Though we do not usually pay attention, the history of enzymology is as old as humanity itself, and dates back to the ancient times. This paper is dedicated to these early moments of this remarkable science that touched our lives in the past and will make life a lot more efficient for humanity in the future. There was almost always a delicate, fundamentally essential relationship between mankind and the enzymes. Challenged by a very alien and hostile Nature full of predators, prehistoric men soon discovered the medicinal properties of the plants, through trial and error. In fact, they accidently discovered the enzyme inhibitors and thus, in crude terms, kindled a sparkling area of research. These plant-derivatives that acted as enzyme inhibitors helped prehistoric men in their pursuit of survival and protection from predators; in hunting and fishing… Later in history, while the underlying purposes of survival and increasing the quality of life stayed intact, the ways and means of enzymology experienced a massive transformation, as the 'trial and error' methodology of the ancients is now replaced with rational scientific theories.

Intracellular Distribution of Enzymes of the Cytidine Diphosphate Choline Pathway in Castor Bean Endosperm

PubMed Central

Lord, J. M.; Kagawa, T.; Beevers, Harry

1972-01-01

The occurrence and subcellular distribution of enzymes of the cytidine diphosphate choline pathway of lecithin synthesis have been examined. Choline kinase (EC 2.7.1.32) was completely soluble, while phosphorylcholine-cytidyl transferase (EC 2.7.7.15) and phosphorylcholine-glyceride transferase (EC 2.7.8.2) were associated with particulate fractions. Although components sedimenting at 10,000 to 100,000 × g contained both enzymes, phosphorylcholine-cytidyl transferase and particularly phosphorylcholine-glyceride transferase were present in the 10,000 × g pellet, which contained the major organelles, mitochondria, and glyoxysomes. When the crude homogenate was centrifuged on a sucrose density gradient, four major bands of particulate protein were recovered. A band at density 1.24 g/cm3 contained the glyoxysomes and was devoid of phosphorylcholine-cytidyl transferase and phosphorylcholine-glyceride transferase activity. Enzyme activity was barely detectable in the mitochondria, at density 1.18 g/cm2. Phosphorylcholine-glyceride transferase was found almost exclusively in a sharp band at density 1.12 g/cm3, and phosphorylcholinecytidyl transferase was found in the uppermost band at density 1.08 g/cm3. Thus, for the synthesis of lecithin in their membranes, the glyoxysomes and mitochondria depend on enzymes elsewhere in the cell; the final two steps in lecithin formation occur, apparently exclusively, in separate particulate cell components. Images PMID:4506764

Production, Characterization of Tannase from Penicillium montanense URM 6286 under SSF Using Agroindustrial Wastes, and Application in the Clarification of Grape Juice (Vitis vinifera L.)

PubMed Central

Cruz, Roberta; Fonseca, Julyanna Cordoville; de Medeiros, Erika Valente; Maciel, Marília de Holanda Cavalcanti; Moreira, Keila Aparecida; Motta, Cristina Maria de Souza

2014-01-01

Tannase is an enzyme that hydrolyzes esters and lateral bonds of tannins, such as tannic acid, releasing glucose and gallic acid and stands out in the clarification of wines and juices. Fungi of the genera Aspergillus and Penicillium are excellent producers of this enzyme. The search for fungi that produce high levels of tannase as well as new substrates for the enzyme production by the SSF is required. The objectives of this study were to evaluate the production of tannase by Aspergillus and Penicillium species through SSF using leaves and agroindustrial waste barbados cherry and mangaba fruit as substrate, select the best producer, optimize production, characterize the crude enzyme extract, and apply it the clarification of grape juice. Selecting the best producer was performed by planning Placket-Burman and RSM. P. montanense showed highest activity with 41.64 U/mL after 72 h of fermentation residue using barbados cherry, with 3.5% tannic acid and 70% moisture. The enzyme showed the highest activity at pH 9.0 and 50°C. The tannase of P. montanense was stable over a wide pH range and temperature and, when applied to grape juice, showed higher efficiency by reducing 46% of the tannin content after incubation 120 m. PMID:25506607

Production, characterization of tannase from Penicillium montanense URM 6286 under SSF using agroindustrial wastes, and application in the clarification of grape juice (Vitis vinifera L.).

PubMed

de Lima, Juliana Silva; Cruz, Roberta; Fonseca, Julyanna Cordoville; de Medeiros, Erika Valente; Maciel, Marília de Holanda Cavalcanti; Moreira, Keila Aparecida; Motta, Cristina Maria de Souza

2014-01-01

Tannase is an enzyme that hydrolyzes esters and lateral bonds of tannins, such as tannic acid, releasing glucose and gallic acid and stands out in the clarification of wines and juices. Fungi of the genera Aspergillus and Penicillium are excellent producers of this enzyme. The search for fungi that produce high levels of tannase as well as new substrates for the enzyme production by the SSF is required. The objectives of this study were to evaluate the production of tannase by Aspergillus and Penicillium species through SSF using leaves and agroindustrial waste barbados cherry and mangaba fruit as substrate, select the best producer, optimize production, characterize the crude enzyme extract, and apply it the clarification of grape juice. Selecting the best producer was performed by planning Placket-Burman and RSM. P. montanense showed highest activity with 41.64 U/mL after 72 h of fermentation residue using barbados cherry, with 3.5% tannic acid and 70% moisture. The enzyme showed the highest activity at pH 9.0 and 50°C. The tannase of P. montanense was stable over a wide pH range and temperature and, when applied to grape juice, showed higher efficiency by reducing 46% of the tannin content after incubation 120 m.

In vitro and in silico Studies of Mangiferin from Aphloia theiformis on Key Enzymes Linked to Diabetes Type 2 and Associated Complications.

PubMed

Picot, Marie C N; Zengin, Gokhan; Mollica, Adriano; Stefanucci, Azzurra; Carradori, Simone; Mahomoodally, Mohamad F

2017-01-01

Mangiferin, was identified in the crude methanol extract, ethyl acetate, and n-butanol fractions of Aphloia theiformis (Vahl.) Benn. This study aimed to analyze the plausible binding modes of mangiferin to key enzymes linked to diabetes type 2 (DT2), obesity, hypertension, Alzheimer's disease, and urolithiasis using molecular docking. Crystallographic structures of α-amylase, α-glucosidase, glycogen phosphorylase (GP), pancreatic lipase, cholesterol esterase (CEase), angiotensin-I-converting enzyme (ACE), acetyl cholinesterase (AChE), and urease available on the Protein Databank database were docked to mangiferin using Gold 6.0 software. We showed that mangiferin bound to all enzymes by π-π and hydrogen bonds mostly. Mangiferin was docked to both allosteric and orthosteric sites of α-glucosidase by π-π interactions. However, several hydrogen bonds were observed at the orthosteric position, suggesting a preference for this site. The docking of mangiferin on AChE with the catalytic pocket occupied by paraoxon could be attributed to π-π stacking involving amino acid residues, Trp341 and Trp124. This study provided an insight of the molecular interaction of mangiferin with the studied enzymes and can be considered as a valuable tool for designing new drugs for better management of these diseases. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Cellulolytic enzymes production by utilizing agricultural wastes under solid state fermentation and its application for biohydrogen production.

PubMed

Saratale, Ganesh D; Kshirsagar, Siddheshwar D; Sampange, Vilas T; Saratale, Rijuta G; Oh, Sang-Eun; Govindwar, Sanjay P; Oh, Min-Kyu

2014-12-01

Phanerochaete chrysosporium was evaluated for cellulase and hemicellulase production using various agricultural wastes under solid state fermentation. Optimization of various environmental factors, type of substrate, and medium composition was systematically investigated to maximize the production of enzyme complex. Using grass powder as a carbon substrate, maximum activities of endoglucanase (188.66 U/gds), exoglucanase (24.22 U/gds), cellobiase (244.60 U/gds), filter paperase (FPU) (30.22 U/gds), glucoamylase (505.0 U/gds), and xylanase (427.0 U/gds) were produced under optimized conditions. The produced crude enzyme complex was employed for hydrolysis of untreated and mild acid pretreated rice husk. The maximum amount of reducing sugar released from enzyme treated rice husk was 485 mg/g of the substrate. Finally, the hydrolysates of rice husk were used for hydrogen production by Clostridium beijerinckii. The maximum cumulative H2 production and H2 yield were 237.97 mL and 2.93 mmoL H2/g of reducing sugar, (or 2.63 mmoL H2/g of cellulose), respectively. Biohydrogen production performance obtained from this work is better than most of the reported results from relevant studies. The present study revealed the cost-effective process combining cellulolytic enzymes production under solid state fermentation (SSF) and the conversion of agro-industrial residues into renewable energy resources.

Regulation of hazardous exposure by protective exposure: modulation of phase II detoxification and lipid peroxidation by Camellia sinensis and Swertia chirata.

PubMed

Saha, Prosenjit; Das, Sukta

2003-01-01

Many natural compounds are now known to have a modulatory role on physiological functions and biotransformation reactions involved in the detoxification process, thereby affording protection from cytotoxic, genotoxic, and metabolic actions of environmental toxicants. As part of a programme on evaluation of food, beverage, and traditional medicinal plants for their anticarcinogenic activity, their effects on detoxification enzymes were also studied. The present report deals with Camellia sinensis and Swertia chirata. The effect of water infusions as well as crude and purified components of these plants on glutathione-S-transferase (GST), glutathione peroxidase (GPx), superoxide dismutase (SOD), and catalase (CAT) was analyzed in mice that were exposed to the chemical carcinogen DMBA. All the four enzymes were found to be activated in different degrees following treatment. The effect of Theaflavin, a component of black tea, was highly significant. The activation of the enzymes was accompanied by significant reduction in lipid peroxidation. The observation suggest the chemopreventive potential of both Camellia sinensis and Swertia chirata. Copyright 2003 Wiley-Liss, Inc.

Environmentally benign process for the preparation of antimicrobial α-methylene-β-hydroxy-γ-butyrolactone (tulipalin B) from tulip biomass.

PubMed

Nomura, Taiji; Hayashi, Emiko; Kawakami, Shohei; Ogita, Shinjiro; Kato, Yasuo

2015-01-01

Tulipalin B (α-methylene-β-hydroxy-γ-butyrolactone, PaB) is an antimicrobial natural product occurring in tulip (Tulipa gesneriana). PaB is directly formed from the precursor glucose ester 6-tuliposide B (PosB) by endogenous Pos-converting enzyme (TCE). Despite the potential usefulness of antibacterial PaB in various industrial applications, lack of facile synthetic schemes hampers its practical use. Herein, we describe an environmentally benign and facile process for the preparation of PaB using tulip biomass materials based on one-step enzyme reaction catalyzed by TCE without the use of petroleum-derived solvents. By screening 115 tulip cultivars, we found three elite cultivars, which accumulated PosB almost exclusively in flower tissues. The flower extracts with aqueous ethanol were partially purified with activated charcoal and subjected to the enzyme reaction with reusable immobilized TCE prepared from bulb crude extracts. The reaction was completed in a few hours at room temperature, and PaB was purified with activated charcoal and ethanol in a batch-wise manner.

Nutrition quality of extraction mannan residue from palm kernel cake on brolier chicken

NASA Astrophysics Data System (ADS)

Tafsin, M.; Hanafi, N. D.; Kejora, E.; Yusraini, E.

2018-02-01

This study aims to find out the nutrient residue of palm kernel cake from mannan extraction on broiler chicken by evaluating physical quality (specific gravity, bulk density and compacted bulk density), chemical quality (proximate analysis and Van Soest Test) and biological test (metabolizable energy). Treatment composed of T0 : palm kernel cake extracted aquadest (control), T1 : palm kernel cake extracted acetic acid (CH3COOH) 1%, T2 : palm kernel cake extracted aquadest + mannanase enzyme 100 u/l and T3 : palm kernel cake extracted acetic acid (CH3COOH) 1% + enzyme mannanase 100 u/l. The results showed that mannan extraction had significant effect (P<0.05) in improving the quality of physical and numerically increase the value of crude protein and decrease the value of NDF (Neutral Detergent Fiber). Treatments had highly significant influence (P<0.01) on the metabolizable energy value of palm kernel cake residue in broiler chickens. It can be concluded that extraction with aquadest + enzyme mannanase 100 u/l yields the best nutrient quality of palm kernel cake residue for broiler chicken.

Radioimmune assay of ganglioside GM/sub 1/ synthase using cholera toxin

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

Honke, K.; Taniguchi, N.; Makita, A.

1986-01-01

A radioimmune assay for uridine 5'-diphosphate-galactose (UDP-Gal):GM/sub 2/ galactosyltransferase, which synthesizes GM/sub 1/, has been developed utilizing cholera toxin. This assay is more sensitive and simpler than previously used assays. Radioactive nucleotide substrate and GM/sub 2/ were incubated with an enzyme sample, and a radiolabeled product, GM/sub 1/, was reacted with cholera toxin. The GM/sub 1/-cholera toxin complex was further reacted with anti-cholera toxin and Staphylococcus aureus cell suspension. The resulting complex was transferred onto a nitrocellulose membrane and quantitated by liquid scintillation counting. This assay was found to be sensitive for the detection of 100 pmol of the reactionmore » product, GM/sub 1/. With this assay method, some properties of the crude enzyme extracts from rat liver were studied. The enzyme had a pH optimum of 6.5-7.0 and required Mn/sup 2 +/. The K/sub m/ values for UDP-Gal and GM/sub 2/ were 0.12 mM and 6 ..mu..M, respectively.« less

Extracellular inulinases from Penicillium janczewskii, a fungus isolated from the rhizosphere of Vernonia herbacea (Asteraceae).

PubMed

Pessoni, R A; Figueiredo-Ribeiro, R C; Braga, M R

1999-07-01

Extracellular inulinases from Penicillium janczewskii were obtained from the filtrate of 12 day-old cultures supplemented with inulin from Vernonia herbacea. Crude filtrates and partially-purified enzyme preparations (peaks I and II) were active on inulin, sucrose and raffinose. The apparent M(r) of the enzymes from peaks I and II were 48 and 66 kDa, respectively. The apparent K(m) (mmol l-1) values of peak I were 0.43 for inulin and 18.7 for sucrose; for peak II they were 0.87 and 18.5 for inulin and sucrose, respectively. Their temperature and pH optima were 55 degrees C and 5.0, respectively. Both peaks catalysed the hydrolysis of beta-(2,1) fructans more rapidly than beta-(2,6) fructans. Free fructose was the predominant product released from inulin, indicating that these enzymes display exo-inulinase activity. In view of these characteristics, the yield and the high specific activity towards beta-(2,1) fructans, inulinases from P. janczewskii can be utilized for the preparation of fructose syrup from inulin.

Antioxidative response of the three macrophytes Ceratophyllum demersum, Egeria densa, and Hydrilla verticillata to a time dependent exposure of cell-free crude extracts containing three microcystins from cyanobacterial blooms of Lake Amatitlán, Guatemala.

PubMed

Romero-Oliva, Claudia Suseth; Contardo-Jara, Valeska; Pflugmacher, Stephan

2015-06-01

Microcystins (MCs) produced by cyanobacteria in natural environments are a potential risk to the integrity of ecosystems. In this study, the effects of cyanobacterial cell-free crude extracts from a Microcystis aeruginosa bloom containing three MC-congeners MC-LR, -RR, and -YR at environmental relevant concentrations of 49.3±2.9, 49.8±5.9, and 6.9±3.8μg/L, respectively, were evaluated on Ceratophyllum demersum (L.), Egeria densa (Planch.), and Hydrilla verticillata (L.f.). Effects on photosynthetic pigments (total chlorophyll (chl), chl a, chl b, and carotenoids), enzymatic defense led by catalase (CAT), peroxidase (POD) and glutathione reductase (GR), and biotransformation enzyme glutathione S-transferase (GST) were measured after 1, 4, and 8h and after 1, 3, 7, and 14 days of exposure. Results show that in all exposed macrophytes, photosynthetic pigments were negatively affected. While chl a and total chl decreased with increasing exposure time, a parallel increase in chl b was observed after 8h. Concomitant increase of ∼5, 16, and 34% of antioxidant carotenoid concentration in exposed C. demersum, E. densa, and H. verticillata, respectively, was also displayed. Enzymatic antioxidant defense systems in all exposed macrophytes were initiated within the first hour of exposure. In exposed E. densa, highest values of CAT and GR activities were observed after 4 and 8h, respectively, while in exposed H. verticillata highest value of POD activity was observed after 8h. An early induction with a significant increase of biotransformation enzyme GST was observed in E. densa after 4h and in C. demersum and H. verticillata after 8h. These results are the first to show rapid induction of stress and further possible MC biotransformation (based on the activation of GST enzymatic activity included in MC metabolization during the biotransformation mechanism) in macrophytes exposed to crude extract containing a mixture of MCs. Copyright © 2015 Elsevier B.V. All rights reserved.

Shelf life determinants and enzyme activities of pearl millet: a comparison of changes in stored flour of hybrids, CMS lines, inbreds and composites.

PubMed

Goyal, Preeti; Chugh, L K

2017-09-01

Shelf life of pearl millet flour is very short because of rapid development of rancidity. This investigation was carried out in view of generating breeding material for development of low rancid pearl millet hybrids/varieties. Flour of twenty-one genotypes; seven hybrids, seven CMS lines, five inbreds and two composites stored in covered aluminium boxes at 37 °C for 30 days along with respective fresh flour was analysed for shelf life indicators/determinants. Crude fat content and fat acidity (FA) of fresh flour of the genotypes varied from 3.8 to 7.2% and 11 to 75 mg KOH/100 g d.m., respectively. FA in stored flour ranged between 180 and 330 mg KOH/100 g d.m. After storage, magnitude of decrease in pH of water extract of flour of the genotypes varied from 0.15 to 0.44. Activity of peroxidase (POX) varied from 378 to 588 units in control flour and irrespective of the genotypes decreased upon storage. Increase in FA (difference between FA of fresh and stored flour) rather total build up of FA was positively associated with crude fat content (r = 0.440*) indicated comparatively more prominent role of lipolytic enzymes. Chemical changes taking place in water soluble fraction of flour were independent of fat content as no correlation was discerned between fat content and decrease in pH. Among the hybrids, HHB 197 had lowest crude fat content (4.7%), lowest total build up FA (212 mg KOH/100 g d.m.), slowest increase in FA (191 mg KOH/100 g d.m.), least decrease in pH (0.31) of water soluble fraction flour during storage and lowest activity of POX in fresh flour (377 units/g d.m). Among all the tested CMS lines, inbreds and composites, HBL 11 showed pattern of quantitative changes in FA, pH and POX activity similar to the hybrid HHB 197 and was identified a promising inbred for developing low-rancid pearl millet variety or hybrid.

Purification and biochemical characterization of ionically unbound polyphenol oxidase from Musa paradisiaca leaf.

PubMed

Diwakar, Sanjeev Kumar; Mishra, Sarad Kumar

2011-01-01

An ionically unbound and thermostable polyphenol oxidase (PPO) was extracted from the leaf of Musa paradisiaca. The enzyme was purified 2.54-fold with a total yield of 9.5% by ammonium sulfate precipitation followed by Sephadex G-100 gel filtration chromatography. The purified enzyme exhibited a clear single band on native polyacrylamide gel electrophoresis (PAGE) and sodium dodecyl sulfate (SDS) PAGE. It was found to be monomeric protein with molecular mass of about 40 kD. The zymographic study using crude extract as enzyme source showed a very clear band around 40 kD and a faint band at around 15 kD, which might be isozymes. The enzyme was optimally active at pH 7.0 and 50°C temperature. The enzyme was active in wide range of pH (4.0-9.0) and temperature (30-90°C). From the thermal inactivation studies in the range 60-75°C, the half-life (t(1/2)) values of the enzyme ranged from 17 to 77 min. The inactivation energy (Ea) value of PPO was estimated to be 91.3 kJ mol(-1). It showed higher specificity with catechol (K(m) = 8 mM) as compared to 4-methylcatechol (K(m) = 10 mM). Among metal ions and reagents tested, Cu(2+), Fe(2+), Hg(2+), Mn(2+), Ni(2+), protocatechuic acid, and ferrulic acid enhanced the enzyme activity, while K(+), Na(+), Co(2+), kojic acid, ascorbic acid, ethylenediamine tetraacetic acid (EDTA), sodium azide, β-mercaptoethanol, and L-cysteine inhibited the activity of the enzyme.

Solution-state NMR analysis of hydroxymethylated resorcinol cured in the presence of crude milled-wood lignin from Acer saccharum

Treesearch

Daniel J. Yelle

2017-01-01

Resorcinol-formaldehyde adhesives can reinforce stress fractures that appear from wood surface preparation. Researchers have found that applying the resorcinol-formaldehyde prepolymer, hydroxymethylated resorcinol is thought to plasticize lignin components and stabilize stress fractures through reactions with lignin subunits and hemicelluloses in wood. In this study, a...

Effect of metallic additives on in situ combustion of Huntington Beach crude experiments

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

Baena, C.J.; Castanier, L.M.; Brigham, W.E.

1990-08-01

The economics and applicability of an in-situ combustion process for the recovery of crude oil are dictated to a large extent by the nature and the amount of fuel formed during the process. The aim of this work is to use combustion tube studies to determine on a quantitative basis, how the nature and the amount of fuel formed could be changed by the presence of metallic additives. These experiments follow from the qualitative observations on the effect of metallic additives on the in-situ combustion of Huntington Beach crude oil made by De los Rios (1987) at SUPRI. He performedmore » kinetic studies on the oxidation of Huntington Beach crude in porous media and showed that the nature of the fuel formed changed when metallic additives were present. Combustion tube runs were performed using the metallic additives: ferrous chloride (FeCl{sub 2{center dot}}4H{sub 2}O), zinc chloride (ZnCl{sub 2}) and stannic chloride (SnCl{sub 4{center dot}}5H{sub 2}O). Unconsolidated cores were prepared by mixing predetermined amounts of an aqueous solution of the metal salt, Huntington Beach crude oil, Ottawa sand and clay in order to achieve the desired fluid saturations. The mixture was then tamped into the combustion tube. Dry air combustion tube runs were performed keeping the conditions of saturation, air flux and injection pressure approximately the same during each run. The nature of the fuel formed and its impact on the combustion parameters were determined and compared with a control run -- an experiment performed with no metallic additive. 30 refs., 33 figs., 6 tabs.« less

Immobilization of a Pleurotus ostreatus Laccase Mixture on Perlite and Its Application to Dye Decolourisation

PubMed Central

Salatino, Piero; Sannia, Giovanni

2014-01-01

In the present study, a crude laccase preparation from Pleurotus ostreatus was successfully immobilized on perlite, a cheap porous silica material, and tested for Remazol Brilliant Blue R (RBBR) decolourisation in a fluidized bed recycle reactor. Results showed that RBBR decolourisation is mainly due to enzyme action despite the occurrence of dye adsorption-related enzyme inhibition. Fine tuning of immobilization conditions allowed balancing the immobilization yield and the resulting rate of decolourisation, with the adsorption capacity of the solid biocatalyst. In the continuous lab scale reactor, a maximum conversion degree of 56.1% was achieved at reactor space-time of 4.2 h. Stability and catalytic parameters of the immobilized laccases were also assessed in comparison with the soluble counterparts, revealing an increase in stability, despite a reduction of the catalytic performances. Both effects are most likely ascribable to the occurrence of multipoint attachment phenomena. PMID:24895564

Optimization and purification of L-asparaginase produced by Streptomyces tendae TK-VL_333.

PubMed

Kavitha, Alapati; Vijayalakshmi, Muvva

2010-01-01

Cultural factors affecting the production of L-asparaginase by Streptomyces tendae isolated from laterite soil samples of Guntur region were investigated on glycerol-asparagine-salts (modified ISP-5) broth. Optimal yields of L-asparaginase were recorded in the culture medium with the initial pH 7.0 incubated at 30 degrees C for 72 h. The strain utilized sucrose (2%) and yeast (2%) extract as carbon and nitrogen sources for L-asparaginase production. The productivity of L-asparaginase was slightly enhanced when the strain was treated with cell-disrupting agents like EDTA. The crude enzyme was purified to homogeneity by ammonium sulfate precipitation, Sephadex G-100 and CM-Sephadex G-50 gel filtration. By employing sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the molecular weight of the enzyme was recorded as 97.4 kDa. This is the first report on production and purification of L-asparaginase from S. tendae.

Circular dichroism of stem bromelain: a third spectral class within the family of cysteine proteinases.

PubMed Central

Arroyo-Reyna, A; Hernandez-Arana, A; Arreguin-Espinosa, R

1994-01-01

Two forms of stem bromelain (EC 3.4.22.4) were isolated from commercial, crude and chromatographically purified preparations of the enzyme by means of gel-filtration and cation-exchange liquid chromatography. These forms possess nearly identical secondary and tertiary structures, as judged from their circular dichroism (c.d.) spectra. The spectral characteristics of stem bromelain suggest that this enzyme belongs to the alpha + beta protein class, as other cysteine proteinases do. In agreement with these results, quantitative estimation of secondary structures yielded amounts similar to those for papain and proteinase omega. However, the bromelain c.d. curve is clearly distinguishable from those reported for papain and proteinase omega, on one hand, and that of chymopapain, on the other. Thus, it is apparent that there are at least three types of c.d. spectra associated with the family of cysteine proteinases. PMID:8198520

Cloning and overexpression of beta-N-acetylglucosaminidase encoding gene nagA from Aspergillus oryzae and enzyme-catalyzed synthesis of human milk oligosaccharide.

PubMed

Matsuo, Ichiro; Kim, Sunhwa; Yamamoto, Yuichi; Ajisaka, Katsumi; Maruyama, Jun-ich; Nakajima, Harushi; Kitamoto, Katsuhiko

2003-03-01

We isolated a beta-N-acetylglucosaminidase encoding gene from the filamentous fungus Aspergillus oryzae, and designated it nagA. The nagA gene encoded a polypeptide of 600 amino acids with significant similarity to glucosaminidases and hexosaminidases of various eukaryotes. A. oryzae strain carrying the nagA gene under the control of the improved glaA promoter produced large amounts of beta-N-acetylglucosaminidase in a wheat bran solid culture. The beta-N-acetylglucosaminidase was purified from crude extracts of the solid culture by column chromatographies on Q-Sepharose and Sephacryl S-200. This enzyme was used for synthesis of lacto-N-triose II, which is contained in human milk. By reverse hydrolysis reaction, lacto-N-triose II and its positional isomer were synthesized from lactose and D-N-acetylglucosamine in 0.21% and 0.15% yield, respectively.

Hydrocarbon activation under sulfate-reducing and methanogenic conditions proceeds by different mechanisms.

NASA Astrophysics Data System (ADS)

Head, Ian; Gray, Neil; Aitken, Caroline; Sherry, Angela; Jones, Martin; Larter, Stephen

2010-05-01

Microbial degradation of alkanes typically involves their conversion to fatty acids which are then catabolised by beta-oxidation. The critical step in this process is activation of the hydrocarbon. Under oxic conditions this is catalyzed by monooxygenase enzymes with the formation of long chain alcohols. In the absence of oxygen alternative alkane activation mechanisms have been observed or proposed. Fumarate addition to alkanes to form alkyl succinates is considered a central process in anaerobic hydrocarbon degradation. Comparative studies of crude oil degradation under sulphate-reducing and methanogenic conditions revealed distinctive patterns of compound class removal and metabolite formation. Alkyl succinates derived from C7 to C26 n-alkanes and branched chain alkanes were found in abundance in sulfate-reducing systems but these were not detected during methanogenic crude oil degradation. Only one other mechanism of alkane activation has been elucidated to date. This involves addition of carbon derived from bicarbonate/CO2 to C-3 of an alkane chain to form a 2-ethylalkane with subsequent removal of the ethyl group leading to the formation of a fatty acid 1 carbon shorter than the original alkane. 2-ethylalkanes have never been detected as metabolites of anaerobic alkane degradation and were not detected in crude oil-degrading methanogenic systems. Due to the range of alkanes present in crude oil it was not possible to infer the generation of C-odd acids from C-even alkanes which is characteristic of the C-3 carboxylation mechanism. Furthermore genes homologous to alkysuccinate synthetases were not detected in the methanogenic hydrocarbon degrading community by pyrosequencing of total DNA extracted from methanogenic enrichments cultures. beta-oxidation genes were detected and intriguingly, alcohol and aldehyde dehydrogenase genes were present. This offers the possibility that alkane activation in the methanogenic system does not proceed via acid metabolites, but may be initiated by an anaerobic hydroxylation reaction. This is not unprecedented and hydroxylation of ethylbenzene has been demonstrated. However the C-H bond dissociation energy of alkanes is typically considered too high to readily permit alkane hydroxylation. It is however clear that alkane activation in these methanogenic crude oil-degrading systems involves mechanisms other than the well-known fumarate-addition reactions.

Immobilization of lambda exonuclease onto polymer micropillar arrays for the solid-phase digestion of dsDNAs.

PubMed

Oliver-Calixte, Nyoté J; Uba, Franklin I; Battle, Katrina N; Weerakoon-Ratnayake, Kumuditha M; Soper, Steven A

2014-05-06

The process of immobilizing enzymes onto solid supports for bioreactions has some compelling advantages compared to their solution-based counterpart including the facile separation of enzyme from products, elimination of enzyme autodigestion, and increased enzyme stability and activity. We report the immobilization of λ-exonuclease onto poly(methylmethacrylate) (PMMA) micropillars populated within a microfluidic device for the on-chip digestion of double-stranded DNA. Enzyme immobilization was successfully accomplished using 3-(3-dimethylaminopropyl) carbodiimide/N-hydroxysuccinimide (EDC/NHS) coupling to carboxylic acid functionalized PMMA micropillars. Our results suggest that the efficiency for the catalysis of dsDNA digestion using λ-exonuclease, including its processivity and reaction rate, were higher when the enzyme was attached to a solid support compared to the free solution digestion. We obtained a clipping rate of 1.0 × 10(3) nucleotides s(-1) for the digestion of λ-DNA (48.5 kbp) by λ-exonuclease. The kinetic behavior of the solid-phase reactor could be described by a fractal Michaelis-Menten model with a catalytic efficiency nearly 17% better than the homogeneous solution-phase reaction. The results from this work will have important ramifications in new single-molecule DNA sequencing strategies that employ free mononucleotide identification.

Application of forward osmosis technology in crude glycerol fermentation biorefinery-potential and challenges.

PubMed

Kalafatakis, S; Braekevelt, S; Lymperatou, A; Zarebska, A; Hélix-Nielsen, C; Lange, L; Skiadas, I V; Gavala, H N

2018-04-24

Forward osmosis (FO) is a low energy-intensive process since the driving force for water transport is the osmotic pressure difference, Δπ, between the feed and draw solutions, separated by the FO membrane, where π draw  > π feed . The potential of FO in wastewater treatment and desalination have been extensively studied; however, regeneration of the draw solution (thereby generating clean water) requires application of an energy-intensive process step like reverse osmosis (RO). In this study, the potential of applying FO for direct water recirculation from diluted fermentation effluent to concentrated feedstock, without the need for an energy-intensive regeneration step (e.g. RO), has been investigated. Butanol production during crude glycerol fermentation by Clostridium pasteurianum, has been selected as a model process and the effect of cross-flow velocity and the dilution of draw solution on the water flux during short-term experiments (200 min), were investigated. Statistical analysis revealed that the dilution of the draw solution is the most influential factor for the water flux. Subsequent modelling of an integrated FO-fermentation process, showed that water recoveries could lead to substantial financial benefits, although the integrated FO-fermentation process demonstrated lower water flux than expected. FTIR analyses of the membrane surface implied that the decrease in water flux was due to the presence of proteins, polysaccharides and other extracellular polymeric substances on the membrane active layer, indicating the presence of a fouling layer. Based on these findings, possible fouling alleviation strategies and future research directions are discussed and proposed.

Alkane hydroxylase genes in psychrophile genomes and the potential for cold active catalysis.

PubMed

Bowman, Jeff S; Deming, Jody W

2014-12-16

Psychrophiles are presumed to play a large role in the catabolism of alkanes and other components of crude oil in natural low temperature environments. In this study we analyzed the functional diversity of genes for alkane hydroxylases, the enzymes responsible for converting alkanes to more labile alcohols, as found in the genomes of nineteen psychrophiles for which alkane degradation has not been reported. To identify possible mechanisms of low temperature optimization we compared putative alkane hydroxylases from these psychrophiles with homologues from nineteen taxonomically related mesophilic strains. Seven of the analyzed psychrophile genomes contained a total of 27 candidate alkane hydroxylase genes, only two of which are currently annotated as alkane hydroxylase. These candidates were mostly related to the AlkB and cytochrome p450 alkane hydroxylases, but several homologues of the LadA and AlmA enzymes, significant for their ability to degrade long-chain alkanes, were also detected. These putative alkane hydroxylases showed significant differences in primary structure from their mesophile homologues, with preferences for specific amino acids and increased flexibility on loops, bends, and α-helices. A focused analysis on psychrophile genomes led to discovery of numerous candidate alkane hydroxylase genes not currently annotated as alkane hydroxylase. Gene products show signs of optimization to low temperature, including regions of increased flexibility and amino acid preferences typical of psychrophilic proteins. These findings are consistent with observations of microbial degradation of crude oil in cold environments and identify proteins that can be targeted in rate studies and in the design of molecular tools for low temperature bioremediation.

Temperature modulates hepatic carbohydrate metabolic enzyme activity and gene expression in juvenile GIFT tilapia (Oreochromis niloticus) fed a carbohydrate-enriched diet.

PubMed

Qiang, J; He, J; Yang, H; Wang, H; Kpundeh, M D; Xu, P; Zhu, Z X

2014-02-01

The effects of rearing temperature on hepatic glucokinase (GK), glucose-6-phosphatase (G6Pase) and Glucose-6-phosphate dehydrogenase (G6PD) activity and gene expression were studied in GIFT (genetically improved farmed tilapia) tilapia fed a high carbohydrate diet containing 28% crude protein, 5% crude lipid and 40% wheat starch. Triplicate groups of fish (11.28 g initial body weight) were fed the diet for 45 days at 22 °C, 28 °C or 34 °C. At the end of the trial, final body weight of juvenile at 28 °C (59.12 g) was higher than that of the fish reared at 22 °C (27.13 g) and 34 °C (43.17 g). Feed intake, feed efficiency and protein efficiency ratio were also better at 28 °C. Liver glycogen levels were higher at 28 °C, while plasma glucose levels were higher in the 22 °C group. Significant (P<0.05) effects of water temperature on enzymes activities and gene expression were observed. Hepatic GK activity and mRNA level were higher at 28 °C than at 34 °C. Higher G6Pase and G6PD activity and gene expression were observed at 22 °C. Overall, the data show that juveniles reared at 28 °C exhibited enhanced liver glycolytic capacity. In contrast, hepatic gluconeogenesis and lipogenesis were increased by low temperature (22 °C). Copyright © 2014. Published by Elsevier Ltd.

Monoclonal antibody-based serological assays and immunocapture-RT-PCR for detecting Rice dwarf virus in field rice plants and leafhopper vectors.

PubMed

Wu, Jianxiang; Ni, Yuequn; Liu, Huan; Ding, Ming; Zhou, Xueping

2014-01-01

Rice dwarf virus (RDV) causes Rice dwarf disease, which leads to considerable losses in rice production in Asia. Purified RDV virions were used as the immunogen to prepare monoclonal antibodies (mAbs). Three murine mAbs against RDV were prepared. Plate-trapped antigen enzyme-linked immunosorbent assay (PTA-ELISA), dot enzyme-linked immunosorbent assay (dot-ELISA) and immunocapture-RT-PCR (IC-RT-PCR) were then developed for sensitive, specific, and rapid detection of RDV in rice and leafhopper samples obtained in the field using the mAbs. The PTA-ELISA, dot-ELISA and IC-RT-PCR detected the virus in infected tissue crude extracts diluted at 1:81,920, 1:10,240 and 1:655,360 (w/v, g mL(-1)), in individual viruliferous rice green leafhopper crude extracts diluted at 1:25,600, 1:6400 and 1:3,276,800 (individual leafhopper/μL), respectively. 878 rice field samples and 531 leafhopper field samples from ten provinces of China were screened for the presence of RDV using the two serological assays and the IC-RT-PCR and the results indicated that 37 of the 878 rice samples and 22 of the 531 leafhopper samples were infected by RDV. All positive samples were from Yunnan Province, indicating that RDV is prevalent in this province, but not in the other nine provinces. The dot-ELISA is suitable for routine detection of large-scale rice and leafhopper samples in field surveys. Copyright © 2013 Elsevier B.V. All rights reserved.

Comparative production of 6-aminopenicillanic acid by different E. coli strains and their acridine orange (AO) induced mutants.

PubMed

Arshad, Rubina; Farooq, Shafqat; Ali, Syed Shahid

2007-11-01

The present study was conducted to see the difference in production of 6-APA I) between wild strains of E. coli collected from local environment and their acridine orange (AO) induced mutants and ii) between mutants and E. coli strains (ATCC 11105 and ATCC 9637) of American Type Culture Collection (ATCC) used commercially for enzymatic production of 6-APA. The optimum conditions for bioconversion were standardized and 6-APA was obtained in crystalline form. Relative PGA activity of local and foreign E. coli strains varied significantly with the highest being 12.7 in mutant strain (BDCS-N-M36) and the lowest 4.3 mg 6-APA h(-1) mg(-1) wet cells in foreign strain (ATCC 11105). The enzyme activity exhibited by mutant strain (BDCS-N-M36) was also two folds higher compared to that in wild parent BDCS-N-W50 (6.3 mg 6-APA h(-1) mg(-1) wet cells). The overall production of 6-APA and conversion ratios ranged between 0.25-0.41 g of 6-APA per 0.5 g of penicillin G and 51-83%, respectively. Maximum conversion ratio (83%) was achieved by using crude cells of mutant strain (BDCS-N-M36) which is the highest value ever reported by crude cells on a shake-flask scale whereas reported 6-APA production by immobilized cells is 60-90% in batch and continuous systems. Results are being discussed with reference to importance of local bacterial strains and their significance for industrially important enzymes.

Distribution of nutrients and antinutrients in milled fractions of chickpea and horse gram: seed coat phenolics and their distinct modes of enzyme inhibition.

PubMed

Sreerama, Yadahally N; Neelam, Dennis A; Sashikala, Vadakkoot B; Pratape, Vishwas M

2010-04-14

Milled fractions of chickpea ( Cicer arietinum L.) and horse gram ( Macrotyloma uniflorum L. Verdc.) were evaluated for their nutritional and antinutritional characteristics. Crude protein content of these fractions ranged from 22.6-23.8 g 100(-1) g in cotyledon to 7.3-9.1 g 100(-1) g in seed coat fractions. The fat content of chickpea fractions (1.6-7.8 g 100(-1) g) was higher than that of horse gram fractions (0.6-2.6 g 100(-1) g). Crude fiber content was higher in seed coat fractions of both legumes than embryonic axe and cotyledon fractions. Seed coat fractions had high dietary fiber content (28.2-36.4 g 100(-1) g), made up of mainly insoluble dietary fiber. Most of the phytic acid and oligosaccharides were located in the cotyledon fractions, whereas phenolic compounds in higher concentrations were found in seed coats. Significantly higher concentrations of proteinaceous and phenolic inhibitors of digestive enzymes were found in cotyledon and seed coat fractions, respectively. The kinetic studies, using Michaelis-Menten and Lineweaver-Burk derivations, revealed that seed coat phenolics inhibit alpha-amylase activity by mixed noncompetitive (chickpea) and noncompetitive (horse gram) inhibition mechanisms. In the case of trypsin, chickpea and horse gram seed coat phenolics showed noncompetitive and uncompetitive modes of inhibition, respectively. These results suggest the wide variability in the nutrient and antinutrient composition in different milled fractions of legumes and potential utility of these fractions as ingredients in functional food product development.

Novel biological properties of Oenothera paradoxa defatted seed extracts: effects on metallopeptidase activity.

PubMed

Kiss, Anna K; Derwińska, Małgorzata; Dawidowska, Anna; Naruszewicz, Marek

2008-09-10

In this study, for the first time, we used the in vitro metallopeptidase model for the identification of a potential novel activity of defatted evening primrose seed extracts. Prepared extracts of different polarity (aqueous, 60% ethanolic, isopropanolic, and 30% isopropanolic) at concentrations of 1.5-100 microg/mL exhibited a significant and dose dependent inhibition of three tested enzymes. The 50% inhibition of enzymes activity showed that aminopeptidase N (APN) was the enzyme affected to the greatest extent with IC50 at the level of 2.8 microg/mL and 2.9 microg/mL for aqueous and 30% isopropanolic extracts, respectively. The activity of neutral endopeptidase (NEP) was quite strongly inhibited by the extracts as well. The HPLC-DAD analysis and bioguided fractionation led to the identification of four active compounds: (-)-epicatechin gallate, proanthocyanidin B3, oenothein B, and penta-O-galloyl-beta-D-glucose (PGG). Oenothein B has been shown previously to inhibit metallopeptidases. The three other compounds are known to inhibit angiotensin-converting enzyme (ACE), but they have not been previously reported to inhibit the NEP and APN activity. PGG and procyanidins with different degrees of polymerization, as the dominating compounds in O. paradoxa seeds, seemed to play a role in the crude extract activity.

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.

Miniaturised enzymatic conductometric biosensor with Nafion membrane for the direct determination of formaldehyde in water samples.

PubMed

Nguyen-Boisse, Thanh-Thuy; Saulnier, Joëlle; Jaffrezic-Renault, Nicole; Lagarde, Florence

2014-02-01

A new conductometric enzyme-based biosensor was developed for the determination of formaldehyde (FA) in aqueous solutions. The biosensor was prepared by cross-linking formaldehyde dehydrogenase from Pseudomonas putida with bovine serum albumin in saturated glutaraldehyde vapours (GA) at the surface of interdigitated gold microelectrodes. Nicotinamide adenine dinucleotide cofactor (NAD(+)) was added in solution at each measurement to maintain enzyme activity. Addition of a Nafion layer over the enzyme modified electrode resulted in a significant increase of biosensor signal due to enhanced accumulation of protons generated by enzymatic reaction at the electrode surface. Different parameters affecting enzyme activity or playing a role in ionic transfer through the Nafion membrane were optimised. In optimal conditions (0.045 mg enzyme, 30 min exposure to GA, 0.3 μL of a 1% (v/v) Nafion solution deposit, measurement in 5 mM phosphate buffer pH 7 containing 20 μM NAD(+)), the biosensor signal was linear up to 10 mM FA, and the detection limit was 18 μM. Relative standard deviations calculated from five consecutive replicates of FA solutions were lower than 5% in the 1-10 mM range. The biosensor was successfully applied to the determination of FA in spiked water samples (tap water and Rhone river water), with recoveries in the 95-110% range.

Evaluation of nanoparticle-immobilized cellulase for improved ethanol yield in simultaneous saccharification and fermentation reactions

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

Lupoi, Jason; Smith, Emily

2011-12-01

Ethanol yields were 2.1 (P = 0.06) to 2.3 (P = 0.01) times higher in simultaneous saccharification and fermentation (SSF) reactions of microcrystalline cellulose when cellulase was physisorbed on silica nanoparticles compared to enzyme in solution. In SSF reactions, cellulose is hydrolyzed to glucose by cellulase while yeast simultaneously ferments glucose to ethanol. The 35 C temperature and the presence of ethanol in SSF reactions are not optimal conditions for cellulase. Immobilization onto solid supports can stabilize the enzyme and promote activity at non-optimum reaction conditions. Mock SSF reactions that did not contain yeast were used to measure saccharification productsmore » and identify the mechanism for the improved ethanol yield using immobilized cellulase. Cellulase adsorbed to 40 nm silica nanoparticles produced 1.6 times (P = 0.01) more glucose than cellulase in solution in 96 h at pH 4.8 and 35 C. There was no significant accumulation (<250 {mu}g) of soluble cellooligomers in either the solution or immobilized enzyme reactions. This suggests that the mechanism for the immobilized enzyme's improved glucose yield compared to solution enzyme is the increased conversion of insoluble cellulose hydrolysis products to soluble cellooligomers at 35 C and in the presence of ethanol. The results show that silica-immobilized cellulase can be used to produce increased ethanol yields in the conversion of lignocellulosic materials by SSF.« less

Effects of oregano essential oil with or without feed enzymes on growth performance, digestive enzyme, nutrient digestibility, lipid metabolism and immune response of broilers fed on wheat-soybean meal diets.

PubMed

Basmacioğlu Malayoğlu, H; Baysal, S; Misirlioğlu, Z; Polat, M; Yilmaz, H; Turan, N

2010-02-01

1. The study was conducted to determine the effects of dietary supplementation of enzyme and oregano essential oil at two levels, alone or together, on performance, digestive enzyme, nutrient digestibility, lipid metabolism and immune response of broilers fed on wheat-soybean meal based diets. 2. The following dietary treatments were used from d 0 to 21. Diet 1 (control, CONT): a commercial diet containing no enzyme or oregano essential oil, diet 2 (ENZY): supplemented with enzyme, diet 3 (EO250): supplemented with essential oil at 250 mg/kg feed, diet 4 (EO500): supplemented with essential oil at 500 mg/kg feed, diet 5 (ENZY + EO250): supplemented with enzyme and essential oil at 250 mg/kg, and diet 6 (ENZY + EO500): supplemented with enzyme and essential oil at 500 mg/kg. 3. Birds fed on diets containing ENZY, EO250 and ENZY + EO250 had significantly higher weight gain than those given CONT diet from d 0 to 7. No significant effects on feed intake, feed conversion ratio, mortality, organ weights except for jejunum weight and intestinal lengths was found with either enzyme or essential oil, alone or in combination, over the 21-d growth period. The supplementation of essential oil together with enzyme decreased jejunum weight compared with essential oil alone. 4. Supplementation with enzyme significantly decreased viscosity and increased dry matter of digesta, but did not alter pH of digesta. There was no effect of essential oil alone at either concentration on viscosity, dry matter or pH of digesta. A significant decrease in viscosity of digesta appeared when essential oil was used with together enzyme. 5. The supplementation of essential oil at both levels with or without enzyme significantly increased chymotrypsin activity in the digestive system, and improved crude protein digestibility. 6. The higher concentration of essential oil with and without enzyme significantly increased serum total cholesterol concentrations. No significant effect on immune response was found with either enzyme or essential oil, alone or together. 7. Enzymes and essential oil had different modes of actions. The supplementation of enzyme with essential oil in diets is likely more effective in view of performance, nutrient digestibility, enzyme activities and immune system.

9 CFR 327.25 - Disposition procedures for product condemned or ordered destroyed under import inspection.

Code of Federal Regulations, 2010 CFR

2010-01-01

... dipping it in a solution of 0.0625 percent tannic acid, followed by immersion in a water bath, then... tannic acid for 1 minute followed by immersion in a water bath, then immersing it for 1 minute in a... establishment: Crude carbolic acid; cresylic disinfectant; a formula consisting of 1 part FD&C green No. 3...

9 CFR 327.25 - Disposition procedures for product condemned or ordered destroyed under import inspection.

Code of Federal Regulations, 2011 CFR

2011-01-01

... dipping it in a solution of 0.0625 percent tannic acid, followed by immersion in a water bath, then... tannic acid for 1 minute followed by immersion in a water bath, then immersing it for 1 minute in a... establishment: Crude carbolic acid; cresylic disinfectant; a formula consisting of 1 part FD&C green No. 3...

Process for tertiary oil recovery using tall oil pitch

DOEpatents

Radke, C.J.

1983-07-25

A process and compositions for enhancing the recovery of acid crudes are disclosed. The process involves injecting caustic solutions into the reservoir to maintain a pH of 11 to 13. The fluid contains an effective amount of multivalent cation for inhibiting alkaline silica dissolution with the reservoir. A tall oil pitch soap is added as a polymeric mobility control agent. (DMC)

Characterization of hydrocarbon-degrading and biosurfactant-producing Pseudomonas sp. P-1 strain as a potential tool for bioremediation of petroleum-contaminated soil.

PubMed

Pacwa-Płociniczak, Magdalena; Płaza, Grażyna Anna; Poliwoda, Anna; Piotrowska-Seget, Zofia

2014-01-01

The Pseudomonas sp. P-1 strain, isolated from heavily petroleum hydrocarbon-contaminated soil, was investigated for its capability to degrade hydrocarbons and produce a biosurfactant. The strain degraded crude oil, fractions A5 and P3 of crude oil, and hexadecane (27, 39, 27 and 13% of hydrocarbons added to culture medium were degraded, respectively) but had no ability to degrade phenanthrene. Additionally, the presence of gene-encoding enzymes responsible for the degradation of alkanes and naphthalene in the genome of the P-1 strain was reported. Positive results of blood agar and methylene blue agar tests, as well as the presence of gene rhl, involved in the biosynthesis of rhamnolipid, confirmed the ability of P-1 for synthesis of glycolipid biosurfactant. 1H and 13C nuclear magnetic resonance, Fourier transform infrared spectrum and mass spectrum analyses indicated that the extracted biosurfactant was affiliated with rhamnolipid. The results of this study indicate that the P-1 and/or biosurfactant produced by this strain have the potential to be used in bioremediation of hydrocarbon-contaminated soils.

Purification, characterisation and protective effects of polysaccharides from alfalfa on hepatocytes.

PubMed

Wang, Shaopu; Dong, Xiaofang; Ma, Hao; Cui, Yaoming; Tong, Jianming

2014-11-04

The objective of this study was to determine the preliminary characteristics and protective effects of alfalfa polysaccharides (APS) on hepatocytes in vitro. The crude APS was purified by DEAE-cellulose and Sephadex G-100 chromatography, resulting in the four purified fractions: APS-1, APS-2, APS-3 and APS-4. The results indicated that APS-3 had higher carbohydrate and uronic acid contents and that APS-4 had a more complicated monosaccharide composition compared to the other purified fractions. The average molecular weights of APS-1, APS-2, APS-3 and APS-4 were 48,536, 6,221, 66,559 and 13,076 Da, respectively. Furthermore, APS (crude and its purified fractions) restored the activities of antioxidant enzymes and increased the total antioxidant capacity of hepatocytes subjected to H2O2-induced oxidative stress. Furthermore, APS treatment counteracted the increases in lactic dehydrogenase and malonaldehyde in the culture supernatant. These results clearly demonstrate that APS possesses a protective effect against oxidative injury in hepatocytes. Copyright © 2014 Elsevier Ltd. All rights reserved.

Methane gas generation from waste water extraction process of crude palm oil in experimental digesters

NASA Astrophysics Data System (ADS)

Dillon, A.; Penafiel, R.; Garzón, P. V.; Ochoa, V.

2015-12-01

Industrial processes to extract crude palm oil, generates large amounts of waste water. High concentrations of COD, ST, SV, NH4 + and low solubility of O2, make the treatment of these effluents starts with anaerobic processes. The anaerobic digestion process has several advantages over aerobic degradation: lower operating costs (not aeration), low sludge production, methane gas generation. The 4 stages of anaerobic digestion are: hydrolysis, acidogenic, acetogenesis and methanogenesis. Through the action of enzymes synthesized by microbial consortia are met. The products of each step to serve as reagents is conducted as follows. The organic load times and cell hydraulic retention, solids content, nutrient availability, pH and temperature are factors that influence directly in biodigesters. The objectives of this presentation is to; characterize the microbial inoculum and water (from palm oil wasted water) to be used in biodigestores, make specific methanogenic activity in bioassays, acclimatize the microorganisms to produce methane gas using basal mineral medium with acetate for the input power, and to determine the production of methane gas digesters high organic load.