Mass Spectrometric Detection of Bacterial Protein Toxins and Their Enzymatic Activity.

PubMed

Kalb, Suzanne R; Boyer, Anne E; Barr, John R

2015-08-31

Mass spectrometry has recently become a powerful technique for bacterial identification. Mass spectrometry approaches generally rely upon introduction of the bacteria into a matrix-assisted laser-desorption time-of-flight (MALDI-TOF) mass spectrometer with mass spectrometric recognition of proteins specific to that organism that form a reliable fingerprint. With some bacteria, such as Bacillus anthracis and Clostridium botulinum, the health threat posed by these organisms is not the organism itself, but rather the protein toxins produced by the organisms. One such example is botulinum neurotoxin (BoNT), a potent neurotoxin produced by C. botulinum. There are seven known serotypes of BoNT, A-G, and many of the serotypes can be further differentiated into toxin variants, which are up to 99.9% identical in some cases. Mass spectrometric proteomic techniques have been established to differentiate the serotype or toxin variant of BoNT produced by varied strains of C. botulinum. Detection of potent biological toxins requires high analytical sensitivity and mass spectrometry based methods have been developed to determine the enzymatic activity of BoNT and the anthrax lethal toxins produced by B. anthracis. This enzymatic activity, unique for each toxin, is assessed with detection of the toxin-induced cleavage of strategically designed peptide substrates by MALDI-TOF mass spectrometry offering unparalleled specificity. Furthermore, activity assays allow for the assessment of the biological activity of a toxin and its potential health risk. Such methods have become important diagnostics for botulism and anthrax. Here, we review mass spectrometry based methods for the enzymatic activity of BoNT and the anthrax lethal factor toxin.

Characterization of selected cellulolytic activities of multi-enzymatic complex system from Penicillium funiculosum.

PubMed

Karboune, Salwa; Geraert, Pierre-André; Kermasha, Selim

2008-02-13

The presence of endo-1,4-beta-D-glucanase, cellobiohydrolase, and beta-glucosidase activities in a multi-enzymatic complex system from Penicillium funiculosum was investigated. The interesting feature of these enzymes is their synergistic action for the hydrolysis of the native cellulose into glucose units. Both endo-1,4-beta-D-glucanase and cellobiohydrolase showed broader pH activity profiles, with pH optima of 4.0 and 4.0-5.0, respectively. However, beta-glucosidase activity showed a narrow pH-activity profile, with an optimum pH of 4.5. The different cellulolytic activities were stable in the acidic pH range of 2.5-6.0 and showed a similar optimal temperature of 60 degrees C. Although beta-glucosidase has shown a close catalytic efficiency as that of endo-1,4-beta-D-glucanase, its thermal stability was lower. However, the thermal stability profile of cellobiohydrolase was close to that of endo-1,4-beta-D-glucanase. The results also revealed the presence of high levels of endo-1,3-1,4-beta-D-glucanase, endo-1,3-beta- d-glucanase, and pectinase activities in the multi-enzymatic cellulolytic complex system. Moreover, the investigated multi-enzymatic complex system was effective in degrading the nonstarch polysaccharides of soybean meal.

Enzymatic Activity of Candida spp. from Oral Cavity and Urine in Children with Nephrotic Syndrome.

PubMed

Olczak-Kowalczyk, Dorota; Roszkowska-Blaim, Maria; Dąbkowska, Maria; Swoboda-Kopeć, Ewa; Gozdowski, Dariusz; Mizerska-Wasiak, Małgorzata; Demkow, Urszula; Pańczyk-Tomaszewska, Małgorzata

2017-01-01

Oral colonization with Candida spp. is not synonymous with a systemic active infection. The aim of the study was to evaluate enzymatic activity of Candida strains isolated from the oral cavity in patients with nephrotic syndrome (NS) and to compare it with the activity determined in urine. We studied 32 children with NS and 26 control healthy children. Children with NS were treated with glucocorticosteroids, cyclosporin A, mycophenolate mofetil or azathioprine. In all children, API-ZYM enzymatic tests were performed to evaluate hydrolytic enzymes of Candida isolated from the oral cavity and in urine. Candida spp. were isolated from the oral cavity in 11 patients with NS (34.4%), all receiving immunosuppressive treatment. All strains produced valine arylamidase, 9 alpha-glucosidase (E16), and 9 N-acetyl-beta-glucosaminidase (E18). A positive correlation between the presence of Candida in the oral cavity and E16 and E18 enzymatic activity in both oral cavity and urine was found. A dose of cyclosporin A had an effect on the enzymatic activity (p < 0.05). We conclude that immunosuppressive treatment of NS in children may predispose to systemic Candida invasion. The results of this study suggest that oral candida infection should be monitored in children with nephrotic syndrome, particularly those treated with immunosuppressive agents.

The Membrane-anchored Serine Protease Prostasin (CAP1/PRSS8) Supports Epidermal Development and Postnatal Homeostasis Independent of Its Enzymatic Activity*

PubMed Central

Peters, Diane E.; Szabo, Roman; Friis, Stine; Shylo, Natalia A.; Uzzun Sales, Katiuchia; Holmbeck, Kenn; Bugge, Thomas H.

2014-01-01

The membrane-anchored serine protease prostasin (CAP1/PRSS8) is part of a cell surface proteolytic cascade that is essential for epithelial barrier formation and homeostasis. Here, we report the surprising finding that prostasin executes these functions independent of its own enzymatic activity. Prostasin null (Prss8−/−) mice lack barrier formation and display fatal postnatal dehydration. In sharp contrast, mice homozygous for a point mutation in the Prss8 gene, which causes the substitution of the active site serine within the catalytic histidine-aspartate-serine triad with alanine and renders prostasin catalytically inactive (Prss8Cat−/Cat− mice), develop barrier function and are healthy when followed for up to 20 weeks. This striking difference could not be explained by genetic modifiers or by maternal effects, as these divergent phenotypes were displayed by Prss8−/− and Prss8Cat−/Cat− mice born within the same litter. Furthermore, Prss8Cat−/Cat− mice were able to regenerate epidermal covering following cutaneous wounding. This study provides the first demonstration that essential in vivo functions of prostasin are executed by a non-enzymatic activity of this unique membrane-anchored serine protease. PMID:24706745

Mass Spectrometric Detection of Bacterial Protein Toxins and Their Enzymatic Activity

PubMed Central

Kalb, Suzanne R.; Boyer, Anne E.; Barr, John R.

2015-01-01

Mass spectrometry has recently become a powerful technique for bacterial identification. Mass spectrometry approaches generally rely upon introduction of the bacteria into a matrix-assisted laser-desorption time-of-flight (MALDI-TOF) mass spectrometer with mass spectrometric recognition of proteins specific to that organism that form a reliable fingerprint. With some bacteria, such as Bacillus anthracis and Clostridium botulinum, the health threat posed by these organisms is not the organism itself, but rather the protein toxins produced by the organisms. One such example is botulinum neurotoxin (BoNT), a potent neurotoxin produced by C. botulinum. There are seven known serotypes of BoNT, A–G, and many of the serotypes can be further differentiated into toxin variants, which are up to 99.9% identical in some cases. Mass spectrometric proteomic techniques have been established to differentiate the serotype or toxin variant of BoNT produced by varied strains of C. botulinum. Detection of potent biological toxins requires high analytical sensitivity and mass spectrometry based methods have been developed to determine the enzymatic activity of BoNT and the anthrax lethal toxins produced by B. anthracis. This enzymatic activity, unique for each toxin, is assessed with detection of the toxin-induced cleavage of strategically designed peptide substrates by MALDI-TOF mass spectrometry offering unparalleled specificity. Furthermore, activity assays allow for the assessment of the biological activity of a toxin and its potential health risk. Such methods have become important diagnostics for botulism and anthrax. Here, we review mass spectrometry based methods for the enzymatic activity of BoNT and the anthrax lethal factor toxin. PMID:26404376

A Survey of Enzymatic Activity in Commercially Available Pool and Spa Products

USDA-ARS?s Scientific Manuscript database

Many pool water treatment products currently available commercially claim that they work effectively by possessing enzyme activity (specifically lipase) that degrades common oil (lipid) contaminants found in pool water. Currently, there is no standard in measuring the enzymatic activity of these enz...

Enzymatic hydrolysis of oleuropein from Olea europea (olive) leaf extract and antioxidant activities.

PubMed

Yuan, Jiao-Jiao; Wang, Cheng-Zhang; Ye, Jian-Zhong; Tao, Ran; Zhang, Yu-Si

2015-02-11

Oleuropein (OE), the main polyphenol in olive leaf extract, is likely to decompose into hydroxytyrosol (HT) and elenolic acid under the action of light, acid, base, high temperature. In the enzymatic process, the content of OE in olive leaf extract and enzyme are key factors that affect the yield of HT. A selective enzyme was screened from among 10 enzymes with a high OE degradation rate. A single factor (pH, temperature, time, enzyme quantity) optimization process and a Box-Behnken design were studied for the enzymatic hydrolysis of 81.04% OE olive leaf extract. Additionally, enzymatic hydrolysis results with different substrates (38.6% and 81.04% OE) were compared and the DPPH antioxidant properties were also evaluated. The result showed that the performance of hydrolysis treatments was best using hemicellulase as a bio-catalyst, and the high purity of OE in olive extract was beneficial to biotransform OE into HT. The optimal enzymatic conditions for achieving a maximal yield of HT content obtained by the regression were as follows: pH 5, temperature 55 °C and enzyme quantity 55 mg. The experimental result was 11.31% ± 0.15%, and the degradation rate of OE was 98.54%. From the present investigation of the antioxidant activity determined by the DPPH method, the phenol content and radical scavenging effect were both decreased after enzymatic hydrolysis by hemicellulase. However, a high antioxidant activity of the ethyl acetate extract enzymatic hydrolysate (IC50 = 41.82 μg/mL) was demonstated. The results presented in this work suggested that hemicellulase has promising and attractive properties for industrial production of HT, and indicated that HT might be a valuable biological component for use in pharmaceutical products and functional foods.

The Impact of Marine Enzymatic Activity on Sea Spray Aerosol Properties

NASA Astrophysics Data System (ADS)

Ryder, O. S.; Michaud, J. M.; Sauer, J. S.; Lee, C.; Förster, J. D.; Pöhlker, C.; Andreae, M. O.; Prather, K. A.

2016-12-01

The composition of sea spray aerosol (SSA) and the relationship between its organic fraction and biological ocean conditions is not well understood, resulting in considerable disagreement in the literature linking biological markers to SSA chemical composition. Recent work suggests that enzymatic activity in seawater may play a key role in dictating aerosol composition by changing the organic pool from which SSA is formed. Here we investigate the role of enzymatic activity on SSA spatial chemical composition, aerosol phase and morphological microstructure. In these experiments, SSA was generated using a novel mini-Marine Aerosol Reference Tank system. SSA collected onto substrates was generated from artificial salt water that had been doped with either 1) unsaturated triglycerides or 2) diatom cellular lysate, both followed by lipase. Results from analysis including morphological studies via atomic force microscopy, and chemical composition investigations both under dry and RH conditions via STXM-NEXAFS are presented.

Neurotoxicity and other pharmacological activities of the snake venom phospholipase A2 OS2: The N-terminal region is more important than enzymatic activity

PubMed Central

Rouault, Morgane; Rash, Lachlan D.; Escoubas, Pierre; Boilard, Eric; Bollinger, James; Lomonte, Bruno; Maurin, Thomas; Guillaume, Carole; Canaan, Stéphane; Deregnaucourt, Christiane; Schrével, Joseph; Doglio, Alain; Gutiérrez, José María; Lazdunski, Michel; Gelb, Michael H.; Lambeau, Gérard

2009-01-01

Several snake venom secreted phospholipases A2 (sPLA2s) including OS2 exert a variety of pharmacological effects ranging from central neurotoxicity to anti-HIV activity by mechanisms that are not yet fully understood. To conclusively address the role of enzymatic activity and map the key structural elements of OS2 responsible for its pharmacological properties, we have prepared single point OS2 mutants at the catalytic site and large chimeras between OS2 and OS1, an homologous but non toxic sPLA2. Most importantly, we found that the enzymatic activity of the active site mutant H48Q is 500-fold lower than that of the wild-type protein, while central neurotoxicity is only 16-fold lower, providing convincing evidence that catalytic activity is at most a minor factor that determines central neurotoxicity. The chimera approach has identified the N-terminal region (residues 1–22) of OS2, but not the central one (residues 58–89), as crucial for both enzymatic activity and pharmacological effects. The C-terminal region of OS2 (residues 102–119) was found to be critical for enzymatic activity, but not for central neurotoxicity and anti-HIV activity, allowing us to further dissociate enzymatic activity and pharmacological effects. Finally, direct binding studies with the C-terminal chimera which poorly binds to phospholipids while it is still neurotoxic, led to the identification of a subset of brain N-type receptors which may be directly involved in central neurotoxicity. PMID:16669624

Enzymatic activity necessary to restore the lethality due to Escherichia coli RNase E deficiency is distributed among bacteria lacking RNase E homologues

PubMed Central

Kageyama, Daisuke; Honda, Naoko; Fujimoto, Hirofumi; Kato, Atsushi

2017-01-01

Escherichia coli RNase E (Eco-RNase E), encoded by rne (Eco-rne), is considered the global RNA decay initiator. Although Eco-RNase E is an essential gene product in E. coli, some bacterial species, such as Bacillus subtilis, do not possess Eco-RNase E sequence homologues. B. subtilis instead possesses RNase J1/J2 (Bsu-RNase J1/J2) and RNase Y (Bsu-RNase Y) to execute RNA decay. Here we found that E. coli lacking the Eco-rne gene (Δrne E. coli) was viable conditional on M9 minimal media by introducing Bsu-RNase J1/J2 or Bsu-RNase Y. We also cloned an extremely short Eco-RNase E homologue (Wpi-RNase E) and a canonical sized Bsu-RNase J1/J2 homologue (Wpi-RNase J) from Wolbachia pipientis, an α-proteobacterial endosymbiont of arthropods. We found that Wpi-RNase J restored the colony-forming ability (CFA) of Δrne E. coli, whereas Wpi-RNase E did not. Unexpectedly, Wpi-RNase E restored defective CFA due to lack of Eco-RNase G, a paralogue of Eco-RNase E. Our results indicate that bacterial species that lack Eco-RNase E homologues or bacterial species that possess Eco-RNase E homologues which lack Eco-RNase E-like activities have a modest Eco-RNase E-like function using RNase J and/or RNase Y. These results suggest that Eco-RNase E-like activities might distribute among a wide array of bacteria and that functions of RNases may have changed dynamically during evolutionary divergence of bacterial lineages. PMID:28542621

Analysis of the link between the redox state and enzymatic activity of the HtrA (DegP) protein from Escherichia coli.

PubMed

Koper, Tomasz; Polit, Agnieszka; Sobiecka-Szkatula, Anna; Wegrzyn, Katarzyna; Scire, Andrea; Figaj, Donata; Kadzinski, Leszek; Zarzecka, Urszula; Zurawa-Janicka, Dorota; Banecki, Bogdan; Lesner, Adam; Tanfani, Fabio; Lipinska, Barbara; Skorko-Glonek, Joanna

2015-01-01

Bacterial HtrAs are proteases engaged in extracytoplasmic activities during stressful conditions and pathogenesis. A model prokaryotic HtrA (HtrA/DegP from Escherichia coli) requires activation to cleave its substrates efficiently. In the inactive state of the enzyme, one of the regulatory loops, termed LA, forms inhibitory contacts in the area of the active center. Reduction of the disulfide bond located in the middle of LA stimulates HtrA activity in vivo suggesting that this S-S bond may play a regulatory role, although the mechanism of this stimulation is not known. Here, we show that HtrA lacking an S-S bridge cleaved a model peptide substrate more efficiently and exhibited a higher affinity for a protein substrate. An LA loop lacking the disulfide was more exposed to the solvent; hence, at least some of the interactions involving this loop must have been disturbed. The protein without S-S bonds demonstrated lower thermal stability and was more easily converted to a dodecameric active oligomeric form. Thus, the lack of the disulfide within LA affected the stability and the overall structure of the HtrA molecule. In this study, we have also demonstrated that in vitro human thioredoxin 1 is able to reduce HtrA; thus, reduction of HtrA can be performed enzymatically.

Show Yourself, Asparaginase: An Enzymatic Reaction Explained through a Hands-On Interactive Activity

PubMed Central

2017-01-01

Determining the catalytic activity of an enzyme can be the perfect method for its identification, for example during purification procedures or for isolation purposes. Herein, we used a pharmaceutically relevant protein to bring the concept of enzymatic activity to the classroom. We designed a hands-on interactive activity in which a medically relevant enzyme, asparaginase, was distinguished from a nonenzymatic protein based on its specific enzymatic activity. The experiment was carried out in the classroom, designed to impact different educational levels from elementary to high school. Our main purposes were to promote the emerging field of protein-based drugs as a source of scientific careers in bionanotechnology and to show the students an image of a “scientist” as that of a common and educated person working in an exciting profession. In addition of being inexpensive, this activity proved to be adaptable for various educational levels and can be easily implemented in different scenarios, for example, scientific fairs, some schools, and so forth. PMID:29599566

Binding of Nickel to Testicular Glutamate–Ammonia Ligase Inhibits Its Enzymatic Activity

PubMed Central

SUN, YINGBIAO; OU, YOUNG; CHENG, MIN; RUAN, YIBING; VAN DER HOORN, FRANS A.

2016-01-01

SUMMARY Exposure to nickel has been shown to cause damage to the testis in several animal models. It is not known if the testis expresses protein(s) that can bind nickel. To test this, we used a nickel-binding assay to isolate testicular nickel-binding proteins. We identified glutamate–ammonia ligase (GLUL) as a prominent nickel-binding protein by mass spectrometry. Protein analysis and reverse transcriptase polymerase chain reaction showed that GLUL is expressed in the testis, predominantly in interstitial cells. We determined that GLUL has a higher affinity for nickel than for its regular co-factor manganese. We produced an enzymatically active, recombinant GLUL protein. Upon binding, nickel interferes with the manganese-catalyzed enzymatic activity of recombinant GLUL protein. We also determined that GLUL activity in testes of animals exposed to nickel sulfate is reduced. Our results identify testicular GLUL as the first testicular protein shown to be affected by nickel exposure. PMID:21254280

Constraints imposed by transmembrane domains affect enzymatic activity of membrane-associated human CD39/NTPDase1 mutants.

PubMed

Musi, Elgilda; Islam, Naziba; Drosopoulos, Joan H F

2007-05-01

Human CD39/NTPDase1 is an endothelial cell membrane-associated nucleotidase. Its large extracellular domain rapidly metabolizes nucleotides, especially ADP released from activated platelets, inhibiting further platelet activation/recruitment. Previous studies using our recombinant soluble CD39 demonstrated the importance of residues S57, D54, and D213 for enzymatic/biological activity. We now report effects of S57A, D54A, and D213A mutations on full-length (FL)CD39 function. Enzymatic activity of alanine modified FLCD39s was less than wild-type, contrasting the enhanced activity of their soluble counterparts. Furthermore, conservative substitutions D54E and D213E led to enzymes with activities greater than the alanine modified FLCD39s, but less than wild-type. Reductions in mutant activities were primarily associated with reduced catalytic rates. Differences in enzymatic activity were not attributable to gross changes in the nucleotide binding pocket or the enzyme's ability to multimerize. Thus, composition of the active site of wild-type CD39 appears optimized for ADPase function in the context of the transmembrane domains.

A scalable lysyl hydroxylase 2 expression system and luciferase-based enzymatic activity assay

PubMed Central

Guo, Hou-Fu; Cho, Eun Jeong; Devkota, Ashwini K.; Chen, Yulong; Russell, William; Phillips, George N.; Yamauchi, Mitsuo; Dalby, Kevin; Kurie, Jonathan M.

2017-01-01

Hydroxylysine aldehyde-derived collagen cross-links (HLCCs) accumulate in fibrotic tissues and certain types of cancer and are thought to drive the progression of these diseases. HLCC formation is initiated by lysyl hydroxylase 2 (LH2), an Fe(II) and α-ketoglutarate (αKG)-dependent oxygenase that hydroxylates telopeptidyl lysine residues on collagen. Development of LH2 antagonists for the treatment of these diseases will require a reliable source of recombinant LH2 protein and a non-radioactive LH2 enzymatic activity assay that is amenable to high throughput screens of small molecule libraries. However, LH2 protein generated previously using E coli– or insect-based expression systems was either insoluble or enzymatically unstable, and LH2 enzymatic activity assays have measured radioactive CO2 released from 14C-labeled αKG during its conversion to succinate. To address these deficiencies, we have developed a scalable process to purify human LH2 protein from Chinese hamster ovary cell-derived conditioned media samples and a luciferase-based assay that quantifies LH2-dependent conversion of αKG to succinate. These methodologies may be applicable to other Fe(II) and αKG-dependent oxygenase systems. PMID:28216326

Continuous monitoring of enzymatic activity within native electrophoresis gels: Application to mitochondrial oxidative phosphorylation complexes

PubMed Central

Covian, Raul; Chess, David; Balaban, Robert S.

2012-01-01

Native gel electrophoresis allows the separation of very small amounts of protein complexes while retaining aspects of their activity. In-gel enzymatic assays are usually performed by using reaction-dependent deposition of chromophores or light scattering precipitates quantified at fixed time points after gel removal and fixation, limiting the ability to analyze enzyme reaction kinetics. Herein, we describe a custom reaction chamber with reaction media recirculation and filtering and an imaging system that permits the continuous monitoring of in-gel enzymatic activity even in the presence of turbidity. Images were continuously collected using time-lapse high resolution digital imaging, and processing routines were developed to obtain kinetic traces of the in-gel activities and analyze reaction time courses. This system also permitted the evaluation of enzymatic activity topology within the protein bands of the gel. This approach was used to analyze the reaction kinetics of two mitochondrial complexes in native gels. Complex IV kinetics showed a short initial linear phase where catalytic rates could be calculated, whereas Complex V activity revealed a significant lag phase followed by two linear phases. The utility of monitoring the entire kinetic behavior of these reactions in native gels, as well as the general application of this approach, is discussed. PMID:22975200

Continuous monitoring of enzymatic activity within native electrophoresis gels: application to mitochondrial oxidative phosphorylation complexes.

PubMed

Covian, Raul; Chess, David; Balaban, Robert S

2012-12-01

Native gel electrophoresis allows the separation of very small amounts of protein complexes while retaining aspects of their activity. In-gel enzymatic assays are usually performed by using reaction-dependent deposition of chromophores or light-scattering precipitates quantified at fixed time points after gel removal and fixation, limiting the ability to analyze the enzyme reaction kinetics. Herein, we describe a custom reaction chamber with reaction medium recirculation and filtering and an imaging system that permits the continuous monitoring of in-gel enzymatic activity even in the presence of turbidity. Images were continuously collected using time-lapse high-resolution digital imaging, and processing routines were developed to obtain kinetic traces of the in-gel activities and analyze reaction time courses. This system also permitted the evaluation of enzymatic activity topology within the protein bands of the gel. This approach was used to analyze the reaction kinetics of two mitochondrial complexes in native gels. Complex IV kinetics showed a short initial linear phase in which catalytic rates could be calculated, whereas Complex V activity revealed a significant lag phase followed by two linear phases. The utility of monitoring the entire kinetic behavior of these reactions in native gels, as well as the general application of this approach, is discussed. Published by Elsevier Inc.

Changes in the enzymatic activity of soil samples upon their storage

NASA Astrophysics Data System (ADS)

Dadenko, E. V.; Kazeev, K. Sh.; Kolesnikov, S. I.; Val'Kov, V. F.

2009-12-01

The influence of the duration and conditions of storage of soil samples on the activity of soil enzymes (catalase, β-fructofuranosidase, and dehydrogenase) was studied for the main soils of southern Russia (different subtypes of chernozems, chestnut soils, brown forest soils, gray forest soils, solonetzes, and solonchaks). The following soil storage conditions were tested: (1) the air-dry state at room temperature, (2) the airdry state at a low positive (in a refrigerator, +4°C) temperature, (3) naturally moist samples at a low positive temperature, and (4) naturally moist samples at a negative (in a freezer, -5°C) temperature. It was found that the sample storing caused significant changes in the enzymatic activities, which depended on the soil type, the land use, the type of enzyme, and the duration and conditions of the sample storage. In the course of the storage, the changes in the enzymatic activity had a nonlinear character. The maximum changes were observed in the initial period (up to 12 weeks). Then, a very gradual decrease in the activity of the studied enzymes was observed. Upon the long-term (>12 weeks) storage under the different conditions, the difference in the activities of the soil enzymes became less pronounced. The storage of soil samples in the air-dried state at room temperature can be recommended for mass investigations.

Respiration and enzymatic activities as indicators of stabilization of sewage sludge composting.

PubMed

Nikaeen, Mahnaz; Nafez, Amir Hossein; Bina, Bijan; Nabavi, BiBi Fatemeh; Hassanzadeh, Akbar

2015-05-01

The objective of this work was to study the evolution of physico-chemical and microbial parameters in the composting process of sewage sludge (SS) with pruning wastes (PW) in order to compare these parameters with respect to their applicability in the evaluation of organic matter (OM) stabilization. To evaluate the composting process and organic matter stability, different microbial activities were compared during composting of anaerobically digested SS with two volumetric ratios, 1:1 and 3:1 of PW:SS and two aeration techniques including aerated static piles (ASP) and turned windrows (TW). Dehydrogenase activity, fluorescein diacetate hydrolysis, and specific oxygen uptake rate (SOUR) were used as microbial activity indices. These indices were compared with traditional parameters, including temperature, pH, moisture content, organic matter, and C/N ratio. The results showed that the TW method and 3:1 (PW:SS) proportion was superior to the ASP method and 1:1 proportion, since the former accelerate the composting process by catalyzing the OM stabilization. Enzymatic activities and SOUR, which reflect microbial activity, correlated well with temperature fluctuations. Based on these results it appears that SOUR and the enzymatic activities are useful parameters to monitor the stabilization of SS compost. Copyright © 2015 Elsevier Ltd. All rights reserved.

Enzymatic properties of a GH19 chitinase isolated from rice lacking a major loop structure involved in chitin binding.

PubMed

Tanaka, Jun; Fukamizo, Tamo; Ohnuma, Takayuki

2017-05-01

The catalytic domains of family GH19 chitinases have been found to consist of a conserved, α-helical core-region and different numbers (1-6) of loop structures, located at both ends of the substrate-binding groove and which extend over the glycon- and aglycon-binding sites. We expressed, purified and enzymatically characterized a GH19 chitinase from rice, Oryza sativa L. cv. Nipponbare (OsChia2a), lacking a major loop structure (loop III) connected to the functionally important β-stranded region. The new enzyme thus contained the five remaining loop structures (loops I, II, IV, V and C-term). The OsChia2a recombinant protein catalyzed hydrolysis of chitin oligosaccharides, (GlcNAc)n (n = 3-6), with inversion of anomeric configuration, indicating that OsChia2a correctly folded without loop III. From thermal unfolding experiments and calorimetric titrations using the inactive OsChia2a mutant (OsChia2a-E68Q), in which the catalytic residue Glu68 was mutated to glutamine, we found that the binding affinities towards (GlcNAc)n (n = 2-6) were almost proportional to the degree of polymerization of (GlcNAc)n, but were much lower than those obtained for a moss GH19 chitinase having only loop III [Ohnuma T, Sørlie M, Fukuda T, Kawamoto N, Taira T, Fukamizo T. 2011. Chitin oligosaccharide binding to a family GH19 chitinase from the moss, Bryum coronatum. FEBS J. 278:3991-4001]. Nevertheless, OsChia2a exhibited significant antifungal activity. It appears that loop III connected to the β-stranded region is important for (GlcNAc)n binding, but is not essential for antifungal activity. © The Author 2017. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Yeasts from sub-Antarctic region: biodiversity, enzymatic activities and their potential as oleaginous microorganisms.

PubMed

Martinez, A; Cavello, I; Garmendia, G; Rufo, C; Cavalitto, S; Vero, S

2016-09-01

Various microbial groups are well known to produce a range of extracellular enzymes and other secondary metabolites. However, the occurrence and importance of investment in such activities have received relatively limited attention in studies of Antarctic soil microbiota. Sixty-one yeasts strains were isolated from King George Island, Antarctica which were characterized physiologically and identified at the molecular level using the D1/D2 region of rDNA. Fifty-eight yeasts (belonging to the genera Cryptococcus, Leucosporidiella, Rhodotorula, Guehomyces, Candida, Metschnikowia and Debaryomyces) were screened for extracellular amylolytic, proteolytic, esterasic, pectinolytic, inulolytic xylanolytic and cellulolytic activities at low and moderate temperatures. Esterase activity was the most common enzymatic activity expressed by the yeast isolates regardless the assay temperature and inulinase was the second most common enzymatic activity. No cellulolytic activity was detected. One yeast identified as Guehomyces pullulans (8E) showed significant activity across six of seven enzymes types tested. Twenty-eight yeast isolates were classified as oleaginous, being the isolate 8E the strain that accumulated the highest levels of saponifiable lipids (42 %).

In situ nanoplasmonic probing of enzymatic activity of monolayer-confined glucose oxidase on colloidal nanoparticles.

PubMed

He, Haili; Xu, Xiaolong; Wu, Haoxi; Zhai, Yujuan; Jin, Yongdong

2013-05-07

In situ probing protein-particle interactions and activities of proteins on colloidal nanoparticle (NP) surfaces is a long-standing key challenge in understanding the nanobio interfaces and virtually important for a variety of biological and biomedical applications. The interactions of NPs with proteins, for instance, are known to form NP bioconjugates or protein coronas; protein surface immobilization and molecular layer-by-layer deposition techniques are widely used, but a clear understanding of the confinement effect on protein activity by molecular coating, at the monolayer level, remains poorly understood. We explore here a novel approach, using colloidal plasmonic nanocomplexes coated with glucose oxidase (GOx) as self-sensing nanoprobes for in situ optical probing of surface-confined enzymatic activity, which is at least 1-2 orders of magnitude more sensitive than standard colorimetric assays for detecting GOx activity. We found that enzymatic activity of monolayer-confined GOx on colloidal NPs was significantly enhanced as compared with free GOx (also proved by conformational changes from circular dichroism studies), with a low apparent Michaelis-Menten constant Km of ~0.115 mM and high turnover kcat/Km of ~8394 M(-1)·s(-1); compared with the "anchored-type" suspending GOx, the outmost polyelectrolyte monolayer-protected "sandwiched-type" GOx exhibits significantly improved enzymatic activities toward higher temperatures and wider pH range. This finding is of fundamental important and instructive for safe use of such nanomaterials for bioapplications.

Ship-borne measurements of microbial enzymatic activity: A rapid biochemical indicator for microbial water quality monitoring

NASA Astrophysics Data System (ADS)

Stadler, Philipp; Loken, Luke; Crawford, John; Schramm, Paul; Sorsa, Kirsti; Kuhn, Catherine; Savio, Domenico; Striegl, Rob; Butman, David; Stanley, Emily; Farnleitner, Andreas H.; Zessner, Matthias

2017-04-01

Contamination of aquatic ecosystems by human and animal wastes is a global concern for water quality. Disclosing fate and transport processes of fecal indicator organism (FIO) in large water bodies is a big challenge due to material intensive and time consuming methods used in microbiological water quality monitoring. In respect of utilization of large surface water resources there is a dearth of rapid microbiological methods that allow a near-real time health related water quality monitoring to be implemented into early warning systems. The detection of enzymatic activities has been proposed as a rapid surrogate for microbiological pollution monitoring of water and water resources (Cabral, 2010; Farnleitner et al., 2001, 2002). Methods such as the beta-D-Glucuronidase assay (GLUC), targeting FIO such as E. coli, were established. New automated enzymatic assays have been implemented during the last years into on-site monitoring stations, ranging from ground- to surface waters (Ryzinska-Paier et al., 2014; Stadler et al., 2017, 2016). While these automated enzymatic methods cannot completely replace assays for culture-based FIO enumeration, they yielded significant information on pollution events and temporal dynamics on a catchment specific basis, but were restricted to stationary measurements. For the first time we conducted ship-borne and automated measurements of enzymatic GLUC activity on large fresh water bodies, including the Columbia River, the Mississippi River and Lake Mendota. Not only are automated enzymatic assays technically feasible from a mobile vessel, but also can be used to localize point sources of potential microbial fecal contamination, such as tributaries or storm drainages. Spatial and temporal patterns of enzymatic activity were disclosed and the habitat specific correlation with microbiological standard assays for FIO determined due to reference samples. The integration of rapid and automated enzymatic assays into well-established systems

Conditional iron and pH-dependent activity of a non-enzymatic glycolysis and pentose phosphate pathway.

PubMed

Keller, Markus A; Zylstra, Andre; Castro, Cecilia; Turchyn, Alexandra V; Griffin, Julian L; Ralser, Markus

2016-01-01

Little is known about the evolutionary origins of metabolism. However, key biochemical reactions of glycolysis and the pentose phosphate pathway (PPP), ancient metabolic pathways central to the metabolic network, have non-enzymatic pendants that occur in a prebiotically plausible reaction milieu reconstituted to contain Archean sediment metal components. These non-enzymatic reactions could have given rise to the origin of glycolysis and the PPP during early evolution. Using nuclear magnetic resonance spectroscopy and high-content metabolomics that allowed us to measure several thousand reaction mixtures, we experimentally address the chemical logic of a metabolism-like network constituted from these non-enzymatic reactions. Fe(II), the dominant transition metal component of Archean oceanic sediments, has binding affinity toward metabolic sugar phosphates and drives metabolism-like reactivity acting as both catalyst and cosubstrate. Iron and pH dependencies determine a metabolism-like network topology and comediate reaction rates over several orders of magnitude so that the network adopts conditional activity. Alkaline pH triggered the activity of the non-enzymatic PPP pendant, whereas gentle acidic or neutral conditions favored non-enzymatic glycolytic reactions. Fe(II)-sensitive glycolytic and PPP-like reactions thus form a chemical network mimicking structural features of extant carbon metabolism, including topology, pH dependency, and conditional reactivity. Chemical networks that obtain structure and catalysis on the basis of transition metals found in Archean sediments are hence plausible direct precursors of cellular metabolic networks.

Conditional iron and pH-dependent activity of a non-enzymatic glycolysis and pentose phosphate pathway

PubMed Central

Keller, Markus A.; Zylstra, Andre; Castro, Cecilia; Turchyn, Alexandra V.; Griffin, Julian L.; Ralser, Markus

2016-01-01

Little is known about the evolutionary origins of metabolism. However, key biochemical reactions of glycolysis and the pentose phosphate pathway (PPP), ancient metabolic pathways central to the metabolic network, have non-enzymatic pendants that occur in a prebiotically plausible reaction milieu reconstituted to contain Archean sediment metal components. These non-enzymatic reactions could have given rise to the origin of glycolysis and the PPP during early evolution. Using nuclear magnetic resonance spectroscopy and high-content metabolomics that allowed us to measure several thousand reaction mixtures, we experimentally address the chemical logic of a metabolism-like network constituted from these non-enzymatic reactions. Fe(II), the dominant transition metal component of Archean oceanic sediments, has binding affinity toward metabolic sugar phosphates and drives metabolism-like reactivity acting as both catalyst and cosubstrate. Iron and pH dependencies determine a metabolism-like network topology and comediate reaction rates over several orders of magnitude so that the network adopts conditional activity. Alkaline pH triggered the activity of the non-enzymatic PPP pendant, whereas gentle acidic or neutral conditions favored non-enzymatic glycolytic reactions. Fe(II)-sensitive glycolytic and PPP-like reactions thus form a chemical network mimicking structural features of extant carbon metabolism, including topology, pH dependency, and conditional reactivity. Chemical networks that obtain structure and catalysis on the basis of transition metals found in Archean sediments are hence plausible direct precursors of cellular metabolic networks. PMID:26824074

Non-enzymatic glycation reduces heparin cofactor II anti-thrombin activity.

PubMed

Ceriello, A; Marchi, E; Barbanti, M; Milani, M R; Giugliano, D; Quatraro, A; Lefebvre, P

1990-04-01

The effects of non-enzymatic glycation on heparin cofactor II activity, at glucose concentrations which might be expected in physiological or diabetic conditions have been evaluated in this study. Radiolabelled glucose incorporation was associated with a loss of heparin cofactor anti-thrombin activity. The heparin cofactor heparin and dermatan sulfate-dependent inhibition of thrombin was significantly reduced, showing a remarkable decrease of the maximum second order rate constant. This study shows that heparin cofactor can be glycated at glucose concentrations found in the blood, and that this phenomenon produces a loss of heparin cofactor-antithrombin activity. These data suggest, furthermore, a possible link between heparin cofactor glycation and the pathogenesis of thrombosis in diabetes mellitus.

Novel, dually radiolabeled peptides for simultaneous monitoring of enzymatic activity and protein targets

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

Efrem Mebrahtu, Suzanne Lapi

2012-12-13

This application investigated a novel imaging approach to develop methods to incorporate multiple radionuclides into a single peptide at chemoselective sites for simultaneous monitoring of cell-bound protein targets as well as specific enzymatic activity, both of which are associated with enhanced tumor growth and metastasis. This imaging construct was synthesized in such a manner so that the PET radionuclide will remain associated with the tumor cells and the SPECT radionuclide was cleaved from the imaging agent. Measurement of the PET agent only will yield information about the tumor marker density while measurement of the amount of co-localization and mismatch ofmore » the two radionuclides will yield information about the enzymatic activity. This coincident measuring technique using both PET and SPECT agents allows us to draw correlations involving the interactions of enzymes (cathepsin, serine-protease urokinase (uPA) and matrix metalloproteases) and other cellular proteins which play a role in cancer growth and metastasis. This technique will allow for studies in xenograft or genetic models of cancer in the same animal at the same time, thus eliminating problems that may occur when trying to invoke comparisons across animals or timepoints. By using radionuclide imaging as opposed to other imaging modalities, this technique has the potential to be translatable and can exploit the high specific activity probes which can be generated with radiotracers. The proof of principle test of this system investigated simultaneous monitoring of matrix metalloprotease (MMP) activity in the extracellular matrix (ECM) as well as density of integrins on the cell surface, both of which can serve as tumor markers. The outcomes/deliverables of this project were as follows: 1. Peptides were synthesized dually labeled at chemospecific sites with PET and SPECT agents. 2. Stability (intrinsic and to radiolysis) and specific activity of these labeled compounds were determined. 3

Membrane Phospholipid Augments Cytochrome P4501a Enzymatic Activity by Modulating Structural Conformation during Detoxification of Xenobiotics

PubMed Central

Ghosh, Manik C.; Ray, Arun K.

2013-01-01

Cytochrome P450 is a superfamily of membrane-bound hemoprotein that gets involved with the degradation of xenobiotics and internal metabolites. Accumulated body of evidence indicates that phospholipids play a crucial role in determining the enzymatic activity of cytochrome P450 in the microenvironment by modulating its structure during detoxification; however, the structure-function relationship of cytochrome P4501A, a family of enzymes responsible for degrading lipophilic aromatic hydrocarbons, is still not well defined. Inducibility of cytochrome P4501A in cultured catfish hepatocytes in response to carbofuran, a widely used pesticide around the world, was studied earlier in our laboratory. In this present investigation, we observed that treating catfish with carbofuran augmented total phospholipid in the liver. We examined the role of phospholipid on the of cytochrome P4501A-marker enzyme which is known as ethoxyresorufin-O-deethylase (EROD) in the context of structure and function. We purified the carbofuran-induced cytochrome P4501A protein from catfish liver. Subsequently, we examined the enzymatic activity of purified P4501A protein in the presence of phospholipid, and studied how the structure of purified protein was influenced in the phospholipid environment. Membrane phospholipid appeared to accelerate the enzymatic activity of EROD by changing its structural conformation and thus controlling the detoxification of xenobiotics. Our study revealed the missing link of how the cytochrome P450 restores its enzymatic activity by changing its structural conformation in the phospholipid microenvironment. PMID:23469105

Membrane phospholipid augments cytochrome P4501a enzymatic activity by modulating structural conformation during detoxification of xenobiotics.

PubMed

Ghosh, Manik C; Ray, Arun K

2013-01-01

Cytochrome P450 is a superfamily of membrane-bound hemoprotein that gets involved with the degradation of xenobiotics and internal metabolites. Accumulated body of evidence indicates that phospholipids play a crucial role in determining the enzymatic activity of cytochrome P450 in the microenvironment by modulating its structure during detoxification; however, the structure-function relationship of cytochrome P4501A, a family of enzymes responsible for degrading lipophilic aromatic hydrocarbons, is still not well defined. Inducibility of cytochrome P4501A in cultured catfish hepatocytes in response to carbofuran, a widely used pesticide around the world, was studied earlier in our laboratory. In this present investigation, we observed that treating catfish with carbofuran augmented total phospholipid in the liver. We examined the role of phospholipid on the of cytochrome P4501A-marker enzyme which is known as ethoxyresorufin-O-deethylase (EROD) in the context of structure and function. We purified the carbofuran-induced cytochrome P4501A protein from catfish liver. Subsequently, we examined the enzymatic activity of purified P4501A protein in the presence of phospholipid, and studied how the structure of purified protein was influenced in the phospholipid environment. Membrane phospholipid appeared to accelerate the enzymatic activity of EROD by changing its structural conformation and thus controlling the detoxification of xenobiotics. Our study revealed the missing link of how the cytochrome P450 restores its enzymatic activity by changing its structural conformation in the phospholipid microenvironment.

Effect of wheat and Miscanthus straw biochars on soil enzymatic activity, ecotoxicity, and plant yield

NASA Astrophysics Data System (ADS)

Mierzwa-Hersztek, Monika; Gondek, Krzysztof; Klimkowicz-Pawlas, Agnieszka; Baran, Agnieszka

2017-07-01

The variety of technological conditions and raw materials from which biochar is produced is the reason why its soil application may have different effects on soil properties and plant growth. The aim of this study was to evaluate the effect of the addition of wheat straw and Miscanthus giganteus straw (5 t DM ha-1) and biochar obtained from this materials in doses of 2.25 and 5 t DM ha-1 on soil enzymatic activity, soil ecotoxicity, and plant yield (perennial grass mixture with red clover). The research was carried out under field conditions on soil with the granulometric composition of loamy sand. No significant effect of biochar amendment on soil enzymatic activity was observed. The biochar-amended soil was toxic to Vibrio fischeri and exhibited low toxicity to Heterocypris incongruens. Application of wheat straw biochar and M. giganteus straw biochar in a dose of 5 t DM ha-1 contributed to an increase in plant biomass production by 2 and 14%, respectively, compared to the soil with mineral fertilisation. Biochars had a more adverse effect on soil enzymatic activity and soil ecotoxicity to H. incongruens and V. fischeri than non-converted wheat straw and M. giganteus straw, but significantly increased the grass crop yield.

Antioxidant and Cytoprotective Activities of Enzymatic Extracts from Rhizoid of Laminaria japonica

PubMed Central

Je, Jae-Young; Park, Soo Yeon; Ahn, Chang-Bum

2017-01-01

Rhizoid of Laminaria japonica was hydrolyzed with proteases and carbohydrases to obtain antioxidant materials. Oxygen radical absorbance capacity (ORAC) of the enzymatic extracts was evaluated and the Protamex extract (PE) exhibited the highest ORAC value. PE also potently scavenged 2,2-diphenyl-1-picrylhydrazyl radical, 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulphonic) acid cation radical, and hydrogen peroxide (H2O2) and had good reducing power. PE inhibited hydroxyl radical-induced DNA scission by measuring the conversion of supercoiled pBR322 plasmid DNA to the open circular form. The cytoprotective effect of PE against H2O2-induced hepatic cell damage was also investigated. PE showed a dose-dependent cytoprotective effect in cultured hepatocytes by inhibiting intracellular reactive oxygen species scavenging activity. In addition, PE up-regulated the expression of heme oxygenase-1, which is a cytoprotective enzyme, by activating translocation of nuclear factor-erythroid 2-related factor 2. Taken together, the enzymatic extract of rhizoid of L. japonica, particularly PE, may be useful for antioxidant additives. PMID:29333384

Unraveling the Enzymatic Activity of Oxygenated Carbon Nanotubes and Their Application in the Treatment of Bacterial Infections.

PubMed

Wang, Huan; Li, Penghui; Yu, Dongqin; Zhang, Yan; Wang, Zhenzhen; Liu, Chaoqun; Qiu, Hao; Liu, Zhen; Ren, Jinsong; Qu, Xiaogang

2018-05-17

Carbon nanotubes (CNTs) and their derivatives have emerged as a series of efficient biocatalysts to mimic the function of natural enzymes in recent years. However, the unsatisfiable enzymatic efficiency usually limits their practical usage ranging from materials science to biotechnology. Here, for the first time, we present the synthesis of several oxygenated-group-enriched carbon nanotubes (o-CNTs) via a facile but green approach, as well as their usage as high-performance peroxidase mimics for biocatalytic reaction. Exhaustive characterizations of the enzymatic activity of o-CNTs have been provided by exploring the accurate effect of various oxygenated groups on their surface including carbonyl, carboxyl, and hydroxyl groups. Because of the "competitive inhibition" effect among all of these oxygenated groups, the catalytic efficiency of o-CNTs is significantly enhanced by weakening the presence of noncatalytic sites. Furthermore, the admirable enzymatic activity of these o-CNTs has been successfully applied in the treatment of bacterial infections, and the results of both in vitro and in vivo nanozyme-mediated bacterial clearance clearly demonstrate the feasibility of o-CNTs as robust peroxidase mimics to effectively decrease the bacterial viability under physiological conditions. We believe that the present study will not only facilitate the construction of novel efficient nanozymes by rationally adjusting the degree of the "competitive inhibition" effect, but also broaden the biological usage of o-CNT-based nanomaterials via their satisfactory enzymatic activity.

Cryptococcus gattii urease as a virulence factor and the relevance of enzymatic activity in cryptococcosis pathogenesis.

PubMed

Feder, Vanessa; Kmetzsch, Lívia; Staats, Charley Christian; Vidal-Figueiredo, Natalia; Ligabue-Braun, Rodrigo; Carlini, Célia Regina; Vainstein, Marilene Henning

2015-04-01

Ureases (EC 3.5.1.5) are Ni(2+) -dependent metalloenzymes produced by plants, fungi and bacteria that hydrolyze urea to produce ammonia and CO2 . The insertion of nickel atoms into the apo-urease is better characterized in bacteria, and requires at least three accessory proteins: UreD, UreF, and UreG. Our group has demonstrated that ureases possess ureolytic activity-independent biological properties that could contribute to the pathogenicity of urease-producing microorganisms. The presence of urease in pathogenic bacteria strongly correlates with pathogenesis in some human diseases. Some medically important fungi also produce urease, including Cryptococcus neoformans and Cryptococcus gattii. C. gattii is an etiological agent of cryptococcosis, most often affecting immunocompetent individuals. The cryptococcal urease might play an important role in pathogenesis. It has been proposed that ammonia produced via urease action might damage the host endothelium, which would enable yeast transmigration towards the central nervous system. To analyze the role of urease as a virulence factor in C. gattii, we constructed knockout mutants for the structural urease-coding gene URE1 and for genes that code the accessory proteins Ure4 and Ure6. All knockout mutants showed reduced multiplication within macrophages. In intranasally infected mice, the ure1Δ (lacking urease protein) and ure4Δ (enzymatically inactive apo-urease) mutants caused reduced blood burdens and a delayed time of death, whereas the ure6Δ (enzymatically inactive apo-urease) mutant showed time and dose dependency with regard to fungal burden. Our results suggest that C. gattii urease plays an important role in virulence, in part possibly through enzyme activity-independent mechanism(s). © 2015 FEBS.

Changes in the amino acid profiles and free radical scavenging activities of Tenebrio molitor larvae following enzymatic hydrolysis.

PubMed

Tang, Yujiao; Debnath, Trishna; Choi, Eun-Ju; Kim, Young Wook; Ryu, Jung Pyo; Jang, Sejin; Chung, Sang Uk; Choi, Young-Jin; Kim, Eun-Kyung

2018-01-01

Tenebrio molitor (T. molitor) larvae provide food at low environmental cost and contribute positively to livelihoods. In this research, we compared the amino acids compositions and antioxidant activities of various extracts of T. molitor to enhance their quality as food. For the comparison, distilled water extracts, enzymatic hydrolysates, and condensed enzymatic hydrolysates of T. molitor larvae were prepared. Their amino acids (AAs) profiles and antioxidant activities, including ferric-reducing antioxidant power, oxygen radical absorption capacity, and DPPH, hydroxyl radical, and hydrogen peroxide radical scavenging properties assay were analyzed. DW extracts had the lowest AAs contents and antioxidant activity compared with enzymatic extracts. Condensed hydrolysates with a combination of alcalase and flavourzyme (C-A+F) exhibited the highest levels of total free AAs (11.1759 g/100 g). C-A+F produced higher total hydrolyzed AAs (32.5292 g/100 g) compared with the other groups. The C-A+F possessed the strongest antioxidant activity. Notably, the antioxidant activities of the hydrolysates and the total hydrolyzed AAs amount were correlated. Taken together, our findings showed that C-A+F was a promising technique for obtaining extracts of T. molitor larvae with antioxidant activity as potential nutritious functional food.

Effect of enzymatic mash treatment and storage on phenolic composition, antioxidant activity, and turbidity of cloudy apple juice.

PubMed

Oszmiański, Jan; Wojdylo, Aneta; Kolniak, Joanna

2009-08-12

The effects of different commercial enzymatic mash treatments on yield, turbidity, color, and polyphenolic and sediment of procyanidins content of cloudy apple juice were studied. Addition of pectolytic enzymes to mash treatment had positive effect on the production of cloud apple juices by improving polyphenolic contents, especially procyanidins and juice yields (68.3% in control samples to 77% after Pectinex Yield Mash). As summary of the effect of enzymatic mash treatment, polyphenol contents in cloudy apple juices significantly increased after Pectinex Yield Mash, Pectinex Smash XXL, and Pectinex XXL maceration were applied but no effect was observed after Pectinex Ultra-SPL I Panzym XXL use, compared to the control samples. The content of polymeric procyanidins represented 50-70% of total polyphenols, but in the present study, polymeric procyanidins were significantly lower in juices than in fruits and also affected by enzymatic treatment (Pectinex AFP L-4 and Panzym Yield Mash) compared to the control samples. The enzymatic treatment decreased procyanidin content in most sediment with the exception of Pectinex Smash XXL and Pectinex AFP L-4. Generally in samples that were treated by pectinase, radical scavenging activity of cloudy apple juices was increased compared to the untreated reference samples. The highest radical scavenging activity was associated with Pectinex Yield Mash, Pectinex Smash XXL, and Pectinex XXL enzyme and the lowest activity with Pectinex Ultra SP-L and Pectinex APFL-4. However, in the case of enzymatic mash treatment cloudy apple juices showed instability of turbidity and low viscosity. These results must be ascribed to the much higher hydrolysis of pectin by enzymatic preparation which is responsible for viscosity. During 6 months of storage at 4 degrees C small changes in analyzed parameters of apple juices were observed.

Polymerase/DNA interactions and enzymatic activity: multi-parameter analysis with electro-switchable biosurfaces

NASA Astrophysics Data System (ADS)

Langer, Andreas; Schräml, Michael; Strasser, Ralf; Daub, Herwin; Myers, Thomas; Heindl, Dieter; Rant, Ulrich

2015-07-01

The engineering of high-performance enzymes for future sequencing and PCR technologies as well as the development of many anticancer drugs requires a detailed analysis of DNA/RNA synthesis processes. However, due to the complex molecular interplay involved, real-time methodologies have not been available to obtain comprehensive information on both binding parameters and enzymatic activities. Here we introduce a chip-based method to investigate polymerases and their interactions with nucleic acids, which employs an electrical actuation of DNA templates on microelectrodes. Two measurement modes track both the dynamics of the induced switching process and the DNA extension simultaneously to quantitate binding kinetics, dissociation constants and thermodynamic energies. The high sensitivity of the method reveals previously unidentified tight binding states for Taq and Pol I (KF) DNA polymerases. Furthermore, the incorporation of label-free nucleotides can be followed in real-time and changes in the DNA polymerase conformation (finger closing) during enzymatic activity are observable.

Controlling enzymatic activity by immobilization on graphene oxide

NASA Astrophysics Data System (ADS)

Bolibok, Paulina; Wiśniewski, Marek; Roszek, Katarzyna; Terzyk, Artur P.

2017-04-01

In this study, graphene oxide (GO) has been applied as a matrix for enzyme immobilization. The protein adsorption capacity of GO is much higher than of other large surface area carbonaceous materials. Its structure and physicochemical properties are reported beneficial also for enzymatic activity modifications. The experimental proof was done here that GO-based biocatalytic systems with immobilized catalase are modifiable in terms of catalyzed reaction kinetic constants. It was found that activity and stability of catalase, considered here as model enzyme, closely depend on enzyme/GO ratio. The changes in kinetic parameters can be related to secondary structure alterations. The correlation between enzyme/GO ratio and kinetic and structure parameters is reported for the first time and enables the conscious control of biocatalytic processes and their extended applications. The biological activity of obtained biocatalytic systems was confirmed in vitro by the use of functional test. The addition of immobilized catalase improved the cells' viability after they were exposed to hydrogen peroxide and tert-butyl-hydroperoxide used as source of reactive oxygen species.

Specific inflammatory response of Anemonia sulcata (Cnidaria) after bacterial injection causes tissue reaction and enzymatic activity alteration.

PubMed

Trapani, M R; Parisi, M G; Parrinello, D; Sanfratello, M A; Benenati, G; Palla, F; Cammarata, M

2016-03-01

The evolution of multicellular organisms was marked by adaptations to protect against pathogens. The mechanisms for discriminating the ''self'' from ''non-self" have evolved into a long history of cellular and molecular strategies, from damage repair to the co-evolution of host-pathogen interactions. We investigated the inflammatory response in Anemonia sulcata (Cnidaria: Anthozoa) following injection of substances that varied in type and dimension, and observed clear, strong and specific reactions, especially after injection of Escherichia coli and Vibrio alginolyticus. Moreover, we analyzed enzymatic activity of protease, phosphatase and esterase, showing how the injection of different bacterial strains alters the expression of these enzymes and suggesting a correlation between the appearance of the inflammatory reaction and the modification of enzymatic activities. Our study shows for the first time, a specific reaction and enzymatic responses following injection of bacteria in a cnidarian. Copyright © 2016 Elsevier Inc. All rights reserved.

Ebselen inhibits QSOX1 enzymatic activity and suppresses invasion of pancreatic and renal cancer cell lines.

PubMed

Hanavan, Paul D; Borges, Chad R; Katchman, Benjamin A; Faigel, Douglas O; Ho, Thai H; Ma, Chen-Ting; Sergienko, Eduard A; Meurice, Nathalie; Petit, Joachim L; Lake, Douglas F

2015-07-30

Quiescin sulfhydryl oxidase 1 (QSOX1) is a highly conserved disulfide bond-generating enzyme that is overexpressed in diverse tumor types. Its enzymatic activity promotes the growth and invasion of tumor cells and alters extracellular matrix composition. In a nude mouse-human tumor xenograft model, tumors containing shRNA for QSOX1 grew significantly more slowly than controls, suggesting that QSOX1 supports a proliferative phenotype in vivo. High throughput screening experiments identified ebselen as an in vitro inhibitor of QSOX1 enzymatic activity. Ebselen treatment of pancreatic and renal cancer cell lines stalled tumor growth and inhibited invasion through Matrigel in vitro. Daily oral treatment with ebselen resulted in a 58% reduction in tumor growth in mice bearing human pancreatic tumor xenografts compared to controls. Mass spectrometric analysis of ebselen-treated QSOX1 mechanistically revealed that C165 and C237 of QSOX1 covalently bound to ebselen. This report details the anti-neoplastic properties of ebselen in pancreatic and renal cancer cell lines. The results here offer a "proof-of-principle" that enzymatic inhibition of QSOX1 may have clinical relevancy.

Identification of Residues That Affect Oligomerization and/or Enzymatic Activity of Influenza Virus H5N1 Neuraminidase Proteins

PubMed Central

Dai, Meiling; Guo, Hongbo; Dortmans, Jos C. F. M.; Dekkers, Jojanneke; Nordholm, Johan; Daniels, Robert; van Kuppeveld, Frank J. M.; de Vries, Erik

2016-01-01

ABSTRACT Influenza A virus (IAV) attachment to and release from sialoside receptors is determined by the balance between hemagglutinin (HA) and neuraminidase (NA). The molecular determinants that mediate the specificity and activity of NA are still poorly understood. In this study, we aimed to design the optimal recombinant soluble NA protein to identify residues that affect NA enzymatic activity. To this end, recombinant soluble versions of four different NA proteins from H5N1 viruses were compared with their full-length counterparts. The soluble NA ectodomains were fused to three commonly used tetramerization domains. Our results indicate that the particular oligomerization domain used does not affect the Km value but may affect the specific enzymatic activity. This particularly holds true when the stalk domain is included and for NA ectodomains that display a low intrinsic ability to oligomerize. NA ectodomains extended with a Tetrabrachion domain, which forms a nearly parallel four-helix bundle, better mimicked the enzymatic properties of full-length proteins than when other coiled-coil tetramerization domains were used, which probably distort the stalk domain. Comparison of different NA proteins and mutagenic analysis of recombinant soluble versions thereof resulted in the identification of several residues that affected oligomerization of the NA head domain (position 95) and therefore the specific activity or sialic acid binding affinity (Km value; positions 252 and 347). This study demonstrates the potential of using recombinant soluble NA proteins to reveal determinants of NA assembly and enzymatic activity. IMPORTANCE The IAV HA and NA glycoproteins are important determinants of host tropism and pathogenicity. However, NA is relatively understudied compared to HA. Analysis of soluble versions of these glycoproteins is an attractive way to study their activities, as they are easily purified from cell culture media and applied in downstream assays. In the

Enzymatic activity in the surface microlayer and subsurface water in the harbour channel

NASA Astrophysics Data System (ADS)

Perliński, Piotr; Mudryk, Zbigniew J.; Antonowicz, Józef

2017-09-01

Hydrolytic activity of eight extracellular enzymes was determined spectrofluorimetric method in the surface microlayer and subsurface water in the harbour channel in Ustka. The ranking order of the potential enzyme activity rates in the studied water layers was as follows: lipase > phosphatase > aminopeptidase > β-glucosidase > α-glucosidase > xylanase > cellulase > chitinase. The level of activity of all studied hydrolases was higher in the surface microlayer than subsurface water. No clear gradients in the level of enzymatic activity were determined along the horizontal profile of the studied channel. Activity of extracellular enzymes was strongly influenced by the season.

The activity of Rhizomuchor miehei lipase as a biocatalyst in enzymatic acylation of cyclic alcohol

NASA Astrophysics Data System (ADS)

Iftitah, Elvina Dhiaul; Srihardyastuti, Arie; Ariefin, Mokhamat

2017-03-01

We report the activity of Rhizomuchor miehei lipase (RML) as a biocatalyst, in particular the investigations concerning the effort of substrate-structure reactivity on the enzymatic acylation. The acylation was studied using acetic anhydride as an acyl donor and performed in n-hexane as a solvent. The selectivity of the enzymatic acylation was revealed by Gas Chromatography-Mass Spectra. We observed that, RML has shown different behavior when catalyzing the acylation of isopulegol and mixture of isopulegol and citronellal (ratio 1:1). The chemoselectivity for the O-acylation was improved when the acyl acceptor included mixture of isopulegol and citronellal

Structure and Activity of a New Low Molecular Weight Heparin Produced by Enzymatic Ultrafiltration

PubMed Central

FU, LI; ZHANG, FUMING; LI, GUOYUN; ONISHI, AKIHIRO; BHASKAR, UJJWAL; SUN, PEILONG; LINHARDT, ROBERT J.

2014-01-01

The standard process for preparing the low molecular weight heparin (LMWH) tinzaparin, through the partial enzymatic depolymerization of heparin, results in a reduced yield due to the formation of a high content of undesired disaccharides and tetrasaccharides. An enzymatic ultrafiltration reactor for LMWH preparation was developed to overcome this problem. The behavior, of the heparin oligosaccharides and polysaccharides using various membranes and conditions, was investigated to optimize this reactor. A novel product, LMWH-II, was produced from the controlled depolymerization of heparin using heparin lyase II in this optimized ultrafiltration reactor. Enzymatic ultrafiltration provides easy control and high yields (>80%) of LMWH-II. The molecular weight properties of LMWH-II were similar to other commercial LMWHs. The structure of LMWH-II closely matched heparin’s core structural features. Most of the common process artifacts, present in many commercial LWMHs, were eliminated as demonstrated by 1D and 2D nuclear magnetic resonance spectroscopy. The antithrombin III and platelet factor-4 binding affinity of LMWH-II were comparable to commercial LMWHs, as was its in vitro anticoagulant activity. PMID:24634007

Allocation of extracellular enzymatic activity in relation to litter composition, N deposition, and mass loss

USGS Publications Warehouse

Sinsabaugh, R. L.; Carreiro, M.M.; Repert, D.A.

2002-01-01

Decomposition of plant material is a complex process that requires interaction among a diversity of microorganisms whose presence and activity is subject to regulation by a wide range of environmental factors. Analysis of extracellular enzyme activity (EEA) provides a way to relate the functional organization of microdecomposer communities to environmental variables. In this study, we examined EEA in relation to litter composition and nitrogen deposition. Mesh bags containing senescent leaves of Quercus borealis (red oak), Acer rubrum (red maple) and Cornus florida (flowering dogwood) were placed on forest floor plots in southeastern New York. One-third of the plots were sprayed monthly with distilled water. The other plots were sprayed monthly with NH4NO3 solution at dose rates equivalent to 2 or 8 g N m-2 y-1. Mass loss, litter composition, fungal mass, and the activities of eight enzymes were measured on 13 dates for each litter type. Dogwood was followed for one year, maple for two, oak for three, For each litter type and treatment, enzymatic turnover activities were calculated from regressions of LN (%mass remaining) vs. cumulative activity. The decomposition of dogwood litter was more efficient than that of maple and oak. Maple litter had the lowest fungal mass and required the most enzymatic work to decompose, even though its mass loss rate was twice that of oak. Across litter types, N amendment reduced apparent enzymatic efficiencies and shifted EEA away from N acquisition and toward P acquisition, and away from polyphenol oxidation and toward polysaccharide hydrolysis. The effect of these shifts on decomposition rate varied with litter composition: dogwood was stimulated, oak was inhibited and maple showed mixed effects. The results show that relatively small shifts in the activity of one or two critical enzymes can significantly alter decomposition rates.

Cold enzymatic bleaching of fluid whey.

PubMed

Campbell, R E; Drake, M A

2013-01-01

Chemical bleaching of fluid whey and retentate with hydrogen peroxide (HP) alone requires high concentrations (100-500 mg of HP/kg) and recent studies have demonstrated that off-flavors are generated during chemical bleaching that carry through to spray-dried whey proteins. Bleaching of fluid whey and retentate with enzymes such as naturally present lactoperoxidase or an exogenous commercial peroxidase (EP) at cold temperatures (4°C) may be a viable alternative to traditional chemical bleaching of whey. The objective of this study was to determine the optimum level of HP for enzymatic bleaching (both lactoperoxidase and EP) at 4°C and to compare bleaching efficacy and sensory characteristics to HP chemical bleaching at 4°C. Selected treatments were subsequently applied for whey protein concentrate with 80% protein (WPC80) manufacture. Fluid Cheddar whey and retentate (80% protein) were manufactured in triplicate from pasteurized whole milk. The optimum concentration of HP (0 to 250 mg/kg) to activate enzymatic bleaching at 4°C was determined by quantifying the loss of norbixin. In subsequent experiments, bleaching efficacy, descriptive sensory analysis, and volatile compounds were monitored at selected time points. A control with no bleaching was also evaluated. Enzymatic bleaching of fluid whey and retentate at 4°C resulted in faster bleaching and higher bleaching efficacy (color loss) than bleaching with HP alone at 250 mg/kg. Due to concentrated levels of naturally present lactoperoxidase, retentate bleached to completion (>80% norbixin destruction in 30 min) faster than fluid whey at 4°C (>80% norbixin destruction in 12h). In fluid whey, the addition of EP decreased bleaching time. Spray-dried WPC80 from bleached wheys, regardless of bleaching treatment, were characterized by a lack of sweet aromatic and buttery flavors, and the presence of cardboard flavor concurrent with higher relative abundance of 1-octen-3-ol and 1-octen-3-one. Among enzymatically

Relationship between Porcine Sperm Motility and Sperm Enzymatic Activity using Paper-based Devices

NASA Astrophysics Data System (ADS)

Matsuura, Koji; Huang, Han-Wei; Chen, Ming-Cheng; Chen, Yu; Cheng, Chao-Min

2017-04-01

Mammalian sperm motility has traditionally been analyzed to determine fertility using computer-assisted semen analysis (CASA) systems. To develop low-cost and robust male fertility diagnostics, we created a paper-based MTT assay and used it to estimate motile sperm concentration. When porcine sperm motility was inhibited using sperm enzyme inhibitors for sperm enzymes related to mitochondrial activity and glycolysis, we simultaneously recorded sperm motility and enzymatic reactivity using a portable motility analysis system (iSperm) and a paper-based MTT assay, respectively. When using our paper-based MTT-assay, we calculated the area mean value signal intensity (AMV) to evaluate enzymatic reactivity. Both sperm motility and AMV decreased following treatment with iodoacetamide (IODO) and 3-bromopyruvic acid (3BP), both of which are inhibitors of glycolytic enzymes including glyceraldehyde-3-phosphate dehydrogenase (GAPDH). We found a correlation between recorded motility using iSperm and AMV from our paper-based assay (P < 0.05), suggesting that a sperm-related enzymatic reaction is involved in sperm motility. Under this protocol, MTT reduction was coupled with catalysis of GAPDH and was promoted by electron transfer from NADH. Based on this inhibitor study, sperm motility can be estimated using our paper-based MTT-assay.

Enzymatic modification of chitosan by cinnamic acids: Antibacterial activity against Ralstonia solanacearum.

PubMed

Yang, Caifeng; Zhou, Yu; Zheng, Yu; Li, Changlong; Sheng, Sheng; Wang, Jun; Wu, Fuan

2016-06-01

This study aimed to identify chitosan polymers that have antibacterial activity against the bacterial wilt pathogen. The chitosan polymers were enzymatically synthesized using chitosan and five cinnamic acids (CADs): caffeic acid (CA), ferulic acid (FA), cinnamic acid (CIA), p-coumaric acid (COA) and chlorogenic acid (CHA), using laccase from Pleurotus ostreatus as a catalyst. The reaction was performed in a phosphate buffered solution under heterogenous reaction conditions. The chitosan derivatives (CTS-g-CADs) were characterized by FT-IR, XRD, TGA and SEM. FT-IR demonstrated that the reaction products bound covalently to the free amino groups or hydroxyl groups of chitosan via band of amide I or ester band. XRD showed a reduced packing density for grafted chitosan comparing to original chitosan. TGA demonstrated that CTS-g-CADs have a higher thermostability than chitosan. Additionally, chitosan and its derivatives showed similar antibacterial activity. However, the IC50 value of the chitosan-caffeic acid derivative (CTS-g-CA) against the mulberry bacterial wilt pathogen RS-5 was 0.23mg/mL, which was two-fifths of the IC50 value of chitosan. Therefore, the enzymatically synthesized chitosan polymers can be used to control plant diseases in biotechnological domains. Copyright © 2016 Elsevier B.V. All rights reserved.

Decomposition rate and enzymatic activity of composted municipal waste and poultry manure in the soil in a biofuel crops field.

PubMed

Cordovil, Cláudia Marques-Dos-Santos; de Varennes, Amarilis; Pinto, Renata Machado Dos Santos; Alves, Tiago Filipe; Mendes, Pedro; Sampaio, Sílvio César

2017-05-01

Biofuel crops are gaining importance because of the need to replace non-renewable sources. Also, due to the increasing amounts of wastes generated, there is the need to recycle them to the soil, both to fertilize crops and to improve soil physical properties through organic matter increase and microbiological changes in the rhizosphere. We therefore studied the influence of six biofuel crops (elephant grass, giant cane, sugarcane, blue gum, black cottonwood, willow) on the decomposition rate and enzymatic activity of composted municipal solid waste and poultry manure. Organic amendments were incubated in the field (litterbag method), buried near each plant or bare soil. Biomass decrease and dehydrogenase, urease and acid phosphatase level in amendments was monitored over a 180-day period. Soil under the litterbags was analysed for the same enzymatic activity and organic matter fractions (last sampling). After 365 days, a fractionation of organic matter was carried out in both amendments and soil under the litterbags. For compost, willow and sugarcane generally led to the greatest enzymatic activity, at the end of the experiment. For manure, dehydrogenase activity decreased sharply with time, the smallest value near sugarcane, while phosphatase and urease generally presented the highest values, at the beginning or after 90 days' incubation. Clustering showed that plant species could be grouped based on biomass and enzymes measured over time. Plant species influenced the decomposition rate and enzymatic activities of the organic amendments. Overall, mineralization of both amendments was associated with a greater urease activity in soils. Dehydrogenase activity in manure was closely associated with urease activity. © 2016 Society of Chemical Industry. © 2016 Society of Chemical Industry.

The dual effects of Maillard reaction and enzymatic hydrolysis on the antioxidant activity of milk proteins.

PubMed

Oh, N S; Lee, H A; Lee, J Y; Joung, J Y; Lee, K B; Kim, Y; Lee, K W; Kim, S H

2013-08-01

The objective of this study was to determine the enhanced effects on the biological characteristics and antioxidant activity of milk proteins by the combination of the Maillard reaction and enzymatic hydrolysis. Maillard reaction products were obtained from milk protein preparations, such as whey protein concentrates and sodium caseinate with lactose, by heating at 55°C for 7 d in sodium phosphate buffer (pH 7.4). The Maillard reaction products, along with untreated milk proteins as controls, were hydrolyzed for 0 to 3h with commercial proteases Alcalase, Neutrase, Protamex, and Flavorzyme (Novozymes, Bagsværd, Denmark). The antioxidant activity of hydrolyzed Maillard reaction products was determined by reaction with 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt, their 1,1-diphenyl-2-picrylhydrazyl radical scavenging activity, and the ability to reduce ferric ions. Further characteristics were evaluated by the o-phthaldialdehyde method and sodium dodecyl sulfate-PAGE. The degree of hydrolysis gradually increased in a time-dependent manner, with the Alcalase-treated Maillard reaction products being the most highly hydrolyzed. Radical scavenging activities and reducing ability of hydrolyzed Maillard reaction products increased with increasing hydrolysis time. The combined products of enzymatic hydrolysis and Maillard reaction showed significantly greater antioxidant activity than did hydrolysates or Maillard reaction products alone. The hydrolyzed Maillard reaction products generated by Alcalase showed significantly higher antioxidant activity when compared with the other protease products and the antioxidant activity was higher for the whey protein concentrate groups than for the sodium caseinate groups. These findings indicate that Maillard reaction products, coupled with enzymatic hydrolysis, could act as potential antioxidants in the pharmaceutical, food, and dairy industries. Copyright © 2013 American Dairy Science Association

Methods for determining enzymatic activity comprising heating and agitation of closed volumes

DOEpatents

Thompson, David Neil; Henriksen, Emily DeCrescenzo; Reed, David William; Jensen, Jill Renee

2016-03-15

Methods for determining thermophilic enzymatic activity include heating a substrate solution in a plurality of closed volumes to a predetermined reaction temperature. Without opening the closed volumes, at least one enzyme is added, substantially simultaneously, to the closed volumes. At the predetermined reaction temperature, the closed volumes are agitated and then the activity of the at least one enzyme is determined. The methods are conducive for characterizing enzymes of high-temperature reactions, with insoluble substrates, with substrates and enzymes that do not readily intermix, and with low volumes of substrate and enzyme. Systems for characterizing the enzymes are also disclosed.

Effects of lead contamination on soil enzymatic activities, microbial biomass, and rice physiological indices in soil-lead-rice (Oryza sativa L.) system.

PubMed

Zeng, Lu S; Liao, Min; Chen, Cheng L; Huang, Chang Y

2007-05-01

The effect of lead (Pb) treatment on the soil enzymatic activities, soil microbial biomass, rice physiological indices and rice biomass were studied in a greenhouse pot experiment. Six levels of Pb viz. 0(CK), 100, 300, 500, 700, 900 mg/kg soil were applied in two types of paddy soils. The results showed that Pb treatment had a stimulating effect on soil enzymatic activities and microbial biomass carbon (Cmic) at low concentration and an inhibitory influence at higher concentration. The degree of influence on enzymatic activities and Cmic by Pb was related to the clay and organic matter contents of the soils. When the Pb treatment was raised to the level of 500 mg/kg, ecological risk appeared both to soil microorganisms and plants. The results also revealed a consistent trend of increased chlorophyll contents and rice biomass initially, maximum at a certain Pb treatment, and then decreased gradually with the increase in Pb concentration. Pb was effective in inducing proline accumulation and its toxicity causes oxidative stress in rice plants. Therefore, it was concluded that soil enzymatic activities, Cmic and rice physiological indices, could be sensitive indicators to reflect environmental stress in soil-lead-rice system.

Structure and activity of a new low-molecular-weight heparin produced by enzymatic ultrafiltration.

PubMed

Fu, Li; Zhang, Fuming; Li, Guoyun; Onishi, Akihiro; Bhaskar, Ujjwal; Sun, Peilong; Linhardt, Robert J

2014-05-01

The standard process for preparing the low-molecular-weight heparin (LMWH) tinzaparin, through the partial enzymatic depolymerization of heparin, results in a reduced yield because of the formation of a high content of undesired disaccharides and tetrasaccharides. An enzymatic ultrafiltration reactor for LMWH preparation was developed to overcome this problem. The behavior, of the heparin oligosaccharides and polysaccharides using various membranes and conditions, was investigated to optimize this reactor. A novel product, LMWH-II, was produced from the controlled depolymerization of heparin using heparin lyase II in this optimized ultrafiltration reactor. Enzymatic ultrafiltration provides easy control and high yields (>80%) of LMWH-II. The molecular weight properties of LMWH-II were similar to other commercial LMWHs. The structure of LMWH-II closely matched heparin's core structural features. Most of the common process artifacts, present in many commercial LWMHs, were eliminated as demonstrated by 1D and 2D nuclear magnetic resonance spectroscopy. The antithrombin III and platelet factor-4 binding affinity of LMWH-II were comparable to commercial LMWHs, as was its in vitro anticoagulant activity. © 2014 Wiley Periodicals, Inc. and the American Pharmacists Association.

Delta-Secretase Phosphorylation by SRPK2 Enhances Its Enzymatic Activity, Provoking Pathogenesis in Alzheimer's Disease.

PubMed

Wang, Zhi-Hao; Liu, Pai; Liu, Xia; Manfredsson, Fredric P; Sandoval, Ivette M; Yu, Shan Ping; Wang, Jian-Zhi; Ye, Keqiang

2017-09-07

Delta-secretase, a lysosomal asparagine endopeptidase (AEP), simultaneously cleaves both APP and tau, controlling the onset of pathogenesis of Alzheimer's disease (AD). However, how this protease is post-translationally regulated remains unclear. Here we report that serine-arginine protein kinase 2 (SRPK2) phosphorylates delta-secretase and enhances its enzymatic activity. SRPK2 phosphorylates serine 226 on delta-secretase and accelerates its autocatalytic cleavage, leading to its cytoplasmic translocation and escalated enzymatic activities. Delta-secretase is highly phosphorylated in human AD brains, tightly correlated with SRPK2 activity. Overexpression of a phosphorylation mimetic (S226D) in young 3xTg mice strongly promotes APP and tau fragmentation and facilitates amyloid plaque deposits and neurofibrillary tangle (NFT) formation, resulting in cognitive impairment. Conversely, viral injection of the non-phosphorylatable mutant (S226A) into 5XFAD mice decreases APP and tau proteolytic cleavage, attenuates AD pathologies, and reverses cognitive defects. Our findings support that delta-secretase phosphorylation by SRPK2 plays a critical role in aggravating AD pathogenesis. Copyright © 2017 Elsevier Inc. All rights reserved.

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

PubMed Central

Guerra, Nelson P.; Pastrana Castro, Lorenzo

2012-01-01

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

Three enzymatically active neurotoxins of Clostridium botulinum strain Af84: BoNT/A2, /F4, and /F5.

PubMed

Kalb, Suzanne R; Baudys, Jakub; Smith, Theresa J; Smith, Leonard A; Barr, John R

2014-04-01

Botulinum neurotoxins (BoNTs) are produced by various species of clostridia and are potent neurotoxins which cause the disease botulism, by cleaving proteins needed for successful nerve transmission. There are currently seven confirmed serotypes of BoNTs, labeled A-G, and toxin-producing clostridia typically only produce one serotype of BoNT. There are a few strains (bivalent strains) which are known to produce more than one serotype of BoNT, producing either both BoNT/A and /B, BoNT/A and /F, or BoNT/B and /F, designated as Ab, Ba, Af, or Bf. Recently, it was reported that Clostridium botulinum strain Af84 has three neurotoxin gene clusters: bont/A2, bont/F4, and bont/F5. This was the first report of a clostridial organism containing more than two neurotoxin gene clusters. Using a mass spectrometry based proteomics approach, we report here that all three neurotoxins, BoNT/A2, /F4, and /F5, are produced by C. botulinum Af84. Label free MS(E) quantification of the three toxins indicated that toxin composition is 88% BoNT/A2, 1% BoNT/F4, and 11% BoNT/F5. The enzymatic activity of all three neurotoxins was assessed by examining the enzymatic activity of the neurotoxins upon peptide substrates, which mimic the toxins' natural targets, and monitoring cleavage of the substrates by mass spectrometry. We determined that all three neurotoxins are enzymatically active. This is the first report of three enzymatically active neurotoxins produced in a single strain of Clostridium botulinum.

An association between Schistosoma mansoni worms and an enzymatically-active protease/peptidase in mouse blood.

PubMed

Darani, H Y; Doenhoff, M J

2008-04-01

An enzyme found previously in extracts of adult Schistosoma mansoni worms, that hydrolysed the chromogenic substrate N-acetyl-DL-phenylalanine beta-naphthyl-ester, has here been further investigated and characterized. Evidence that the molecule found in the parasite was antigenically and enzymatically homologous with a constituent of normal mouse plasma has been consolidated using a monospecific serum in immunoelectrophoresis and Western immunoblotting. The molecular size of the enzyme was found to be approximately 70 kDa and it was inhibited by a serine protease inhibitor, but not by inhibitors of other classes of protease. The enzymatic activity found in normal mouse serum was also found in normal rat serum, but not in sera from several other mammalian species.

Boar spermatozoa encapsulated in barium alginate membranes: a microdensitometric evaluation of some enzymatic activities during storage at 18 degrees C.

PubMed

Faustini, Massimo; Torre, Maria Luisa; Stacchezzini, Simona; Norberti, Roberta; Consiglio, Anna Lange; Porcelli, Franca; Conte, Ubaldo; Munari, Eleonora; Russo, Vincenzo; Vigo, Daniele

2004-01-01

The customary dilution of boar semen for subsequent artificial insemination (AI) procedures damages the cell membrane of spermatozoa, resulting in a loss of enzymes and other cytoplasmic contents and acrosomal reactions. We encapsulated non-diluted boar semen in barium alginate membranes to optimize AI procedures and to improve the functional integrity of spermatozoal membranes during storage. The percentage of non-reacted acrosomes (NRA) and measurements of enzyme leakage (cytochrome c oxidase (COX), lactate dehydrogenase (LDH), and glucose-6-phosphate dehydrogenase (G6PDH)) were used as indices of the functional status of diluted, unencapsulated and encapsulated spermatozoa, stored for 72 h at 18 degrees C. Enzymatic activity was assessed in situ by microdensitometry, and non-reacted acrosomes were microscopically determined by staining. The percentage of acrosome integrity and the intracellular enzymatic activities during storage were different for unencapsulated and encapsulated semen. Semen dilution caused a rapid decline in enzymatic activities and concomitant acrosomal reactions. Encapsulated spermatozoa had significantly higher acrosome integrity (77% versus 55%; P < 0.01 after 72 h) and an overall higher in situ enzymatic activity. For cytochrome c oxidase and lactate dehydrogenase the greatest differences between encapsulated and unencapsulated spermatozoa were present after 72 h whereas for glucose-6-phosphate dehydrogenase significant differences were found within 24h of storage. The encapsulation process maintains a better preservation environment for boar spermatozoa and could be a promising, innovative technique to improve storage of these cells.

Enzymatic digestive activity and absorption efficiency in Tagelus dombeii upon Alexandrium catenella exposure

NASA Astrophysics Data System (ADS)

Fernández-Reiriz, M. J.; Navarro, J. M.; Cisternas, B. A.; Babarro, J. M. F.; Labarta, U.

2013-12-01

We analyzed absorption efficiency (AE) and digestive enzyme activity (amylase, cellulase complex, and laminarinase) of the infaunal bivalve Tagelus dombeii originating from two geographic sites, Corral-Valdivia and Melinka-Aysén, which have different long-term paralytic shellfish poisoning (PSP) exposure rates. We report the effects of past feeding history (origin) on T. dombeii exposed to a mixed diet containing the toxic dinoflagellate Alexandrium catenella and another dinoflagellate-free control diet over a 12-day period in the laboratory. Absorption efficiency values of T. dombeii individuals that experienced PSP exposure in their habitat (Melinka-Aysén) remained unchanged during exposure to toxic food in the laboratory. In contrast, T. dombeii from a non-PSP exposure field site (Corral-Valdivia) showed a significant reduction in AE with toxic exposure time. This study established that the amylase and cellulase complexes were the most important enzymes in the digestive glands of Tagelus from both sites. The temporal evolution of enzymatic activity under toxic diet was fitted to exponential (amylase and cellulase) and to a logarithmic (laminarinase) models. In all fits, we found significant effect of origin in the model parameters. At the beginning of the experiment, higher enzymatic activity was observed for clams from Corral-Valdivia. The amylase activity decreased with time exposure for individuals from Corral and increased for individuals from Melinka. Cellulase activity did not vary over time for clams from Corral, but increased for individuals from Melinka and laminarinase activity decreased over time for individuals from Corral and remained unchanged over time for Melinka. A feeding history of exposure to the dinoflagellate A. catenella was reflected in the digestive responses of both T. dombeii populations.

Dramatic enhancement of enzymatic activity in organic solvents by lyoprotectants

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

Dabulis, K.; Klibanov, A.M.

1993-03-05

When seven different hydrolytic enzymes (four proteases and three lipases) were lyophilized from aqueous solution containing a ligand, N-Ac-L-Phe-NH[sub 2], their catalytic activity in anhydrous solvents was far greater (one to two orders of magnitude) than that of the enzymes lyophilized without the ligand. This ligand-induced activation was expressed regardless of whether the substrate employed in organic solvents structurally resembled the ligand. Furthermore, nonligand lyoprotectants [sorbitol, other sugars, and poly(ethylene glycol)] also dramatically enhanced enzymatic activity in anhydrous solvents when present in enzyme aqueous solution prior to lyophilization. The effects of the ligand and of the lyoprotectants were nonadditive, suggestingmore » the same mechanism of action. Excipient-activated and nonactivated enzymes exhibited identical activities in water. Also, addition of the excipients directly to suspensions of nonactivated enzymes in organic solvents had no appreciable effect on catalytic activity. These observations indicate that the mechanism of the excipient-induced activation is based on the ability of the excipients to alleviate reversible denaturation of enzymes upon lyophilization. Activity enhancement induced by the excipients is displayed even after their removal by washing enzymes with anhydrous solvents. Subtilisin Carlsberg, lyophilized with sorbitol, was found to be a much more efficient practical catalyst than its regular' counterpart.« less

Conformational change results in loss of enzymatic activity of jack bean urease on its interaction with silver nanoparticle.

PubMed

Ponnuvel, Shobana; Subramanian, Balakumar; Ponnuraj, Karthe

2015-10-01

Urease is an enzyme produced by microbes such as bacteria, yeast and fungi. Plants also produce this enzyme. Urease action splits urea into ammonia and carbamate. This action is having important implications in agro-chemical, medicinal and environment. Therefore there is always a constant search for new and novel compounds which could inhibit this enzyme. Here we have studied the interaction of jack bean urease (JBU) with silver nanoparticle to analyze the influence of the resultant protein corona formation on the catalytic property of JBU. Several techniques like UV-Vis, gel shift assay and CD spectroscopy have been used to characterize this interaction. Urease activity assay suggests that the protein corona formation inhibits the enzymatic action of JBU. The loss of enzymatic action could be either due to the nanoparticle blocking the active site of JBU or a conformational change in the protein. The CD spectra of JBU-AgNP complexes clearly revealed significant changes in the secondary structural composition of the JBU and this could be the reason for the loss of enzymatic activity of JBU. This study revealed an interesting observation, where the interaction of AgNP with JBU resulted destabilization of hexameric nature of JBU which is otherwise highly stable. The results of the present study could be useful in the development of nanoparticle based material for inhibiting the ureolytic activity of ureases in different fields.

Enzymatic activities produced by mixed Saccharomyces and non-Saccharomyces cultures: relationship with wine volatile composition.

PubMed

Maturano, Yolanda Paola; Assof, Mariela; Fabani, María Paula; Nally, María Cristina; Jofré, Viviana; Rodríguez Assaf, Leticia Anahí; Toro, María Eugenia; Castellanos de Figueroa, Lucía Inés; Vazquez, Fabio

2015-11-01

During certain wine fermentation processes, yeasts, and mainly non-Saccharomyces strains, produce and secrete enzymes such as β-glucosidases, proteases, pectinases, xylanases and amylases. The effects of enzyme activity on the aromatic quality of wines during grape juice fermentation, using different co-inoculation strategies of non-Saccharomyces and Saccharomyces cerevisiae yeasts, were assessed in the current study. Three strains with appropriate enological performance and high enzymatic activities, BSc562 (S. cerevisiae), BDv566 (Debaryomyces vanrijiae) and BCs403 (Candida sake), were assayed in pure and mixed Saccharomyces/non-Saccharomyces cultures. β-Glucosidase, pectinase, protease, xylanase and amylase activities were quantified during fermentations. The aromatic profile of pure and mixed cultures was determined at the end of each fermentation. In mixed cultures, non-Saccharomyces species were detected until day 4-5 of the fermentation process, and highest populations were observed in MSD2 (10% S. cerevisiae/90% D. vanrijiae) and MSC1 (1% S. cerevisiae/99% C. sake). According to correlation and multivariate analysis, MSD2 presented the highest concentrations of terpenes and higher alcohols which were associated with pectinase, amylase and xylanase activities. On the other hand, MSC1 high levels of β-glucosidase, proteolytic and xylanolytic activities were correlated to esters and fatty acids. Our study contributes to a better understanding of the effect of enzymatic activities by yeasts on compound transformations that occur during wine fermentation.

Sirtuin 1 Enzymatic Activity Is Required for Cartilage Homeostasis In Vivo in a Mouse Model

PubMed Central

Gabay, Odile; Sanchez, Christelle; Dvir-Ginzberg, Mona; Gagarina, Viktoria; Zaal, Kristien J.; Song, Yingjie; He, Xiao Hong; McBurney, Michael W.

2014-01-01

Objective We and others previously demonstrated that sirtuin 1 (SIRT-1) regulates apoptosis and cartilage-specific gene expression in human chondrocytes and mouse models. This study was undertaken to determine if SIRT-1 enzymatic activity plays a protective role in cartilage homeostasis in vivo, by investigating mice with SIRT-1 mutations to characterize their cartilage. Methods Articular cartilage was harvested from the paws and knees of 5- and 6-month-old wild-type (WT) mice and mice homozygous for SIRT-1tm2.1Mcby (SIRT-1y/y), an allele carrying a point mutation that encodes a SIRT-1 protein with no enzymatic activity (y/y mice). Mice ages 2 days old and 6–7 days old were also examined. Mouse joint cartilage was processed for histologic examination or biochemical analyses of chondrocyte cultures. Results We found that articular cartilage tissue sections from y/y mice of up to 6 months of age contained reduced levels of type II collagen, aggrecan, and glycosaminoglycan compared to sections from WT mice. In contrast, protein levels of matrix metalloproteinase 8 (MMP-8), MMP-9, and MMP-13 were elevated in the cartilage of y/y mice. In addition, chondrocyte apoptosis was elevated in SIRT-1 mutant mice as compared to their WT littermates. Consistent with these observations, protein tyrosine phosphatase 1b was elevated in the y/y mice. Conclusion Our in vivo findings in this animal model demonstrate that mice with defective SIRT-1 also have defective cartilage, with elevated rates of cartilage degradation with age. Hence, normal cartilage homeostasis requires enzymatically active SIRT-1 protein. PMID:23124828

Effects of molecular weight of hyaluronic acid on its viscosity and enzymatic activities of lysozyme and peroxidase.

PubMed

Kim, Jihoon; Chang, Ji-Youn; Kim, Yoon-Young; Kim, Moon-Jong; Kho, Hong-Seop

2018-05-01

To investigate the effects of the molecular weight of hyaluronic acid on its viscosity and enzymatic activities of lysozyme and peroxidase in solution and on the hydroxyapatite surface. Hyaluronic acids of four different molecular weights (10 kDa, 100 kDa, 1 MDa, and 2 MDa), hen egg-white lysozyme, bovine lactoperoxidase, and human whole saliva were used. Viscosity values of hyaluronic acids were measured using a cone-and-plate viscometer at six different concentrations (0.1-5.0 mg/mL). Enzymatic activities of lysozyme and peroxidase were examined by hydrolysis of fluorescein-labeled Micrococcus lysodeikticus and oxidation of fluorogenic 2',7'-dichlorofluorescein to fluorescing 2',7'-dichlorofluorescein, respectively. In solution assays, only 2 MDa-hyaluronic acid significantly inhibited lysozyme activities in saliva. In surface assays, hyaluronic acids inhibited lysozyme and peroxidase activities; the inhibitory activities were more apparent with high-molecular-weight ones in saliva than in purified enzymes. The 100 kDa-hyaluronic acid at 5.0 mg/mL, 1 MDa-one at 0.5 mg/mL, and 2 MDa-one at 0.2 mg/mL showed viscosity values similar to those of human whole saliva at a shear rate range required for normal oral functions. The differences among the influences of the three conditions on the enzymatic activities were not statistically significant. High-molecular-weight hyaluronic acids at low concentration and low-molecular-weight ones at high concentration showed viscosity values similar to those of human whole saliva. Inhibitory effects of hyaluronic acids on lysozyme and peroxidase activities were more significant with high-molecular-weight ones on the surface and in saliva compared with in solution and on purified enzymes. Copyright © 2018 Elsevier Ltd. All rights reserved.

Glycosylation site-targeted PEGylation of glucose oxidase retains native enzymatic activity.

PubMed

Ritter, Dustin W; Roberts, Jason R; McShane, Michael J

2013-04-10

Targeted PEGylation of glucose oxidase at its glycosylation sites was investigated to determine the effect on enzymatic activity, as well as the bioconjugate's potential in an optical biosensing assay. Methoxy-poly(ethylene glycol)-hydrazide (4.5kDa) was covalently coupled to periodate-oxidized glycosylation sites of glucose oxidase from Aspergillus niger. The bioconjugate was characterized using gel electrophoresis, liquid chromatography, mass spectrometry, and dynamic light scattering. Gel electrophoresis data showed that the PEGylation protocol resulted in a drastic increase (ca. 100kDa) in the apparent molecular mass of the protein subunit, with complete conversion to the bioconjugate; liquid chromatography data corroborated this large increase in molecular size. Mass spectrometry data proved that the extent of PEGylation was six poly(ethylene glycol) chains per glucose oxidase dimer. Dynamic light scattering data indicated the absence of higher-order oligomers in the PEGylated GOx sample. To assess stability, enzymatic activity assays were performed in triplicate at multiple time points over the course of 29 days in the absence of glucose, as well as before and after exposure to 5% w/v glucose for 24h. At a confidence level of 95%, the bioconjugate's performance was statistically equivalent to native glucose oxidase in terms of activity retention over the 29 day time period, as well as following the 24h glucose exposure. Finally, the bioconjugate was entrapped within a poly(2-hydroxyethyl methacrylate) hydrogel containing an oxygen-sensitive phosphor, and the construct was shown to respond approximately linearly with a 220±73% signal change (n=4, 95% confidence interval) over the physiologically-relevant glucose range (i.e., 0-400mg/dL); to our knowledge, this represents the first demonstration of PEGylated glucose oxidase incorporated into an optical biosensing assay. Copyright © 2013 Elsevier Inc. All rights reserved.

Decipher the dynamic coordination between enzymatic activity and structural modulation at focal adhesions in living cells

NASA Astrophysics Data System (ADS)

Lu, Shaoying; Seong, Jihye; Wang, Yi; Chang, Shiou-Chi; Eichorst, John Paul; Ouyang, Mingxing; Li, Julie Y.-S.; Chien, Shu; Wang, Yingxiao

2014-07-01

Focal adhesions (FAs) are dynamic subcellular structures crucial for cell adhesion, migration and differentiation. It remains an enigma how enzymatic activities in these local complexes regulate their structural remodeling in live cells. Utilizing biosensors based on fluorescence resonance energy transfer (FRET), we developed a correlative FRET imaging microscopy (CFIM) approach to quantitatively analyze the subcellular coordination between the enzymatic Src activation and the structural FA disassembly. CFIM reveals that the Src kinase activity only within the microdomain of lipid rafts at the plasma membrane is coupled with FA dynamics. FA disassembly at cell periphery was linearly dependent on this raft-localized Src activity, although cells displayed heterogeneous levels of response to stimulation. Within lipid rafts, the time delay between Src activation and FA disassembly was 1.2 min in cells seeded on low fibronectin concentration ([FN]) and 4.3 min in cells on high [FN]. CFIM further showed that the level of Src-FA coupling, as well as the time delay, was regulated by cell-matrix interactions, as a tight enzyme-structure coupling occurred in FA populations mediated by integrin αvβ3, but not in those by integrin α5β1. Therefore, different FA subpopulations have distinctive regulation mechanisms between their local kinase activity and structural FA dynamics.

Force-Manipulation Single-Molecule Spectroscopy Studies of Enzymatic Dynamics

NASA Astrophysics Data System (ADS)

Lu, H. Peter; He, Yufan; Lu, Maolin; Cao, Jin; Guo, Qing

2014-03-01

Subtle conformational changes play a crucial role in protein functions, especially in enzymatic reactions involving complex substrate-enzyme interactions and chemical reactions. We applied AFM-enhanced and magnetic tweezers-correlated single-molecule spectroscopy to study the mechanisms and dynamics of enzymatic reactions involved with kinase and lysozyme proteins. Enzymatic reaction turnovers and the associated structure changes of individual protein molecules were observed simultaneously in real-time by single-molecule FRET detections. Our single-molecule spectroscopy measurements of enzymatic conformational dynamics have revealed time bunching effect and intermittent coherence in conformational state change dynamics involving in enzymatic reaction cycles. The coherent conformational state dynamics suggests that the enzymatic catalysis involves a multi-step conformational motion along the coordinates of substrate-enzyme complex formation and product releasing. Our results support a multiple-conformational state model, being consistent with a complementary conformation selection and induced-fit enzymatic loop-gated conformational change mechanism in substrate-enzyme active complex formation.

Quantitative Collection and Enzymatic Activity of Glucose Oxidase Nanotubes Fabricated by Templated Layer-by-Layer Assembly.

PubMed

Zhang, Shouwei; Demoustier-Champagne, Sophie; Jonas, Alain M

2015-08-10

We report on the fabrication of enzyme nanotubes in nanoporous polycarbonate membranes via the layer-by-layer (LbL) alternate assembly of polyethylenimine (PEI) and glucose oxidase (GOX), followed by dissolution of the sacrificial template in CH2Cl2, collection, and final dispersion in water. An adjuvant-assisted filtration methodology is exploited to extract quantitatively the nanotubes without loss of activity and morphology. Different water-soluble CH2Cl2-insoluble adjuvants are tested for maximal enzyme activity and nanotube stability; whereas NaCl disrupts the tubes by screening electrostatic interactions, the high osmotic pressure created by fructose also contributes to loosening the nanotubular structures. These issues are solved when using neutral, high molar mass dextran. The enzymatic activity of intact free nanotubes in water is then quantitatively compared to membrane-embedded nanotubes, showing that the liberated nanotubes have a higher catalytic activity in proportion to their larger exposed surface. Our study thus discloses a robust and general methodology for the fabrication and quantitative collection of enzymatic nanotubes and shows that LbL assembly provides access to efficient enzyme carriers for use as catalytic swarming agents.

Impact of potato cultivation and cattle farming on physicochemical parameters and enzymatic activities of Neotropical high Andean Páramo ecosystem soils.

PubMed

Avellaneda-Torres, Lizeth Manuela; León Sicard, Tomás Enrique; Torres Rojas, Esperanza

2018-08-01

The Andean Páramos are high mountain ecosystems whose soils are essential for the management of South American water resources, but research on anthropic impacts to these soils is currently minimal and insufficient. The objective of this study was to evaluate the impacts of potato (Solanum tuberosum) cultivation and livestock on the physicochemical parameters and enzymatic activities that determine the soil quality of the Neotropical high Andean Páramo ecosystem in the Nevados National Natural Park (Nevados NNP) in Colombia. It was hypothesised that sites with potato crops and livestock farming would exhibit significant changes in soil physicochemical parameters and enzymatic activities compared with Páramo sites that have been conserved without agriculture. Samples were collected from soils under potato cultivation, livestock and Páramo (subject to the lowest degree of human intervention possible), on three farms in the El Bosque District at three different altitudes (Buenos Aires, El Edén and La Secreta) during two seasons (dry and rainy). The results showed that none of the physical parameters under study presented statistically significant differences due to the type of use (livestock, potato crop or Páramo), season of sampling (dry or rainy season) or altitude (different farms). The chemical parameters that statistically significantly differed due to land use were organic carbon, cation exchange capacity, calcium, potassium, and ammonium and those that showed statistically significant differences associated with the sampling timing were organic carbon, nitrogen, cation exchange capacity, total carbon, C/N and nitrate. Additionally, there were differences in organic carbon due to the altitude of the farms. With respect to enzymatic activities, those of β-glucosidase, phosphodiesterase and urease significantly decreased in soils under potato cultivation and livestock relative to those of Páramo, but those of acid phosphatase and protease increased

Tunable Enzymatic Activity and Enhanced Stability of Cellulase Immobilized in Biohybrid Nanogels.

PubMed

Peng, Huan; Rübsam, Kristin; Jakob, Felix; Schwaneberg, Ulrich; Pich, Andrij

2016-11-14

the enzyme. The biohybrid nanogels demonstrated significantly improved stability in preserving enzymatic activity compared with free cellulase. The functional biohybrid nanogels with tunable enzymatic activity and improved stability are promising candidates for applications in biocatalysis, biomass conversion, or energy utilization fields.

Enzymatic vegetable organic extracts as soil biochemical biostimulants and atrazine extenders.

PubMed

García-Martínez, Ana María; Tejada, Manuel; Díaz, Ana Isabel; Rodríguez-Morgado, Bruno; Bautista, Juan; Parrado, Juan

2010-09-08

The purpose of this study was to gather information on the potential effects of organic biostimulants on soil activity and atrazine biodegradation. Carob germ enzymatic extract (CGEE) and wheat condensed distiller solubles enzymatic extract (WCDS-EE) have been obtained using an enzymatic process; their main organic components are soluble carbohydrates and proteins in the form of peptides and free amino acids. Their application to soil results in high biostimulation, rapidly increased dehydrogenase, phosphatase and glucosidase activities, and an observed atrazine extender capacity due to inhibition of its mineralization. The extender capacity of both extracts is proportional to the protein/carbohydrate ratio content. As a result, these enzymatic extracts are highly microbially available, leading to two independent phenomena, fertility and an atrazine persistence that is linked to increased soil activity.

Antioxidative activities of hydrolysates from edible birds nest using enzymatic hydrolysis

NASA Astrophysics Data System (ADS)

Muhammad, Nurul Nadia; Babji, Abdul Salam; Ayub, Mohd Khan

2015-09-01

Edible bird's nest protein hydrolysates (EBN) were prepared via enzymatic hydrolysis to investigate its antioxidant activity. Two types of enzyme (alcalase and papain) were used in this study and EBN had been hydrolysed with different hydrolysis time (30, 60, 90 and 120 min). Antioxidant activities in EBN protein hydrolysate were measured using DPPH, ABTS+ and Reducing Power Assay. From this study, increased hydrolysis time from 30 min to 120 min contributed to higher DH, as shown by alcalase (40.59%) and papain (24.94%). For antioxidant assay, EBN hydrolysed with papain showed higher scavenging activity and reducing power ability compared to alcalase. The highest antioxidant activity for papain was at 120 min hydrolysis time with ABTS (54.245%), DPPH (49.78%) and Reducing Power (0.0680). Meanwhile for alcalase, the highest antioxidant activity was at 30 min hydrolysis time. Even though scavenging activity for EBN protein hydrolysates were high, the reducing power ability was quite low as compared to BHT and ascorbic Acid. This study showed that EBN protein hydrolysate with alcalase and papain treatments potentially exhibit high antioxidant activity which have not been reported before.

Long-term effects of nickel oxide nanoparticles on performance, microbial enzymatic activity, and microbial community of a sequencing batch reactor.

PubMed

Wang, Sen; Li, Zhiwei; Gao, Mengchun; She, Zonglian; Guo, Liang; Zheng, Dong; Zhao, Yangguo; Ma, Bingrui; Gao, Feng; Wang, Xuejiao

2017-02-01

The nitrogen and phosphorus removal, microbial enzymatic activity, and microbial community of a sequencing batch reactor (SBR) were evaluated under long-term exposure to nickel oxide nanoparticles (NiO NPs). High NiO NP concentration (over 5 mg L -1 ) affected the removal of chemical oxygen demand, nitrogen, and phosphorus. The presence of NiO NP inhibited the microbial enzymatic activities and reduced the nitrogen and phosphorus removal rates of activated sludge. The microbial enzymatic activities of the activated sludge showed a similar variation trend to the nitrogen and phosphorus removal rates with the increase in NiO NP concentration from 0 to 60 mg L -1 . The Ni content in the effluent and activated sludge showed an increasing trend with the increase in NiO NP concentration. Some NiO NPs were absorbed on the sludge surface or penetrate the cell membrane into the interior of microbial cells in the activated sludge. NiO NP facilitated the increase in reactive oxygen species by disturbing the balance between the oxidation and anti-oxidation processes, and the variation in lactate dehydrogenase demonstrated that NiO NP could destroy the cytomembrane and cause variations in the microbial morphology and physiological function. High-throughput sequencing demonstrated that the microbial community of SBR had some obvious changes at 0-60 mg L -1 NiO NPs at the phyla, class and genus levels. Copyright © 2016 Elsevier Ltd. All rights reserved.

Solid-State Treatment of Castor Cake Employing the Enzymatic Cocktail Produced from Pleurotus djamor Fungi.

PubMed

Sánchez-Cantú, Manuel; Ortiz-Moreno, Liliana; Ramos-Cassellis, María E; Marín-Castro, Marco; De la Cerna-Hernández, C

2018-06-01

In this work, the enzymatic cocktail produced by Pleurotus djamor fungi extracted at pH of 4.8 and 5.3 was employed for castor cake solid-state treatment. Proximal, X-ray powder diffraction and scanning electron microscopy analysis of the pristine castor cake were carried out. First, Pleurotus djamor stain was inoculated in castor cake for the enzymatic production and the enzymatic activity was determined. The maximum enzymatic activity was identified at days 14 (65.9 UI/gss) and 11 (140.3 UI/gss) for the enzymatic cocktail obtained at pH 5.3 and 4.8, respectively. Then, the enzymatic cocktail obtained at the highest enzymatic activity days was employed directly over castor cake. Lignin was degraded throughout incubation time achieving a 47 and 45% decrease for the cocktail produced at pH 4.8 and 5.3, correspondingly. These results were corroborated by the SEM and XRD analysis where a higher porosity and xylan degradation were perceived throughout the enzymatic treatment.

Mapping of Functional Domains of the Lipid Kinase Phosphatidylinositol 4-Kinase Type III Alpha Involved in Enzymatic Activity and Hepatitis C Virus Replication

PubMed Central

Harak, Christian; Radujkovic, Danijela; Taveneau, Cyntia; Reiss, Simon; Klein, Rahel; Bressanelli, Stéphane

2014-01-01

ABSTRACT The lipid kinase phosphatidylinositol 4-kinase III alpha (PI4KIIIα) is an endoplasmic reticulum (ER)-resident enzyme that synthesizes phosphatidylinositol 4-phosphate (PI4P). PI4KIIIα is an essential host factor for hepatitis C virus (HCV) replication. Interaction with HCV nonstructural protein 5A (NS5A) leads to kinase activation and accumulation of PI4P at intracellular membranes. In this study, we investigated the structural requirements of PI4KIIIα in HCV replication and enzymatic activity. Therefore, we analyzed PI4KIIIα mutants for subcellular localization, reconstitution of HCV replication in PI4KIIIα knockdown cell lines, PI4P induction in HCV-positive cells, and lipid kinase activity in vitro. All mutants still interacted with NS5A and localized in a manner similar to that of the full-length enzyme, suggesting multiple regions of PI4KIIIα are involved in NS5A interaction and subcellular localization. Interestingly, the N-terminal 1,152 amino acids were dispensable for HCV replication, PI4P induction, and enzymatic function, whereas further N-terminal or C-terminal deletions were deleterious, thereby defining the minimal PI4KIIIα core enzyme at a size of ca. 108 kDa. Additional deletion of predicted functional motifs within the C-terminal half of PI4KIIIα also were detrimental for enzymatic activity and for the ability of PI4KIIIα to rescue HCV replication, with the exception of a proposed nuclear localization signal, suggesting that the entire C-terminal half of PI4KIIIα is involved in the formation of a minimal enzymatic core. This view was supported by structural modeling of the PI4KIIIα C terminus, suggesting a catalytic center formed by an N- and C-terminal lobe and an armadillo-fold motif, which is preceded by three distinct alpha-helical domains probably involved in regulation of enzymatic activity. IMPORTANCE The lipid kinase PI4KIIIα is of central importance for cellular phosphatidylinositol metabolism and is a key host cell

Pressure Modulation of the Enzymatic Activity of Phospholipase A2, A Putative Membrane-Associated Pressure Sensor.

PubMed

Suladze, Saba; Cinar, Suleyman; Sperlich, Benjamin; Winter, Roland

2015-10-07

Phospholipases A2 (PLA2) catalyze the hydrolysis reaction of sn-2 fatty acids of membrane phospholipids and are also involved in receptor signaling and transcriptional pathways. Here, we used pressure modulation of the PLA2 activity and of the membrane's physical-chemical properties to reveal new mechanistic information about the membrane association and subsequent enzymatic reaction of PLA2. Although the effect of high hydrostatic pressure (HHP) on aqueous soluble and integral membrane proteins has been investigated to some extent, its effect on enzymatic reactions operating at the water/lipid interface has not been explored, yet. This study focuses on the effect of HHP on the structure, membrane binding and enzymatic activity of membrane-associated bee venom PLA2, covering a pressure range up to 2 kbar. To this end, high-pressure Fourier-transform infrared and high-pressure stopped-flow fluorescence spectroscopies were applied. The results show that PLA2 binding to model biomembranes is not significantly affected by pressure and occurs in at least two kinetically distinct steps. Followed by fast initial membrane association, structural reorganization of α-helical segments of PLA2 takes place at the lipid water interface. FRET-based activity measurements reveal that pressure has a marked inhibitory effect on the lipid hydrolysis rate, which decreases by 75% upon compression up to 2 kbar. Lipid hydrolysis under extreme environmental conditions, such as those encountered in the deep sea where pressures up to the kbar-level are encountered, is hence markedly affected by HHP, rendering PLA2, next to being a primary osmosensor, a good candidate for a sensitive pressure sensor in vivo.

A pilot study of the modulation of sirtuins on arylamine N-acetyltransferase 1 and 2 enzymatic activity.

PubMed

Turiján-Espinoza, Eneida; Salazar-González, Rául Alejandro; Uresti-Rivera, Edith Elena; Hernández-Hernández, Gloria Estela; Ortega-Juárez, Montserrat; Milán, Rosa; Portales-Pérez, Diana

2018-03-01

Arylamine N -acetyltransferase (NAT; E.C. 2.3.1.5) enzymes are responsible for the biotransformation of several arylamine and hydrazine drugs by acetylation. In this process, the acetyl group transferred to the acceptor substrate produces NAT deacetylation and, in consequence, it is susceptible of degradation. Sirtuins are protein deacetylases, dependent on nicotine adenine dinucleotide, which perform post-translational modifications on cytosolic proteins. To explore possible sirtuin participation in the enzymatic activity of arylamine NATs, the expression levels of NAT1, NAT2, SIRT1 and SIRT6 in peripheral blood mononuclear cells (PBMC) from healthy subjects were examined by flow cytometry and Western blot. The in situ activity of the sirtuins on NAT enzymatic activity was analyzed by HPLC, in the presence or absence of an agonist (resveratrol) and inhibitor (nicotinamide) of sirtuins. We detected a higher percentage of positive cells for NAT2 in comparison with NAT1, and higher numbers of SIRT1+ cells compared to SIRT6 in lymphocytes. In situ NAT2 activity in the presence of NAM inhibitors was higher than in the presence of its substrate, but not in the presence of resveratrol. In contrast, the activity of NAT1 was not affected by sirtuins. These results showed that NAT2 activity might be modified by sirtuins.

Enzymatic activities and effects of mycovirus infection on the virulence of Metarhizium anisopliae in Rhipicephalus microplus.

PubMed

Perinotto, Wendell M S; Golo, Patricia S; Coutinho Rodrigues, Caio J B; Sá, Fillipe A; Santi, Lucélia; Beys da Silva, Walter O; Junges, Angela; Vainstein, Marilene H; Schrank, Augusto; Salles, Cristiane M C; Bittencourt, Vânia R E P

2014-06-16

The present study aimed to evaluate the pathogenic potential of different Metarhizium anisopliae s.l. isolates and to determine whether differences in enzymatic activities of proteases, lipases and chitinases and infection with mycoviruses affect the control of Rhipicephalus microplus achieved by these fungal isolates. Engorged female ticks were exposed to fungal suspensions. The lipolytic and proteolytic activities in the isolates were evaluated using chromogenic substrates and the chitinolytic activity was determined using fluorescent substrates. A gel zymography was performed to determine the approximate size of serine proteases released by M. anisopliae isolates. To detect mycoviral infections, dsRNA was digested using both RNAse A and S1 endonuclease; samples were analyzed on an agarose gel. Four of the five isolates tested were infected with mycovirus; however, the level of control of R. microplus ticks achieved with the only isolate free of infection (isolate CG 347) was low. This finding suggests that mycoviral infection does not affect the virulence of fungi against ticks. Although all five isolates were considered pathogenic to R. microplus, the best tick control and the highest levels of enzymatic activity were achieved with the isolates CG 629 and CG 148. The in vitro activities of lipases, proteases and chitinases produced by M. anisopliae s.l. differed among isolates and may be related to their virulence. Copyright © 2014 Elsevier B.V. All rights reserved.

Association between enzymatic and non-enzymatic antioxidant defense mechanism with apolipoprotein E genotypes in Alzheimer disease.

PubMed

Kharrazi, Hadi; Vaisi-Raygani, Asad; Rahimi, Zohreh; Tavilani, Haidar; Aminian, Mahdi; Pourmotabbed, Tayebeh

2008-08-01

There are evidence suggesting that APOE-varepsilon4 allele play an important role in the pathogenesis of Alzheimer's disease (AD) by reducing peripheral levels and activities of a broad spectrum of nonenzymatic and enzymatic antioxidants systems. However, the link between APOE genotype, oxidative stress, and AD has yet to be established. In this study we examined whether antioxidant defense mechanism exacerbates the risk of AD in individual carrying APOE-varepsilon4 allele in a population from Tehran, Iran. We determined the enzymatic activities of the erythrocyte Cu-Zn superoxide dismutase (Cu-Zn SOD), glutathione peroxidase (GSH-Px), catalase (CAT) and serum level of total antioxidant status(TAS) in various APOE genotypes in 91 patients with AD and 91 healthy subjects as control group (age and sex-matched). The results showed that the TAS level and the activities of enzymatic antioxidants CAT and GSH-Px were significantly lower and the SOD activity was significantly higher in AD patients compared to controls. The AD patients with APOE-varepsilon4 allele genotype had significantly lower serum TAS concentration and lower erythrocytes GSH-Px and CAT activities (p=0.001) but significantly higher erythrocytes Cu-Zn SOD activity (p=0.001) than the non-APOE-varepsilon4 carrier AD and the control group. In addition, the association observed between the factors involved in an antioxidant defense mechanism and APOE-varepsilon4 allele in AD increased with age of the subjects. These data indicate that the reduced serum level of TAS and activity of CAT, GSH-Px and increased SOD exacerbate the risk of AD in individuals carrying APOE-varepsilon4 allele. The reduced antioxidants defense in APOE-varepsilon4 allele carrier may contribute to beta-amyloidosis. This effect, however, is more pronounced in the AD patients older than 75 years of age. This suggests that a therapeutic modality should be considered for these subjects.

Influence of nitrogen sources on the enzymatic activity and grown by Lentinula edodes in biomass Eucalyptus benthamii.

PubMed

Pedri, Z C; Lozano, L M S; Hermann, K L; Helm, C V; Peralta, R M; Tavares, L B B

2015-11-01

Lignocellulose is the most abundant environmental component and a renewable organic resource in soil. There are some filamentous fungi which developed the ability to break down and use cellulose, hemicellulose and lignin as an energy source. The objective of this research was to analyze the effect of three nitrogen resources (ammonium sulfate, saltpetre, soybean) in the holocellulolitic activity of Lentinula edodes EF 50 using as substrate sawdust E. benthamii. An experimental design mixture was applied with repetition in the central point consisting of seven treatments (T) of equal concentrations of nitrogen in ammonium sulfate, potassium nitrate and soybean. The enzymatic activity of avicelase, carboxymetilcellulase, β-glucosidase, xylanases and manganese peroxidase was determined. The humidity, pH, water activity (aw) and qualitative analysis of mycelial growth in 8 times of cultivation were evaluated. The results showed negative effect on enzyme production in treatments with maximum concentration of ammonium sulfate and potassium nitrate. The treatments with cooked soybean flour expressed higher enzymatic activities in times of 3, 6 and 9 days of culture, except in the activity of manganese peroxidase. The highest production was observed in the treatment with ammonium sulfate, and soybean (83.86 UI.L-1) at 20 days of cultivation.

Enzymatic Activity Detection via Electrochemistry for Enceladus

NASA Technical Reports Server (NTRS)

Studemeister, Lucy; Koehne, Jessica; Quinn, Richard

2017-01-01

Electrochemical detection of biological molecules is a pertinent topic and application in many fields such as medicine, environmental spills, and life detection in space. Proteases, a class of molecules of interest in the search for life, catalyze the hydrolysis of peptides. Trypsin, a specific protease, was chosen to investigate an optimized enzyme detection system using electrochemistry. This study aims at providing the ideal functionalization of an electrode that can reliably detect a signal indicative of an enzymatic reaction from an Enceladus sample.

Phenotypic characterization of enzymatic activity of clinical dermatophyte isolates from animals with and without skin lesions and humans.

PubMed

Gnat, Sebastian; Łagowski, Dominik; Nowakiewicz, Aneta; Zięba, Przemysław

2018-05-20

The pathogenesis of dermatophytoses is associated with the secretion of enzymes degrading the infected tissue components. Although many studies on enzymatic activity of dermatophytes have been conducted over the years, there have been no concrete proposals on construction of the profile of enzymes characteristic of individual species, genus, or ecological types of dermatophytes. The aim of this study was to assess the capability of clinical dermatophyte isolates from both symptomatic and asymptomatic animals and humans to produce different enzymes. Clinical isolates of 234 dermatophyte strains collected during routine examination of animal health were used in this study. The enzymatic production of keratinase, elastase, phospholipase, lipase, protease, DNase, and gelatinase as well as the haemolytic activity were evaluated using specific test media. The overall degree of enzymatic activity of the analysed clinical isolates of the dermatophytes was 67%. All tested clinical isolates of different species of dermatophytes showed keratinase activity and 96% additionally exhibited phospholipase activity. The weakest activity among the tested enzymes was demonstrated for elastase and gelatinase. 83% of the isolates of the dermatophytes showed haemolytic activity. Our data indicate that clinical isolates of dermatophytes from different species produce enzymes with different levels of activities. Profile of enzymes characteristic of individual species, genus, or ecological types of dermatophytes is possibly dependent upon factors related to the host. The relationship between each enzyme and the occurrence of skin lesions in animals and humans or asymptomatic animal carriers varies on whether the infection is caused by T. mentagrophytes, T. verrucosum, or M. canis. Interestingly, only keratinase seems to be correlated with the appearance of dermatophyte infections, irrespective of the pathogen species, and elastase is a characteristic enzyme for dermatophyte strains

Real-time ESI-MS of enzymatic conversion: impact of organic solvents and multiplexing.

PubMed

Scheerle, Romy K; Grassmann, Johanna; Letzel, Thomas

2012-01-01

Different enzymatic assays were characterized systematically by real-time electrospray ionization mass spectrometry (ESI-MS) in the presence of organic solvents as well as in multiplex approaches and in a combination of both. Typically, biological enzymatic reactions are studied in aqueous solutions, since most enzymes show their full activity solely in aqueous solutions. However, in recent years, the use of organic solvents in combination with enzymatic reactions has gained increasing interest due to biotechnological advantages in chemical synthesis, development of online coupled setups screening for enzyme regulatory compounds, advantages regarding mass spectrometric detection and others. In the current study, the influence of several common organic solvents (methanol, ethanol, isopropanol, acetone, acetonitrile) on enzymatic activity (hen egg white lysozyme, chitinase, α-chymotrypsin, elastase from human neutrophils and porcine pancreas, acetylcholinesterase) was tested. Moreover, multiplexing is a promising approach enabling fast and cost-efficient screening methods, e.g. for determination of inhibitors in complex mixtures or in the field of biomedical research. Although in multiplexed setups the enzymatic activity may be affected by the presence of other substrates and/or enzymes, the expected advantages possibly will predominate. To investigate those effects, we measured multiple enzymatic assays simultaneously. For all conducted measurements, the conversion rate of the substrate(s) was calculated, which reflects the enzymatic activity. The results provide an overview about the susceptibility of the selected enzymes towards diverse factors and a reference point for many applications in analytical chemistry and biotechnology.

Photoelectrochemical enzymatic biosensors.

PubMed

Zhao, Wei-Wei; Xu, Jing-Juan; Chen, Hong-Yuan

2017-06-15

Enzymatic biosensors have been valuable bioanalytical devices for analysis of diverse targets in disease diagnosis, biological and biomedical research, etc. Photoelectrochemical (PEC) bioanalysis is a recently emerged method that promptly becoming a subject of new research interests due to its attractive potential for future bioanalysis with high sensitivity and specificity. PEC enzymatic biosensors integrate the inherent sensitivities of PEC bioanalysis and the selectivity of enzymes and thus share their both advantages. Currently, PEC enzymatic biosensors have become a hot topic of significant research and the recent impetus has grown rapidly as demonstrated by increased research papers. Given the pace of advances in this area, this review will make a thorough discussion and survey on the fundamentals, sensing strategies, applications and the state of the art in PEC enzymatic biosensors, followed by future prospects based on our own opinions. We hope this work could provide an accessible introduction to PEC enzymatic biosensors for any scientist. Copyright © 2016 Elsevier B.V. All rights reserved.

Effect of gypsum application on enzymatic browning activity in lettuce.

PubMed

Chutichudet, Prasit; Chutichudet, B; Kaewsit, S

2009-09-15

A comprehensive study to evaluate calcium, in terms of gypsum (CaSO4.2H2O) by soil dressing application, on enzymatic browning activity of Polyphenol oxidase (PPO) and internal qualities was tested on lettuce var. Grand Rapids under field conditions. A factorial in completely randomized design was arranged with four replications. The results showed that plants-treated with 50 mg kg(-1) gypsum applied at 40 DAP had the maximal fresh weight of 25.83 g plant(-1). The internal qualities of the lettuce at harvest showed that plants treated with 50 mg kg(-1) gypsum had the maximal chlorophyll content (26.80 mg m(-2)), while all gypsum concentrations applied in this study, had less content of ascorbic acid than the control plants. Plants-treated with 100 mg kg(-1) gypsum affected to the lowest level of PPO activity at week 3 after transplanting. Furthermore, gypsum application had no effect to biomass, leaf colour, the contents of phenolic and quinone in lettuce at harvesting stage.

Biocolloids with ordered urease multilayer shells as enzymatic reactors.

PubMed

Lvov, Y; Caruso, F

2001-09-01

The preparation of biocolloids with organized enzyme-containing multilayer shells for exploitation as colloidal enzymatic nanoreactors is described. Urease multilayers were assembled onto submicrometer-sized polystyrene spheres by the sequential adsorption of urease and polyelectrolyte, in a predetermined order, utilizing electrostatic interactions for layer growth. The catalytic activity of the biocolloids increased proportionally with the number of urease layers deposited on the particles, demonstrating that biocolloid particles with tailored enzymatic activities can be produced. It was further found that precoating the latex spheres with nanoparticles (40-nm silica or 12-nm magnetite) enhanced both the stability (with respect to adsorption) and enzymatic activity of the urease multilayers. The presence of the magnetite nanoparticle coating also provided a magnetic function that allowed the biocolloids to be easily and rapidly separated with a permanent magnet. The fabrication of such colloids opens new avenues for the application of bioparticles and represents a promising route for the creation of complex catalytic particles.

Enzymatic biofuel cell-based self-powered biosensing of protein kinase activity and inhibition via thiophosphorylation-mediated interface engineering.

PubMed

Gu, Chengcheng; Gai, Panpan; Han, Lei; Yu, Wen; Liu, Qingyun; Li, Feng

2018-05-24

We developed a facile and ultrasensitive enzymatic biofuel cell (EBFC)-based self-powered biosensor of protein kinase A (PKA) activity and inhibition via thiophosphorylation-mediated interface engineering. The detection limit was down to 0.00022 U mL-1 (S/N = 3). In addition, the PKA activities from MCF-7 and A549 cell lysates were analyzed and achieved reliable results.

Herbivore species identity and composition affect soil enzymatic activity through altered plant composition in a coastal tallgrass prairie

USDA-ARS?s Scientific Manuscript database

Although single species of herbivores are known to affect soil microbial communities, the effects of herbivore species identity and functional composition on soil microbes is unknown. We tested the effects of single species of orthopterans and multiple species combinations on soil enzymatic activity...

Activity-Dependent Enzymatic Assay for the Detection of Toluene-Oxidizing Bacteria Capable of Trichloroethylene Degradation

NASA Astrophysics Data System (ADS)

Kauffman, M. E.; Kauffman, M. E.; Keener, W. K.; Watwood, M. E.; Lehman, R. M.

2001-12-01

Toluene-oxidizing bacteria produce enzymes that cometabolically degrade trichloroethylene (TCE). These inducible enzymes are produced only in the presence of certain aromatic substrates such as toluene or phenol. Recent laboratory studies have utilized analog chemical substrates to identify production of bacterial enzymes capable of degrading trichloroethylene. These analog substrates produce chromogenic and/or fluorescent products when biotransformed by the enzymes of interest. In this study, 3-hydroxyphenylacetylene (3-HPA) was identified as an activity-dependent enzymatic probe for the detection of three of the four known toluene oxygenase enzymes capable of TCE degradation. Laboratory studies were conducted using pure cultures of Burkholderia cepacia G4, Burkholderia pickettii PKO1, and Pseudomonas putida F1. Cell cultures grown on lactate (non-enzyme inducing) or lactate and toluene (inducing) were trapped trapped on black polycarbonate filters, exposed to 3-HPA, and examined for fluorescence using an epifluorescent microscope. Additionally, B. cepacia G4 cells were grown under the same conditions, but in the presence of mineral and basalt specimens to allow for bacterial attachment. The specimens were then exposed to 3-HPA and examined under an epifluorescent microscope. Our results demonstrate that cells induced for the production of oxygenase enzymes, both unattached and attached, are able to transform 3-HPA to a fluorescent product, although cells attached to geologic materials, such as basalt, take substantially longer to transform the probe. Cells grown under non-inducing conditions do not transform the probe, regardless of their attachment status. Additionally, well water samples taken from a TCE-contaminated aquifer were successfully assayed using the 3-HPA enzymatic probe. The development of this enzyme activity-dependent enzymatic assay provides a fast and reliable method to assess the potential for TCE and aromatic contaminant bioremediation.

Self-Assembly of Spider Silk-Fusion Proteins Comprising Enzymatic and Fluorescence Activity.

PubMed

Humenik, Martin; Mohrand, Madeleine; Scheibel, Thomas

2018-04-18

The recombinant spider silk protein eADF4(C16) was genetically fused either with esterase 2 (EST2) or green fluorescent protein (GFP). The fusions EST-eADF4(C16) and GFP-eADF4(C16) were spectroscopically investigated and showed native structures of EST and GFP. The structural integrity was confirmed by the enzymatic activity of EST and the fluorescence of GFP. The spider silk moiety retained its intrinsically unstructured conformation in solution and the self-assembly into either nanofibrils or nanoparticles could be controlled by the concentration of phosphate. Particles, however, showed significantly lower activity of the EST and GFP domains likely caused by a steric hindrance. However, upon self-assembly of EST-eADF4(C16) and GFP-eADF4(C16) into fibrils the protein activities were retained. In general, the fusion of globular enzymes with the spider silk domain allows the generation of fibrous biomaterials with catalytic or light emitting properties.

Vancomycin analogues containing monosaccharides exhibit improved antibiotic activity: a combined one-pot enzymatic glycosylation and chemical diversification strategy.

PubMed

Thayer, Desiree A; Wong, Chi-Huey

2006-09-18

Many natural products contain carbohydrate moieties that contribute to their biological activity. Manipulation of the carbohydrate domain of natural products through multiple glycosylations to identify new derivatives with novel biological activities has been a difficult and impractical process. We report a practical one-pot enzymatic approach with regeneration of cosubstrates to synthesize analogues of vancomycin that contain an N-alkyl glucosamine, which exhibited marked improvement in antibiotic activity against a vancomycin-resistant strain of Enterococcus.

Multi-Scale Computational Enzymology: Enhancing Our Understanding of Enzymatic Catalysis

PubMed Central

Gherib, Rami; Dokainish, Hisham M.; Gauld, James W.

2014-01-01

Elucidating the origin of enzymatic catalysis stands as one the great challenges of contemporary biochemistry and biophysics. The recent emergence of computational enzymology has enhanced our atomistic-level description of biocatalysis as well the kinetic and thermodynamic properties of their mechanisms. There exists a diversity of computational methods allowing the investigation of specific enzymatic properties. Small or large density functional theory models allow the comparison of a plethora of mechanistic reactive species and divergent catalytic pathways. Molecular docking can model different substrate conformations embedded within enzyme active sites and determine those with optimal binding affinities. Molecular dynamics simulations provide insights into the dynamics and roles of active site components as well as the interactions between substrate and enzymes. Hybrid quantum mechanical/molecular mechanical (QM/MM) can model reactions in active sites while considering steric and electrostatic contributions provided by the surrounding environment. Using previous studies done within our group, on OvoA, EgtB, ThrRS, LuxS and MsrA enzymatic systems, we will review how these methods can be used either independently or cooperatively to get insights into enzymatic catalysis. PMID:24384841

Improvement of antioxidant and moisture-preserving activities of Sargassum horneri polysaccharide enzymatic hydrolyzates.

PubMed

Shao, Ping; Chen, Xiaoxiao; Sun, Peilong

2015-03-01

In the previous study, we have found that polysaccharides isolated from Sargassum horneri exhibited bioactivities. The aim of this study was to investigate the antioxidant and moisture-preserving activities of molecular weight alteration of Sargassum horneri polysaccharide in vitro. For this purpose, the homogeneous active polysaccharide SHP was isolated from Sargassum horneri, and response surface methodology was employed to optimize the enzymatic degradation conditions to get SHP-derived fragments with different molecular weight. Results proved that the polysaccharide is capable of scavenging both ABTS and DPPH radicals in vitro. The study revealed that the polysaccharides had strong moisture-absorption and -retention capacities as compared to propanediol and glycerin. Furthermore, these data demonstrated that molecular weight had a certain effect on antioxidant activities and strong moisture-retention capacities of the polysaccharide from Sargassum horneri. Copyright © 2014 Elsevier B.V. All rights reserved.

Dissecting the link between the enzymatic activity and the SaPI inducing capacity of the phage 80α dUTPase.

PubMed

Alite, Christian; Humphrey, Suzanne; Donderis, Jordi; Maiques, Elisa; Ciges-Tomas, J Rafael; Penadés, José R; Marina, Alberto

2017-09-11

The trimeric staphylococcal phage-encoded dUTPases (Duts) are signalling molecules that induce the cycle of some Staphylococcal pathogenicity islands (SaPIs) by binding to the SaPI-encoded Stl repressor. To perform this regulatory role, these Duts require an extra motif VI, as well as the Dut conserved motifs IV and V. While the apo form of Dut is required for the interaction with the Stl repressor, usually only those Duts with normal enzymatic activity can induce the SaPI cycle. To understand the link between the enzymatic activities and inducing capacities of the Dut protein, we analysed the structural, biochemical and physiological characteristics of the Dut80α D95E mutant, which loses the SaPI cycle induction capacity despite retaining enzymatic activity. Asp95 is located at the threefold central channel of the trimeric Dut where it chelates a divalent ion. Here, using state-of-the-art techniques, we demonstrate that D95E mutation has an epistatic effect on the motifs involved in Stl binding. Thus, ion binding in the central channel correlates with the capacity of motif V to twist and order in the SaPI-inducing disposition, while the tip of motif VI is disturbed. These alterations in turn reduce the affinity for the Stl repressor and the capacity to induce the SaPI cycle.

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

Selection and validation of enzymatic activities as functional markers in wood biotechnology and fungal ecology.

PubMed

Mathieu, Yann; Gelhaye, Eric; Dumarçay, Stéphane; Gérardin, Philippe; Harvengt, Luc; Buée, Marc

2013-02-15

The dead wood and forest soils are sources of diversity and under-explored fungal strains with biotechnological potential, which require to be studied. Numerous enzymatic tests have been proposed to investigate the functional potential of the soil microbial communities or to test the functional abilities of fungal strains. Nevertheless, the diversity of these functional markers and their relevance in environmental studies or biotechnological screening does still have not been demonstrated. In this work, we assessed ten different extracellular enzymatic activities involved in the wood decaying process including β-etherase that specifically cleaves the β-aryl ether linkages in the lignin polymer. For this purpose, a collection of 26 fungal strains, distributed within three ecological groups (white, brown and soft rot fungi), has been used. Among the ten potential functional markers, the combinatorial use of only six of them allowed separation between the group of white and soft rot fungi from the brown rot fungi. Moreover, our results suggest that extracellular β-etherase is a rare and dispensable activity among the wood decay fungi. Finally, we propose that this set of markers could be useful for the analysis of fungal communities in functional and environmental studies, and for the selection of strains with biotechnological interests. Copyright © 2012 Elsevier B.V. All rights reserved.

A high-throughput assay for enzymatic polyester hydrolysis activity by fluorimetric detection.

PubMed

Wei, Ren; Oeser, Thorsten; Billig, Susan; Zimmermann, Wolfgang

2012-12-01

A fluorimetric assay for the fast determination of the activity of polyester-hydrolyzing enzymes in a large number of samples has been developed. Terephthalic acid (TPA) is a main product of the enzymatic hydrolysis of polyethylene terephthalate (PET), a synthetic polyester. Terephthalate has been quantified following its conversion to the fluorescent 2-hydroxyterephthalate by an iron autoxidation-mediated generation of free hydroxyl radicals. The assay proved to be robust at different buffer concentrations, reaction times, pH values, and in the presence of proteins. A validation of the assay was performed by analyzing TPA formation from PET films and nanoparticles catalyzed by a polyester hydrolase from Thermobifida fusca KW3 in a 96-well microplate format. The results showed a close correlation (R(2) = 0.99) with those obtained by a considerably more tedious and time-consuming HPLC method, suggesting the aptness of the fluorimetric assay for a high-throughput screening for polyester hydrolases. The method described in this paper will facilitate the detection and development of biocatalysts for the modification and degradation of synthetic polymers. The fluorimetric assay can be used to quantify the amount of TPA obtained as the final degradation product of the enzymatic hydrolysis of PET. In a microplate format, this assay can be applied for the high-throughput screening of polyester hydrolases. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Substitutions in PBP2b from β-Lactam-resistant Streptococcus pneumoniae Have Different Effects on Enzymatic Activity and Drug Reactivity*

PubMed Central

Calvez, Philippe; Breukink, Eefjan; Roper, David I.; Dib, Mélanie; Contreras-Martel, Carlos; Zapun, André

2017-01-01

Pneumococcus resists β-lactams by expressing variants of its target enzymes, the penicillin-binding proteins (PBPs), with many amino acid substitutions. Up to 10% of the sequence can be modified. These altered PBPs have a much reduced reactivity with the drugs but retain their physiological activity of cross-linking the peptidoglycan, the major constituent of the bacterial cell wall. However, because β-lactams are chemical and structural mimics of the natural substrate, resistance mediated by altered PBPs raises the following paradox: how PBPs that react poorly with the drugs maintain a sufficient level of activity with the physiological substrate. This question is addressed for the first time in this study, which compares the peptidoglycan cross-linking activity of PBP2b from susceptible and resistant strains with their inhibition by different β-lactams. Unexpectedly, the enzymatic activity of the variants did not correlate with their antibiotic reactivity. This finding indicates that some of the numerous amino acid substitutions were selected to restore a viable level of enzymatic activity by a compensatory molecular mechanism. PMID:28062575

Chromophoric dissolved organic matter and microbial enzymatic activity. A biophysical approach to understand the marine carbon cycle.

PubMed

Gonnelli, Margherita; Vestri, Stefano; Santinelli, Chiara

2013-12-01

This study reports the first information on extracellular enzymatic activity (EEA) combined with a study of DOM dynamics at the Arno River mouth. DOM dynamics was investigated from both a quantitative (dissolved organic carbon, DOC) and a qualitative (absorption and fluorescence of chromophoric DOM, CDOM) perspective. The data here reported highlight that the Arno River was an important source of both DOC and CDOM for this coastal area. CDOM optical properties suggested that terrestrial DOM did not undergo simple dilution at the river mouth but, other physical-chemical and biological processes were probably at work to change its molecular characteristics. This observation was further supported by the "potential" enzymatic activity of β-glucosidase (BG) and leucine aminopeptidase (LAP). Their Vmax values were markedly higher in the river water than in the seawater and their ratio suggested that most of the DOM used by microbes in the Arno River was polysaccharide-like, while in the seawater it was mainly protein-like. © 2013. Published by Elsevier B.V. All rights reserved.

Urease immobilized polymer hydrogel: Long-term stability and enhancement of enzymatic activity.

PubMed

Kutcherlapati, S N Raju; Yeole, Niranjan; Jana, Tushar

2016-02-01

A method has been developed in which an enzyme namely urease was immobilized inside hydrogel matrix to study the stability and enzymatic activity in room temperature (∼27-30°C). This urease coupled hydrogel (UCG) was obtained by amine-acid coupling reaction and this procedure is such that it ensured the wider opening of mobile flap of enzyme active site. A systematic comparison of urea-urease assay and the detailed kinetic data clearly revealed that the urease shows activity for more than a month when stored at ∼27-30°C in case of UCG whereas it becomes inactive in case of free urease (enzyme in buffer solution). The aqueous microenvironment inside the hydrogel, unusual morphological features and thermal behaviour were believed to be the reasons for unexpected behaviour. UCG displayed enzyme activity at basic pH and up to 60°C. UCG showed significant enhancement in activity against thermal degradation compared to free urease. In summary, this method is a suitable process to stabilize the biomacromolecules in standard room temperature for many practical uses. Copyright © 2015 Elsevier Inc. All rights reserved.

Enzymatic activity of anthropogenic proto-organic soils in soilless farming

NASA Astrophysics Data System (ADS)

Bireescu, Geanina; Dazzi, Carmelo; Laudicina, Vito Armando; Lo Papa, Giuseppe

2017-04-01

In soilless agriculture and horticulture coir is the more used substratum to grow plants because it is widely available and more environmentally friendly than sphagnum or peat. In Italy, soilless agriculture concerns an area of about 1,000 hectares, particularly concentrated in Sicily. The southern coastal belt of this region is the area interested by the most significant experiences in the application of techniques of soilless cultivation that, recently, has been used also for growing table grapes. Starting from the above consideration we suppose that the features of the coconut fiber underlay an evident transformation and that even after few years of table grape cultivation, such organic material undergone to a transformation that allows for the formation of a proto-organic soil (a proto-Histosol, we supposed). If this is true, we believe that, in this case, to speak about soilless cultivation is for sure misleading for the common people, as we should define this cultivation "on anthropogenic soils" instead. To fit the aims of this survey we used a big greenhouse devoted to soilless cultivation of table grape in a farm in the Southern Sicily We have considered the enzymatic activity that characterized the coconut fiber after 3 cycles of cultivation of table grapes. We used as a control the coconut fiber that the farmer used to prepare pots for soilless cultivation and coconut fiber of: 6 pots at the end of the first productive cycle 6 pots at the end of the second cycle and 3 pots at the end of the third cycle. On these organic samples we investigated three enzymes, belonging to oxydoreductase (catalase and dehydrogenase) and hydrolase (urease) classes. Statistical analysis of the investigated enzymes was developed using IBM Statistic SPSS v20 by ANOVA, Tukey test HSD for p ≤ 0.01 and Multivariate Statistical Analysis. Results have shown significant differences in enzymes content and quality among coir tests. The use of the coco fiber, as nutritive substratum

Differential Expression and Enzymatic Activity of DPPIV/CD26 Affects Migration Ability of Cervical Carcinoma Cells

PubMed Central

Beckenkamp, Aline; Willig, Júlia Biz; Santana, Danielle Bertodo; Nascimento, Jéssica; Paccez, Juliano Domiraci; Zerbini, Luiz Fernando; Bruno, Alessandra Nejar; Pilger, Diogo André; Wink, Márcia Rosângela; Buffon, Andréia

2015-01-01

Dipeptidyl peptidase IV (DPPIV/CD26) is a transmembrane glycoprotein that inactivates or degrades some bioactive peptides and chemokines. For this reason, it regulates cell proliferation, migration and adhesion, showing its role in cancer processes. This enzyme is found mainly anchored onto the cell membrane, although it also has a soluble form, an enzymatically active isoform. In the present study, we investigated DPPIV/CD26 activity and expression in cervical cancer cell lines (SiHa, HeLa and C33A) and non-tumorigenic HaCaT cells. The effect of the DPPIV/CD26 inhibitor (sitagliptin phosphate) on cell migration and adhesion was also evaluated. Cervical cancer cells and keratinocytes exhibited DPPIV/CD26 enzymatic activity both membrane-bound and in soluble form. DPPIV/CD26 expression was observed in HaCaT, SiHa and C33A, while in HeLa cells it was almost undetectable. We observed higher migratory capacity of HeLa, when compared to SiHa. But in the presence of sitagliptin SiHa showed an increase in migration, indicating that, at least in part, cell migration is regulated by DPPIV/CD26 activity. Furthermore, in the presence of sitagliptin phosphate, SiHa and HeLa cells exhibited a significant reduction in adhesion. However this mechanism seems to be mediated independent of DPPIV/CD26. This study demonstrates, for the first time, the activity and expression of DPPIV/CD26 in cervical cancer cells and the effect of sitagliptin phosphate on cell migration and adhesion. PMID:26222679

Differential Expression and Enzymatic Activity of DPPIV/CD26 Affects Migration Ability of Cervical Carcinoma Cells.

PubMed

Beckenkamp, Aline; Willig, Júlia Biz; Santana, Danielle Bertodo; Nascimento, Jéssica; Paccez, Juliano Domiraci; Zerbini, Luiz Fernando; Bruno, Alessandra Nejar; Pilger, Diogo André; Wink, Márcia Rosângela; Buffon, Andréia

2015-01-01

Dipeptidyl peptidase IV (DPPIV/CD26) is a transmembrane glycoprotein that inactivates or degrades some bioactive peptides and chemokines. For this reason, it regulates cell proliferation, migration and adhesion, showing its role in cancer processes. This enzyme is found mainly anchored onto the cell membrane, although it also has a soluble form, an enzymatically active isoform. In the present study, we investigated DPPIV/CD26 activity and expression in cervical cancer cell lines (SiHa, HeLa and C33A) and non-tumorigenic HaCaT cells. The effect of the DPPIV/CD26 inhibitor (sitagliptin phosphate) on cell migration and adhesion was also evaluated. Cervical cancer cells and keratinocytes exhibited DPPIV/CD26 enzymatic activity both membrane-bound and in soluble form. DPPIV/CD26 expression was observed in HaCaT, SiHa and C33A, while in HeLa cells it was almost undetectable. We observed higher migratory capacity of HeLa, when compared to SiHa. But in the presence of sitagliptin SiHa showed an increase in migration, indicating that, at least in part, cell migration is regulated by DPPIV/CD26 activity. Furthermore, in the presence of sitagliptin phosphate, SiHa and HeLa cells exhibited a significant reduction in adhesion. However this mechanism seems to be mediated independent of DPPIV/CD26. This study demonstrates, for the first time, the activity and expression of DPPIV/CD26 in cervical cancer cells and the effect of sitagliptin phosphate on cell migration and adhesion.

Is enzymatic hydrolysis a reliable analytical strategy to quantify glucuronidated and sulfated polyphenol metabolites in human fluids?

PubMed

Quifer-Rada, Paola; Martínez-Huélamo, Miriam; Lamuela-Raventos, Rosa M

2017-07-19

Phenolic compounds are present in human fluids (plasma and urine) mainly as glucuronidated and sulfated metabolites. Up to now, due to the unavailability of standards, enzymatic hydrolysis has been the method of choice in analytical chemistry to quantify these phase II phenolic metabolites. Enzymatic hydrolysis procedures vary in enzyme concentration, pH and temperature; however, there is a lack of knowledge about the stability of polyphenols in their free form during the process. In this study, we evaluated the stability of 7 phenolic acids, 2 flavonoids and 3 prenylflavanoids in urine during enzymatic hydrolysis to assess the suitability of this analytical procedure, using three different concentrations of β-glucuronidase/sulfatase enzymes from Helix pomatia. The results indicate that enzymatic hydrolysis negatively affected the recovery of the precursor and free-form polyphenols present in the sample. Thus, enzymatic hydrolysis does not seem an ideal analytical strategy to quantify glucuronidated and sulfated polyphenol metabolites.

Enzymatic Processes in Marine Biotechnology.

PubMed

Trincone, Antonio

2017-03-25

In previous review articles the attention of the biocatalytically oriented scientific community towards the marine environment as a source of biocatalysts focused on the habitat-related properties of marine enzymes. Updates have already appeared in the literature, including marine examples of oxidoreductases, hydrolases, transferases, isomerases, ligases, and lyases ready for food and pharmaceutical applications. Here a new approach for searching the literature and presenting a more refined analysis is adopted with respect to previous surveys, centering the attention on the enzymatic process rather than on a single novel activity. Fields of applications are easily individuated: (i) the biorefinery value-chain, where the provision of biomass is one of the most important aspects, with aquaculture as the prominent sector; (ii) the food industry, where the interest in the marine domain is similarly developed to deal with the enzymatic procedures adopted in food manipulation; (iii) the selective and easy extraction/modification of structurally complex marine molecules, where enzymatic treatments are a recognized tool to improve efficiency and selectivity; and (iv) marine biomarkers and derived applications (bioremediation) in pollution monitoring are also included in that these studies could be of high significance for the appreciation of marine bioprocesses.

Enzymatic Processes in Marine Biotechnology

PubMed Central

Trincone, Antonio

2017-01-01

In previous review articles the attention of the biocatalytically oriented scientific community towards the marine environment as a source of biocatalysts focused on the habitat-related properties of marine enzymes. Updates have already appeared in the literature, including marine examples of oxidoreductases, hydrolases, transferases, isomerases, ligases, and lyases ready for food and pharmaceutical applications. Here a new approach for searching the literature and presenting a more refined analysis is adopted with respect to previous surveys, centering the attention on the enzymatic process rather than on a single novel activity. Fields of applications are easily individuated: (i) the biorefinery value-chain, where the provision of biomass is one of the most important aspects, with aquaculture as the prominent sector; (ii) the food industry, where the interest in the marine domain is similarly developed to deal with the enzymatic procedures adopted in food manipulation; (iii) the selective and easy extraction/modification of structurally complex marine molecules, where enzymatic treatments are a recognized tool to improve efficiency and selectivity; and (iv) marine biomarkers and derived applications (bioremediation) in pollution monitoring are also included in that these studies could be of high significance for the appreciation of marine bioprocesses. PMID:28346336

Enzymatic Synthesis of Amino Acids Endcapped Polycaprolactone: A Green Route Towards Functional Polyesters.

PubMed

Duchiron, Stéphane W; Pollet, Eric; Givry, Sébastien; Avérous, Luc

2018-01-30

ε-caprolactone (CL) has been enzymatically polymerized using α-amino acids based on sulfur (methionine and cysteine) as (co-)initiators and immobilized lipase B of Candida antarctica (CALB) as biocatalyst. In-depth characterizations allowed determining the corresponding involved mechanisms and the polymers thermal properties. Two synthetic strategies were tested, a first one with direct polymerization of CL with the native amino acids and a second one involving the use of an amino acid with protected functional groups. The first route showed that mainly polycaprolactone (PCL) homopolymer could be obtained and highlighted the lack of reactivity of the unmodified amino acids due to poor solubility and affinity with the lipase active site. The second strategy based on protected cysteine showed higher monomer conversion, with the amino acids acting as (co-)initiators, but their insertion along the PCL chains remained limited to chain endcapping. These results thus showed the possibility to synthesize enzymatically polycaprolactone-based chains bearing amino acids units. Such cysteine endcapped PCL materials could then find application in the biomedical field. Indeed, subsequent functionalization of these polyesters with drugs or bioactive molecules can be obtained, by derivatization of the amino acids, after removal of the protecting group.

Micromechanical Modeling Study of Mechanical Inhibition of Enzymatic Degradation of Collagen Tissues

PubMed Central

Tonge, Theresa K.; Ruberti, Jeffrey W.; Nguyen, Thao D.

2015-01-01

This study investigates how the collagen fiber structure influences the enzymatic degradation of collagen tissues. We developed a micromechanical model of a fibrous collagen tissue undergoing enzymatic degradation based on two central hypotheses. The collagen fibers are crimped in the undeformed configuration. Enzymatic degradation is an energy activated process and the activation energy is increased by the axial strain energy density of the fiber. We determined the intrinsic degradation rate and characteristic energy for mechanical inhibition from fibril-level degradation experiments and applied the parameters to predict the effect of the crimped fiber structure and fiber properties on the degradation of bovine cornea and pericardium tissues under controlled tension. We then applied the model to examine the effect of the tissue stress state on the rate of tissue degradation and the anisotropic fiber structures that developed from enzymatic degradation. PMID:26682825

Biofunctional Properties of Enzymatic Squid Meat Hydrolysate

PubMed Central

Choi, Joon Hyuk; Kim, Kyung-Tae; Kim, Sang Moo

2015-01-01

Squid is one of the most important commercial fishes in the world and is mainly utilized or consumed as sliced raw fish or as processed products. The biofunctional activities of enzymatic squid meat hydrolysate were determined to develop value-added products. Enzymatic squid hydrolysate manufactured by Alcalase effectively quenched 1,1-diphenyl-2-picrylhydrazyl radical, hydroxyl radical, and hydrogen peroxide radical with IC50 values of 311, 3,410, and 111.5 μg/mL, respectively. Angiotensin I-converting enzyme inhibitory activity of squid hydrolysate was strong with an IC50 value of 145.1 μg/mL, while tyrosinase inhibitory activity with an IC50 value of 4.72 mg/mL was moderately low. Overall, squid meat hydrolysate can be used in food or cosmetic industries as a bioactive ingredient and possibly be used in the manufacture of seasoning, bread, noodle, or cosmetics. PMID:25866752

Enhanced enzymatic stability and antitumor activity of daunorubicin-GnRH-III bioconjugates modified in position 4.

PubMed

Manea, Marilena; Leurs, Ulrike; Orbán, Erika; Baranyai, Zsuzsa; Öhlschläger, Peter; Marquardt, Andreas; Schulcz, Ákos; Tejeda, Miguel; Kapuvári, Bence; Tóvári, József; Mezo, Gábor

2011-07-20

Here, we report on the synthesis, enzymatic stability, and antitumor activity of novel bioconjugates containing the chemotherapeutic agent daunorubicin attached through an oxime bond to various gonadotropin-releasing hormone-III (GnRH-III) derivatives. In order to increase the enzymatic stability of the bioconjugates (in particular against chymotrypsin), (4)Ser was replaced by N-Me-Ser or Lys(Ac). A compound in which (4)Lys was not acetylated was also prepared, with the aim of investigating the influence of the free ε-amino group on the biochemical properties. The in vitro cytostatic effect of the bioconjugates was determined on MCF-7 human breast, HT-29 human colon, and LNCaP human prostate cancer cells by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Their stability/degradation (1) in human serum, (2) in the presence of rat liver lysosomal homogenate, and (3) in the presence of digestive enzymes (trypsin, chymotrypsin, and pepsin) was analyzed by liquid chromatography in combination with mass spectrometry. The results showed that (1) all synthesized bioconjugates had in vitro cytostatic effect, (2) they were stable in human serum at least for 24 h, and (3) they were hydrolyzed in the presence of lysosomal homogenate. All compounds were stable in the presence of (1) pepsin and (2) trypsin (except for the (4)Lys containing bioconjugate). In the presence of chymotrypsin, all bioconjugates were digested; the degradation rate strongly depending on their structure. The bioconjugates in which (4)Ser was replaced by N-Me-Ser or Lys(Ac) had the highest enzymatic stability, making them potential candidates for oral administration. In vivo tumor growth inhibitory effect of two selected bioconjugates was evaluated on orthotopically developed C26 murine colon carcinoma bearing mice. The results indicated that the compound containing Lys(Ac) in position 4 had significantly higher antitumor activity than the parent bioconjugate.

Improved antimelanogenesis and antioxidant effects of polysaccharide from Cuscuta chinensis Lam seeds after enzymatic hydrolysis.

PubMed

Liu, Zi-Jun; Wang, Ya-Lan; Li, Qi-Ling; Yang, Liu

2018-01-01

Cuscuta chinensis polysaccharide (CPS) was extracted using hot water and enzymatically hydrolyzed C. chinensis polysaccharide (ECPS) was produced by the mannase enzymatic hydrolysis process. The purpose of this research was to investigate the antimelanogenic activity of ECPS and CPS in B16F10 melanoma cells. The in vitro antioxidant activity was assessed by their ferric iron reducing power and DPPH free radical scavenging activities. The molecular mass distribution of polysaccharides was determined using SEC-MALLS-RI. CPS was successfully enzymatically degraded using mannase and the weighted average molecular weights of CPS and ECPS were 434.6 kDa and 211.7 kDa. The results of biological activity assays suggested that the enzymatically hydrolyzed polysaccharide had superior antimelanogenic activity and antioxidant effect than the original polysaccharide. ECPS exhibited antimelanogenic activity by down-regulating the expression of tyrosinase, MITF, and TRP-1 without cytotoxic effects in B16F10 melanoma cells. In conclusion, ECPS have the potential to become a skin whitening product.

Mass spectrometric real-time monitoring of an enzymatic phosphorylation assay using internal standards and data-handling freeware.

PubMed

Krappmann, Michael; de Boer, Arjen R; Kool, Daniël R W; Irth, Hubertus; Letzel, Thomas

2016-04-30

Continuous-flow reaction detection systems (monitoring enzymatic reactions with mass spectrometry (MS)) lack quantitative values so far. Therefore, two independent internal standards (IS) are implemented in a way that the online system stability can be observed, quantitative conversion values for substrate and product can be obtained and they can be used as mass calibration standards for high MS accuracy. An application previously developed for the MS detection of peptide phosphorylation by cAMP-dependent protein kinase A (PKA) (De Boer et al., Anal. Bioanal. Chem. 2005, 381, 647-655) was transferred to a continuous-flow reaction detection system. This enzymatic reaction, involving enzyme activation as well as the transfer of a phosphate group from ATP to a peptide substrate, was used to prove the compatibility of a quantitative enzymatic assay in a continuous-flow real-time system (connected to MS). Moreover (using internal standards), the critical parameter reaction temperature (including solution density variations depending on temperature) was studied in the continuous-flow mixing system. Furthermore, two substrates (malantide and kemptide), two enzyme types (catalytic subunit of PKA and complete PKA) and one inhibitor were tested to determine system robustness and long-term availability. Even spraying solutions that contained significant amount of MS contaminants (e.g. the polluted catalytic subunit) resulted in quantifiable MS signal intensities. Subsequent recalculations using the internal standards led to results representing the power of this application. The presented methodology and the data evaluation with available Achroma freeware enable the direct coupling of biochemical assays with quantitative MS detection. Monitoring changes such as temperature, reaction time, inhibition, or compound concentrations can be observed quantitatively and thus enzymatic activity can be calculated. Copyright © 2016 John Wiley & Sons, Ltd.

Enzymatic activity profile of a Brazilian culture collection of Candida albicans isolated from diabetics and non-diabetics with oral candidiasis.

PubMed

Sanitá, Paula Volpato; Zago, Chaiene Evelin; Pavarina, Ana Cláudia; Jorge, Janaina Habib; Machado, Ana Lúcia; Vergani, Carlos Eduardo

2014-06-01

The secretion of hydrolytic enzymes is a fundamental virulence factor of Candida albicans to develop disease. The objective of this study was to characterise the virulence of 148 clinical isolates of C. albicans from oral candidiasis by assessing the expression of phospholipase (PL) and secreted aspartyl proteinase (SAP). Isolates were obtained from healthy subjects (HS) and diabetics (DOC) and non-diabetics with oral candidiasis (NDOC). An aliquot (5 μl) of each cell suspension was inoculated on PL and SAP agar plates and incubated. Enzymes secretion was detected by the formation of an opaque halo around the colonies and enzymatic activity (PZ) was determined by the ratio between colony diameter and colony diameter plus the halo zone. Statistical comparisons were made by a one-way anova followed by Tukey's post hoc test (α = 0.05). The clinical sources of C. albicans had significant effect (P < 0.001) on the PZ values of both enzymes. For PL, clinical isolates from NDOC and DOC had highest enzymatic activity than those from HS (P < 0.05), with no significant differences between them (P = 0.506). For SAP, C. albicans from NDOC showed the lower enzymatic activity (P < 0.001). There were no significant differences between isolates from HS and DOC (P = 0.7051). C. albicans isolates from NDOC and DOC patients showed an increased production of PL. © 2013 Blackwell Verlag GmbH.

Expression of the enzymatically active legumain-like cysteine proteinase TvLEGU-1 of Trichomonas vaginalis in Pichia pastoris.

PubMed

Reséndiz-Cardiel, Gerardo; Arroyo, Rossana; Ortega-López, Jaime

2017-06-01

The legumain-like cysteine proteinase TvLEGU-1 from Trichomonas vaginalis plays a major role in trichomonal cytoadherence. However, its structure-function characterization has been limited by the lack of a reliable recombinant expression platform to produce this protein in its native folded conformation. TvLEGU-1 has been expressed in Escherichia coli as inclusion bodies and all efforts to refold it have failed. Here, we describe the expression of the synthetic codon-optimized tvlegu-1 (tvlegu-1-opt) gene in Pichia pastoris strain X-33 (Mut+) under the inducible AOX1 promoter. The active TvLEGU-1 recombinant protein (rTvLEGU-1) was secreted into the medium when tvlegu-1-opt was fused to the Aspergillus niger alpha-amylase signal peptide. The rTvLEGU-1 secretion was influenced by the gene copy number and induction temperature. Data indicate that increasing tvlegu-1-opt gene copy number was detrimental for heterologous expression of the enzymatically active TvLEGU-1. Indeed, expression of TvLEGU-1 had a greater impact on cell viability for those clones with 26 or 29 gene copy number, and cell lysis was observed when the induction was carried out at 30 °C. The enzyme activity in the medium was higher when the induction was carried out at 16 °C and in P. pastoris clones with lower gene copy number. The results presented here suggest that both copy number and induction temperature affect the rTvLEGU-1 expression in its native-like and active conformation. Copyright © 2017 Elsevier Inc. All rights reserved.

Enzymatic mechanisms of biological magnetic sensitivity.

PubMed

Letuta, Ulyana G; Berdinskiy, Vitaly L; Udagawa, Chikako; Tanimoto, Yoshifumi

2017-10-01

Primary biological magnetoreceptors in living organisms is one of the main research problems in magnetobiology. Intracellular enzymatic reactions accompanied by electron transfer have been shown to be receptors of magnetic fields, and spin-dependent ion-radical processes can be a universal mechanism of biological magnetosensitivity. Magnetic interactions in intermediate ion-radical pairs, such as Zeeman and hyperfine (HFI) interactions, in accordance with proposed strict quantum mechanical theory, can determine magnetic-field dependencies of reactions that produce biologically important molecules needed for cell growth. Hyperfine interactions of electrons with nuclear magnetic moments of magnetic isotopes can explain the most important part of biomagnetic sensitivities in a weak magnetic field comparable to the Earth's magnetic field. The theoretical results mean that magnetic-field dependencies of enzymatic reaction rates in a weak magnetic field that can be independent of HFI constant a, if H < a, and are determined by the rate constant of chemical transformations in the enzyme active site. Both Zeeman and HFI interactions predict strong magnetic-field dependence in weak magnetic fields and magnetic-field independence of enzymatic reaction rate constants in strong magnetic fields. The theoretical results can explain the magnetic sensitivity of E. coli cell and demonstrate that intracellular enzymatic reactions are primary magnetoreceptors in living organisms. Bioelectromagnetics. 38:511-521, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Hyposialylation of neprilysin possibly affects its expression and enzymatic activity in hereditary inclusion-body myopathy muscle.

PubMed

Broccolini, Aldobrando; Gidaro, Teresa; De Cristofaro, Raimondo; Morosetti, Roberta; Gliubizzi, Carla; Ricci, Enzo; Tonali, Pietro A; Mirabella, Massimiliano

2008-05-01

Autosomal recessive hereditary inclusion-body myopathy (h-IBM) is caused by mutations of the UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase gene, a rate-limiting enzyme in the sialic acid metabolic pathway. Previous studies have demonstrated an abnormal sialylation of glycoproteins in h-IBM. h-IBM muscle shows the abnormal accumulation of proteins including amyloid-beta (Abeta). Neprilysin (NEP), a metallopeptidase that cleaves Abeta, is characterized by the presence of several N-glycosylation sites, and changes in these sugar moieties affect its stability and enzymatic activity. In the present study, we found that NEP is hyposialylated and its expression and enzymatic activity reduced in all h-IBM muscles analyzed. In vitro, the experimental removal of sialic acid by Vibrio Cholerae neuraminidase in cultured myotubes resulted in reduced expression of NEP. This was most likely because of a post-translational modification consisting in an abnormal sialylation of the protein that leads to its reduced stability. Moreover, treatment with Vibrio Cholerae neuraminidase was associated with an increased immunoreactivity for Abeta mainly in the form of distinct cytoplasmic foci within myotubes. We hypothesize that, in h-IBM muscle, hyposialylated NEP has a role in hampering the cellular Abeta clearing system, thus contributing to its abnormal accumulation within vulnerable fibers and possibly promoting muscle degeneration.

Determination of myoglobin based on its enzymatic activity by stopped-flow spectrophotometry

NASA Astrophysics Data System (ADS)

Zheng, Qi; Liu, Zhihong; Cai, Ruxiu

2005-04-01

A new method has been developed for the determination of myoglobin (Mb) based on its enzymatic activity for the oxidation of o-phenylenediamine (OPDA) with hydrogen peroxide. Stopped-flow spectrophotometry was used to study the kinetic behavior of the oxidation reaction. The catalytic activity of Mb was compared to other three kinds of catalyst. The time dependent absorbance of the reaction product, 2,3-diamimophenazine (DAPN), at a wavelength of 426 nm was recorded. The initial reaction rate obtained at 40 °C was found to be proportional to the concentration of Mb in the range of 1.0 × 10 -6 to 4.0 × 10 -9 mol L -1. The detection limit of Mb was found to be 9.93 × 10 -10 mol L -1. The relative standard deviations were within 5% for the determination of different concentrations of Mb. Excess of bovine serum albumin (BSA), Ca(II), Mg(II), Cu(II), glucose, caffeine, lactose and uric acid did not interfere.

Asparatic acid 221 is critical in the calcium-induced modulation of the enzymatic activity of human aminopeptidase A.

PubMed

Goto, Yoshikuni; Hattori, Akira; Mizutani, Shigehiko; Tsujimoto, Masafumi

2007-12-21

Aminopeptidase A (APA) plays an important role in the regulation of blood pressure by mediating angiotensin II degradation in the renin-angiotensin system. The Ca2+-induced modulation of enzymatic activity is the most characteristic feature of APA among the M1 family of aminopeptidases. In this study, we used site-directed mutagenesis for any residues responsible for the Ca2+ modulation of human APA. Alignment of sequences of the M1 family members led to the identification of Asp-221 as a significant residue of APA among the family members. Replacement of Asp-221 with Asn or Gln resulted in a loss of Ca2+ responsiveness toward synthetic substrates. These enzymes were also unresponsive to Ca2+ when peptide hormones, such as angiotensin II, cholecystokinin-8, neurokinin B, and kallidin, were employed as substrates. These results suggest that the negative charge of Asp-221 is essential for Ca2+ modulation of the enzymatic activity of APA and causes preferential cleavage of acidic amino acid at the N-terminal end of substrate peptides.

Mitochondrial intermediate peptidase: Expression in Escherichia coli and improvement of its enzymatic activity detection with FRET substrates

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

Marcondes, Marcelo F.; Torquato, Ricardo J.S.; Assis, Diego M.

2010-01-01

In the present study, soluble, functionally-active, recombinant human mitochondrial intermediate peptidase (hMIP), a mitochondrial metalloendoprotease, was expressed in a prokaryotic system. The hMIP fusion protein, with a poly-His-tag (6x His), was obtained by cloning the coding region of hMIP cDNA into the pET-28a expression vector, which was then used to transform Escherichia coli BL21 (DE3) pLysS. After isolation and purification of the fusion protein by affinity chromatography using Ni-Sepharose resin, the protein was purified further using ion exchange chromatography with a Hi-trap resource Q column. The recombinant hMIP was characterized by Western blotting using three distinct antibodies, circular dichroism, andmore » enzymatic assays that used the first FRET substrates developed for MIP and a series of protease inhibitors. The successful expression of enzymatically-active hMIP in addition to the FRET substrates will contribute greatly to the determination of substrate specificity of this protease and to the development of specific inhibitors that are essential for a better understanding of the role of this protease in mitochondrial functioning.« less

Chemical, enzymatic and cellular antioxidant activity studies of Agaricus blazei Murrill.

PubMed

Hakime-Silva, Ricardo A; Vellosa, José C R; Khalil, Najeh M; Khalil, Omar A K; Brunetti, Iguatemy L; Oliveira, Olga M M F

2013-09-01

Mushrooms possess nutritional and medicinal properties that have long been used for human health preservation and that have been considered by researchers as possible sources of free radical scavengers. In this work, the antioxidant properties of water extracts from Agaricus blazei Murill, produced by maceration and decoction, are demonstrated in vitro. Resistance to oxidation is demonstrated through three mechanisms: i) inhibition of enzymatic oxidative process, with 100% inhibition of HRP (horseradish peroxidase) and MPO (myeloperoxidase); ii) inhibition of cellular oxidative stress, with 80% inhibition of the oxidative burst of polymorphonuclear neutrophils (PMNs); and iii) direct action over reactive species, with 62% and 87% suppression of HOCl and superoxide anion radical (O2• -), respectively. From the data, it was concluded that the aqueous extract of A. blazei has significant antioxidant activity, indicating its possible application for nutraceutical and medicinal purposes.

A Comparison of Protein Kinases Inhibitor Screening Methods Using Both Enzymatic Activity and Binding Affinity Determination

PubMed Central

Rudolf, Amalie Frederikke; Skovgaard, Tine; Knapp, Stefan; Jensen, Lars Juhl; Berthelsen, Jens

2014-01-01

Binding assays are increasingly used as a screening method for protein kinase inhibitors; however, as yet only a weak correlation with enzymatic activity-based assays has been demonstrated. We show that the correlation between the two types of assays can be improved using more precise screening conditions. Furthermore a marked improvement in the correlation was found by using kinase constructs containing the catalytic domain in presence of additional domains or subunits. PMID:24915177

Prediction and characterization of enzymatic activities guided by sequence similarity and genome neighborhood networks

DOE PAGES

Zhao, Suwen; Sakai, Ayano; Zhang, Xinshuai; ...

2014-06-30

Metabolic pathways in eubacteria and archaea often are encoded by operons and/or gene clusters (genome neighborhoods) that provide important clues for assignment of both enzyme functions and metabolic pathways. We describe a bioinformatic approach (genome neighborhood network; GNN) that enables large scale prediction of the in vitro enzymatic activities and in vivo physiological functions (metabolic pathways) of uncharacterized enzymes in protein families. We demonstrate the utility of the GNN approach by predicting in vitro activities and in vivo functions in the proline racemase superfamily (PRS; InterPro IPR008794). The predictions were verified by measuring in vitro activities for 51 proteins inmore » 12 families in the PRS that represent ~85% of the sequences; in vitro activities of pathway enzymes, carbon/nitrogen source phenotypes, and/or transcriptomic studies confirmed the predicted pathways. The synergistic use of sequence similarity networks3 and GNNs will facilitate the discovery of the components of novel, uncharacterized metabolic pathways in sequenced genomes.« less

The mechanism of action of lymphokines. IX. The enzymatic basis of hydrogen peroxide production by lymphokine-activated macrophages.

PubMed

Freund, M; Pick, E

1986-08-15

The purpose of this study was to elucidate the biochemical basis of the enhanced hydrogen peroxide (H2O2) production by guinea pig peritoneal macrophages (MP) cultured in lymphokine (LK)-containing medium. The markedly augmented H2O2 generation by these cells, demonstrable by the horseradish peroxidase (HRP)-catalyzed oxidation of phenol red, is distinguished by its lack of dependence on a second stimulus. We demonstrate that H2O2 production is truly spontaneous and is not caused by a stimulant present among the H2O2 assay reagents. The principal candidate for such a role was HRP type II (a mixture of five isoenzymes) that was reported to be capable of eliciting an oxidative burst in MP. Four distinct HRP isoenzymes that were found incapable of provoking an oxidative response were nevertheless adequate for demonstrating H2O2 production by LK-activated MP. Blocking the MP receptor for mannose by the addition of mannan to the assay system resulted in enhanced detection of H2O2 by low concentrations of HRP type II and by three out of four HRP isoenzymes. Treatment of MP with LK-containing medium for 72 hr did not result in a significant change in the activity of cellular superoxide dismutase (SOD) compared with MP cultured for the same length of time in control medium. By using the specific inhibitor of copper, zinc-containing SOD, sodium diethyldithiocarbamate (DDC), and the universal SOD inhibitor, sodium nitroprusside, we found that the predominant enzyme in guinea pig peritoneal MP is probably manganese-containing SOD. Incubation of LK-activated MP with nitroprusside resulted in almost total inhibition of H2O2 production and a simultaneous switch to superoxide (O2-) liberation. Similar exposure to DDC had no effect. These data indicate that H2O2 produced by LK-activated MP is derived exclusively by enzymatic dismutation of O2- mediated by a manganese-containing SOD. The increase in spontaneous H2O2 production induced by LK is therefore secondary to augmented O2

Proinflammatory Actions of Visfatin/Nicotinamide Phosphoribosyltransferase (Nampt) Involve Regulation of Insulin Signaling Pathway and Nampt Enzymatic Activity*

PubMed Central

Jacques, Claire; Holzenberger, Martin; Mladenovic, Zvezdana; Salvat, Colette; Pecchi, Emilie; Berenbaum, Francis; Gosset, Marjolaine

2012-01-01

Visfatin (also termed pre-B-cell colony-enhancing factor (PBEF) or nicotinamide phosphoribosyltransferase (Nampt)) is a pleiotropic mediator acting on many inflammatory processes including osteoarthritis. Visfatin exhibits both an intracellular enzymatic activity (nicotinamide phosphoribosyltransferase, Nampt) leading to NAD synthesis and a cytokine function via the binding to its hypothetical receptor. We recently reported the role of visfatin in prostaglandin E2 (PGE2) synthesis in chondrocytes. Here, our aim was to characterize the signaling pathways involved in this response in exploring both the insulin receptor (IR) signaling pathway and Nampt activity. IR was expressed in human and murine chondrocytes, and visfatin triggered Akt phosphorylation in murine chondrocytes. Blocking IR expression with siRNA or activity using the hydroxy-2-naphthalenyl methyl phosphonic acid tris acetoxymethyl ester (HNMPA-(AM)3) inhibitor diminished visfatin-induced PGE2 release in chondrocytes. Moreover, visfatin-induced IGF-1R−/− chondrocytes released higher concentration of PGE2 than IGF-1R+/+ cells, a finding confirmed with an antibody that blocked IGF-1R. Using RT-PCR, we found that visfatin did not regulate IR expression and that an increased insulin release was also unlikely to be involved because insulin was unable to increase PGE2 release. Inhibition of Nampt activity using the APO866 inhibitor gradually decreased PGE2 release, whereas the addition of exogenous nicotinamide increased it. We conclude that the proinflammatory actions of visfatin in chondrocytes involve regulation of IR signaling pathways, possibly through the control of Nampt enzymatic activity. PMID:22399297

Vertical and horizontal distributions of microbial abundances and enzymatic activities in propylene-glycol-affected soils.

PubMed

Biró, Borbála; Toscano, Giuseppe; Horváth, Nikoletta; Matics, Heléna; Domonkos, Mónika; Scotti, Riccardo; Rao, Maria A; Wejden, Bente; French, Helen K

2014-01-01

The natural microbial activity in the unsaturated soil is vital for protecting groundwater in areas where high loads of biodegradable contaminants are supplied to the surface, which usually is the case for airports using aircraft de-icing fluids (ADF) in the cold season. Horizontal and vertical distributions of microbial abundance were assessed along the western runway of Oslo Airport (Gardermoen, Norway) to monitor the effect of ADF dispersion with special reference to the component with the highest chemical oxygen demand (COD), propylene glycol (PG). Microbial abundance was evaluated by several biondicators: colony-forming units (CFU) of some physiological groups (aerobic and anaerobic heterotrophs and microscopic fungi), most probable numbers (MPN) of PG degraders, selected catabolic enzymatic activities (fluorescein diacetate (FDA) hydrolase, dehydrogenase, and β-glucosidase). High correlations were found between the enzymatic activities and microbial counts in vertical soil profiles. All microbial abundance indicators showed a steep drop in the first meter of soil depth. The vertical distribution of microbial abundance can be correlated by a decreasing exponential function of depth. The horizontal trend of microbial abundance (evaluated as total aerobic CFU, MPN of PG-degraders, and FDA hydrolase activity) assessed in the surface soil at an increasing distance from the runway is correlated negatively with the PG and COD loads, suggesting the relevance of other chemicals in the modulation of microbial growth. The possible role of potassium formate, component of runway de-icers, has been tested in the laboratory by using mixed cultures of Pseudomonas spp., obtained by enrichment with a selective PG medium from soil samples taken at the most contaminated area near the runway. The inhibitory effect of formate on the growth of PG degraders is proven by the reduction of biomass yield on PG in the presence of formate.

Formation of marine snow and enhanced enzymatic activities in oil-contaminated seawater

NASA Astrophysics Data System (ADS)

Ziervogel, K.; McKay, L.; Yang, T.; Rhodes, B.; Nigro, L.; Gutierrez, T.; Teske, A.; Arnosti, C.

2010-12-01

The fate of oil spilled into the ocean depends on its composition, as well as on biological, chemical, and physical characteristics of the spill site. We investigated the effects of oil addition from the Deepwater Horizon (DH) spill on otherwise uncontaminated water collected close to the spill site. Incubation on a roller table mimicked the physical dynamics of natural seawater, leading to the formation of marine snow-oil aggregates. We measured the enzymatic activities of heterotrophic microbes associated with the aggregates and in the surrounding water, and assessed microbial population and community composition as oil-marine snow aggregates formed and aged in the water. Surface seawater taken near the spill site in May 2010 that had no visible crude oil was incubated in 1-l glass bottles with (oil-bottles) and without (no-oil bottles) a seawater-oil mixture collected from the same site. In the oil-bottles formation of brownish, densely packed marine snow (2-3 cm diameter) was observed within the first hour of the roller table incubation. In contrast no-oil bottles showed aggregate formation only after 3 days, and aggregates were almost transparent, less abundant, and smaller in size (< 1cm diameter). Subsamples of the water surrounding the aggregates were taken throughout 21 days of the roller table incubation, and analyzed for bacterial abundance and community structure as well as the activities of hydrolytic enzymes that are used by heterotrophic bacteria to degrade organic matter. We monitored oil-degrading activities with MUF-stearate and -butyrate, and also measured b-glucosidase, alkaline phosphatase, aminopeptidase, and six different polysaccharide hydrolase activities. Enzymatic activities were up to one order of magnitude higher in the oil-bottles compared with the no-oil bottles throughout the entire incubation time. Butyrate hydrolysis was elevated throughout the time course of the incubation, and stearate hydrolysis was particularly high over the

Phospholipase C produced by Clostridium botulinum types C and D: comparison of gene, enzymatic, and biological activities with those of Clostridium perfringens alpha-toxin.

PubMed

Fatmawati, Ni Nengah Dwi; Sakaguchi, Yoshihiko; Suzuki, Tomonori; Oda, Masataka; Shimizu, Kenta; Yamamoto, Yumiko; Sakurai, Jun; Matsushita, Osamu; Oguma, Keiji

2013-01-01

Clostridium botulinum type C and D strains recently have been found to produce PLC on egg yolk agar plates. To characterize the gene, enzymatic and biological activities of C. botulinum PLCs (Cb-PLCs), the cb-plc genes from 8 strains were sequenced, and 1 representative gene was cloned and expressed as a recombinant protein. The enzymatic and hemolytic activities of the recombinant Cb-PLC were measured and compared with those of the Clostridium perfringens alpha-toxin. Each of the eight cb-plc genes encoded a 399 amino acid residue protein preceded by a 27 residue signal peptide. The protein consists of 2 domains, the N- and C-domains, and the overall amino acid sequence identity between Cb-PLC and alpha-toxin was greater than 50%, suggesting that Cb-PLC is homologous to the alpha-toxin. The key residues in the N-domain were conserved, whereas those in the C-domain which are important in membrane interaction were different than in the alpha-toxin. As expected, Cb-PLC could hydrolyze egg yolk phospholipid, p-nitrophenylphosphorylcholine, and sphingomyelin, and also exhibited hemolytic activity;however, its activities were about 4- to over 200-fold lower than those of alpha-toxin. Although Cb-PLC showed weak enzymatic and biological activities, it is speculated that Cb-PLC might play a role in the pathogenicity of botulism or for bacterial survival.

[Enzymatic characteristics of peroxidase from Chrysanthemum morifolium cv. Bo-ju].

PubMed

Zhu, Yu-Yun; Lyu, Xin-Lin; Li, Xiang-Wei; Zhang, Dong; Dong, Li-Hua; Zhu, Jing-Jing; Wang, Zhi-Min; Zhang, Jin-Zhen

2018-04-01

The enzymatic browning is one of the main reasons for affecting the quality of medicinal flowers. In the process of chrysanthemum harvesting and processing, improper treatment will lead to the browning and severely impact the appearance and quality of chrysanthemum. Peroxidase enzyme is one of the oxidoreductases that cause enzymatic browning of fresh chrysanthemum. The enzymatic characteristics of peroxidase (POD) in chrysanthemum were studied in this paper. In this experiment, the effects of different reaction substrates and their concentrations, PH value of buffer and reaction temperatures on the activity of POD enzyme were investigated. The results showed that the optimal substrate of POD was guaiacol, and the optimal concentration of POD was 50 mmol·L⁻¹. The optimal pH value and reaction temperature were 4.4 and 30-35 °C, respectively. Michaelis-Menten equation was obtained to express the kinetics of enzyme-catalyzed reaction of POD, Km=0.193 mol·L⁻¹, Vmax=0.329 D·min⁻¹. In addition, the results of POD enzyme thermal stability test showed that the POD enzyme activity was inhibited when being treated at 80 °C for 4 min or at 100 °C for 2 min. The above results were of practical significance to reveal the enzymatic browning mechanism, control the enzymatic browning and improve the quality of chrysanthemum, and can also provide the basis for the harvesting and processing of medicinal materials containing polyphenols. Copyright© by the Chinese Pharmaceutical Association.

Swimming training attenuates oxidative damage and increases enzymatic but not non-enzymatic antioxidant defenses in the rat brain.

PubMed

Nonato, L F; Rocha-Vieira, E; Tossige-Gomes, R; Soares, A A; Soares, B A; Freitas, D A; Oliveira, M X; Mendonça, V A; Lacerda, A C; Massensini, A R; Leite, H R

2016-09-29

Although it is well known that physical training ameliorates brain oxidative function after injuries by enhancing the levels of neurotrophic factors and oxidative status, there is little evidence addressing the influence of exercise training itself on brain oxidative damage and data is conflicting. This study investigated the effect of well-established swimming training protocol on lipid peroxidation and components of antioxidant system in the rat brain. Male Wistar rats were randomized into trained (5 days/week, 8 weeks, 30 min; n=8) and non-trained (n=7) groups. Forty-eight hours after the last session of exercise, animals were euthanized and the brain was collected for oxidative stress analysis. Swimming training decreased thiobarbituric acid reactive substances (TBARS) levels (P<0.05) and increased the activity of the antioxidant enzyme superoxide dismutase (SOD) (P<0.05) with no effect on brain non-enzymatic total antioxidant capacity, estimated by FRAP (ferric-reducing antioxidant power) assay (P>0.05). Moreover, the swimming training promoted metabolic adaptations, such as increased maximal workload capacity (P<0.05) and maintenance of body weight. In this context, the reduced TBARS content and increased SOD antioxidant activity induced by 8 weeks of swimming training are key factors in promoting brain resistance. In conclusion, swimming training attenuated oxidative damage and increased enzymatic antioxidant but not non-enzymatic status in the rat brain.

Enzymatic hydrolysis of potato pulp.

PubMed

Lesiecki, Mariusz; Białas, Wojciech; Lewandowicz, Grażyna

2012-01-01

Potato pulp constitutes a complicated system of four types of polysaccharides: cellulose, hemicellulose, pectin and starch. Its composition makes it a potential and attractive raw material for the production of the second generation bioethanol. The aim of this research project was to assess the usefulness of commercial enzymatic preparations for the hydrolysis of potato pulp and to evaluate the effectiveness of hydrolysates obtained in this way as raw materials for ethanol fermentation. Sterilised potato pulp was subjected to hydrolysis with commercial enzymatic preparations. The effectiveness of the preparations declared as active towards only one fraction of potato pulp (separate amylase, pectinase and cellulase activity) and mixtures of these preparations was analysed. The monomers content in hydrolysates was determined using HPLC method. The application of amylolytic enzymes for potato pulp hydrolysis resulted in the release of only 18% of raw material with glucose as the dominant (77%) constituent of the formed product. In addition, 16% galactose was also determined in it. The hydrolysis of the cellulose fraction yielded up to 35% raw material and the main constituents of the obtained hydrolysate were glucose (46%) and arabinose (40%). Simultaneous application of amylolytic, cellulolytic and pectinolytic enzymes turned out to be the most effective way of carrying out the process as its efficiency in this case reached 90%. The obtained hydrolysate contained 63% glucose, 25% arabinose and 12% other simple substances. The application of commercial enzymatic preparations made it possible to perform potato pulp hydrolysis with 90% effectiveness. This was achieved by the application of a complex of amylolytic, cellulolytic and pectinolytic enzymes and the hydrolysate obtained in this way contained, primarily, glucose making it a viable substrate for ethanol fermentation.

Allergenicity, trypsin inhibitor activity and nutritive quality of enzymatically modified soy proteins.

PubMed

De La Barca, Ana María Calderón; Wall, Abraham; López-Díaz, José Alberto

2005-05-01

Two ultrafiltered soy flour protein fractions were evaluated; the first was obtained by hydrolysis (0.5-3 kDa, F(0.5-3)), and the second was an enzymatically methionine-enriched fraction (1-10 kDa, F(1-10)E). Amino acid profiles, protein quality, allergenicity (against soy-sensitive infant sera) and trypsin inhibitor activity were determined. Fraction F(1-10)E fulfilled amino acid requirements for infants, whereas the F(0.5-3) fraction was methionine deficient. Both fractions were similar in net protein utilization, and F(1-10)E digestibility was comparable with casein and higher (P?activity with respect to soy flour was 8.1%, 3.3% and 1% for hydrolysate, F(1-10)E and F(0.5-3), respectively. Both fractions presented high nutritive quality and reduced or null allergenicity. The trypsin inhibitor activity decreased along processing and could be a useful indicator for production of hypoallergenic proteins.

Role of low density lipoprotein in the activation of plasma lysolecithin acyltransferase activity. Effect of chemical and enzymatic modifications of the lipoprotein on enzyme activity.

PubMed

Subbaiah, P V; Chen, C H; Bagdade, J D; Albers, J J

1985-01-01

The effect of various chemical and enzymatic modifications of low density lipoprotein (LDL) on its ability to activate the isolated human plasma lysolecithin acyltransferase (LAT) was studied. Removal of all lipids from LDL resulted in the complete loss of LAT activation. Removal of only neutral lipids by extraction with heptane retained up to 50% of the original activity, which was not increased further by reconstitution of the LDL with the extracted lipids. Hydrolysis of the diacylphosphoglycerides of the LDL with phospholipases resulted in complete loss of LAT activation which was partially restored by the addition of egg lecithin. Hydrolysis of more than 4% of LDL protein by trypsin led to a linear decrease in activity with complete loss of activity occurring when about 25% of the LDL protein is hydrolyzed. Modification of the arginine groups of LDL reversibly inhibited the activation of LAT. Modification of lysine residues of LDL by acetylation, acetoacetylation or succinylation also abolished its ability to activate lysolecithin acylation.

Downregulation of Rubisco Activity by Non-enzymatic Acetylation of RbcL.

PubMed

Gao, Xiang; Hong, Hui; Li, Wei-Chao; Yang, Lili; Huang, Jirong; Xiao, You-Li; Chen, Xiao-Ya; Chen, Gen-Yun

2016-07-06

Atmospheric carbon dioxide (CO2) is assimilated by the most abundant but sluggish enzyme, ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). Here we show that acetylation of lysine residues of the Rubisco large subunit (RbcL), including Lys201 and Lys334 in the active sites, may be an important mechanism in the regulation of Rubisco activities. It is well known that Lys201 reacts with CO2 for carbamylation, a prerequisite for both carboxylase and oxygenase activities of Rubisco, and Lys334 contacts with ribulose-1,5-bisphosphate (RuBP). The acetylation level of RbcL in plants is lower during the day and higher at night, inversely correlating with the Rubisco carboxylation activity. A search of the chloroplast proteome database did not reveal a canonical acetyltransferase; instead, we found that a plant-derived metabolite, 7-acetoxy-4-methylcoumarin (AMC), can non-enzymatically acetylate both native Rubisco and synthesized RbcL peptides spanning Lys334 or Lys201. Furthermore, lysine residues were modified by synthesized 4-methylumbelliferone esters with different electro- and stereo-substitutes, resulting in varied Rubisco activities. 1-Chloroethyl 4-methylcoumarin-7-yl carbonate (ClMC) could transfer the chloroethyl carbamate group to lysine residues of RbcL and completely inactivate Rubisco, whereas bis(4-methylcoumarin-7-yl) carbonate (BMC) improved Rubisco activity through increasing the level of Lys201 carbamylation. Our findings indicate that RbcL acetylation negatively regulates Rubisco activity, and metabolic derivatives can be designed to dissect and improve CO2 fixation efficiency of plants through lysine modification. Copyright © 2016 The Author. Published by Elsevier Inc. All rights reserved.

Ultrasound-assisted enzymatic extraction and antioxidant activity of polysaccharides from pumpkin (Cucurbita moschata).

PubMed

Wu, Hao; Zhu, Junxiang; Diao, Wenchao; Wang, Chengrong

2014-11-26

An efficient ultrasound-assisted enzymatic extraction (UAEE) of Cucurbita moschata polysaccharides (CMCP) was established and the CMCP antioxidant activities were studied. The UAEE operating parameters (extraction temperature, ultrasonic power, pH, and liquid-to-material ratio) were optimized using the central composite design (CCD) and the mass transfer kinetic study in UAEE procedure was used to select the optimal extraction time. Enzymolysis and ultrasonication that were simultaneously conducted was selected as the UAEE synergistic model and the optimum extraction conditions with a maximum polysaccharide yield of 4.33 ± 0.15% were as follows: extraction temperature, 51.5 °C; ultrasonic power, 440 W; pH, 5.0; liquid-to-material ratio, 5.70:1 mL/g; and extraction time, 20 min. Evaluation of the antioxidant activity in vitro suggested that CMCP has good potential as a natural antioxidant used in the food or medicine industry because of their high reducing power and positive radical scavenging activity for DPPH radical. Copyright © 2014 Elsevier Ltd. All rights reserved.

Beneficial effect of mixture of additives amendment on enzymatic activities, organic matter degradation and humification during biosolids co-composting.

PubMed

Awasthi, Mukesh Kumar; Wang, Quan; Chen, Hongyu; Awasthi, Sanjeev Kumar; Wang, Meijing; Ren, Xiuna; Zhao, Junchao; Zhang, Zengqiang

2018-01-01

The objective of this study was to identify the effect of mixture of additives to improve the enzymatic activities, organic matter humification and diminished the bioavailability of heavy metals (HMs) during biosolids co-composting. In this study, zeolite (Z) (10%, 15% and 30%) with 1%lime (L) (dry weight basis of biosolids) was blended into the mixture of biosolids and wheat straw, respectively. The without any amendment and 1%lime applied treatments were run for comparison (Control). The Z+L addition resulted rapid organic matter degradation and humification with maximum enzymatic activities. In addition, higher dosage of Z+1%L amendment reduced the bioavailability of HMs (Cu and Zn) and improved the end product quality as compared to control and 1%L applied treatments. However, the 30%Z+1%L applied treatment showed maximum humification and low bioavailability of HMs but considering the economic feasibility and compost quality results, the treatment with 10%Z+1%L is recommended for biosolids co-composting. Copyright © 2017 Elsevier Ltd. All rights reserved.

A Multiplex Enzymatic Machinery for Cellular Protein S-nitrosylation.

PubMed

Seth, Divya; Hess, Douglas T; Hausladen, Alfred; Wang, Liwen; Wang, Ya-Juan; Stamler, Jonathan S

2018-02-01

S-nitrosylation, the oxidative modification of Cys residues by nitric oxide (NO) to form S-nitrosothiols (SNOs), modifies all main classes of proteins and provides a fundamental redox-based cellular signaling mechanism. However, in contrast to other post-translational protein modifications, S-nitrosylation is generally considered to be non-enzymatic, involving multiple chemical routes. We report here that endogenous protein S-nitrosylation in the model organism E. coli depends principally upon the enzymatic activity of the hybrid cluster protein Hcp, employing NO produced by nitrate reductase. Anaerobiosis on nitrate induces both Hcp and nitrate reductase, thereby resulting in the S-nitrosylation-dependent assembly of a large interactome including enzymes that generate NO (NO synthase), synthesize SNO-proteins (SNO synthase), and propagate SNO-based signaling (trans-nitrosylases) to regulate cell motility and metabolism. Thus, protein S-nitrosylation by NO in E. coli is essentially enzymatic, and the potential generality of the multiplex enzymatic mechanism that we describe may support a re-conceptualization of NO-based cellular signaling. Copyright © 2017 Elsevier Inc. All rights reserved.

Enzymatic processing of pigmented and non pigmented rice bran on changes in oryzanol, polyphenols and antioxidant activity.

PubMed

Prabhu, Ashish A; Jayadeep, A

2015-10-01

Bran from different rice varieties is a treasure of nutrients and nutraceuticals, and its use is limited due to the poor sensory and functional properties. Application of enzymes can alter the functional and phytochemical properties. So the effect of endo-xylanase, cellulase and their combination on microstructural, nutraceutical and antioxidant properties of pigmented (Jyothi) and non-pigmented (IR64) rice bran were investigated. Scanning electron micrograph revealed micro structural changes in fibre structures on processing. All the enzymatic processing methods resulted in an increase in the content of oryzanol, soluble, bound and total polyphenols, flavonoid and tannin. It also showed an increase in the bioactivity with respect to free radical scavenging activity and total antioxidant activity. However, extent of the increase in bio-actives varied with the type of bran and enzyme application method. Endo-xylanase showed higher percentage difference compared to controls of Jyothi and IR64 bran extracts respectively in the content of the bound (10 & 19 %) and total (20 & 14 %) polyphenols. Combination of both the enzymes resulted in higher percentage increase of bioactive components and properties. It resulted in greater percentage difference compared to controls of Jyothi and IR64 extracts respectively in the content of soluble (58 & 17 %) and total (21 & 14 %) polyphenols, flavonoids (12 & 38 %), γ-oryzanol (10 & 12 %), free radical scavenging activity (64 & 30 %) and total antioxidant activity (82 & 136 %). It may be concluded that enzymatic bio-processing of bran with cellulose and hemicellulose degrading enzymes can improve its nutraceutical properties, and it may be used for development of functional foods.

Proximity-activated nanoparticles: in vitro performance of specific structural modification by enzymatic cleavage

PubMed Central

Adam Smith, R; Sewell, Sarah L; Giorgio, Todd D

2008-01-01

The development and in vitro performance of a modular nanoscale system capable of specific structural modification by enzymatic activity is described in this work. Due to its small physical size and adaptable characteristics, this system has the potential for utilization in targeted delivery systems and biosensing. Nanoparticle probes were synthesized containing two distinct fluorescent species including a quantum dot base particle and fluorescently labeled cleavable peptide substrate. Activity of these probes was monitored by gel electrophoresis with quantitative cleavage measurements made by fluorometric analysis. The model proximity-activated nanoparticles studied here exhibit significant susceptibility to cleavage by matrix metalloprotease-7 (MMP-7) at physiologically relevant concentrations, with nearly complete cleavage of available substrate molecules after 24 hours. This response is specific to MMP-7 enzyme activity, as cleavage is completely inhibited with the addition of EDTA. Utilization of enzyme-specific modification is a sensitive approach with broad applications for targeted therapeutics and biosensing. The versatility of this nanoparticle system is highlighted in its modular design, as it has the capability to integrate characteristics for detection, biosensing, targeting, and payload delivery into a single, multifunctional nanoparticle structure. PMID:18488420

Improved measurement of extracellular enzymatic activities in subsurface sediments using competitive desorption treatment

NASA Astrophysics Data System (ADS)

Hoarfrost, Adrienne; Snider, Rachel; Arnosti, Carol

2017-02-01

Extracellular enzymatic activities initiate microbially-driven heterotrophic carbon cycling in subsurface sediments. While measurement of hydrolytic activities in sediments is fundamental to our understanding of carbon cycling, these measurements are often technically difficult due to sorption of organic substrates to the sediment matrix. Most methods that measure hydrolysis of organic substrates in sediments rely on recovery of a fluorophore or fluorescently-labeled target substrate from a sediment incubation. The tendency for substrates to sorb to sediments results in lower recovery of an added substrate, and can result in data that are unusable or difficult to interpret. We developed a treatment using competitive desorption of a fluorescently-labeled, high molecular weight organic substrate that improves recovery of the labeled substrate from sediment subsamples. Competitive desorption treatment improved recovery of the fluorescent substrate by a median of 66%, expanded the range of sediments for which activity measurements could be made, and was effective in sediments from a broad range of geochemical contexts. More reliable measurements of hydrolytic activities in sediments will yield usable and more easily interpretable data from a wider range of sedimentary environments, enabling better understanding of microbially-catalyzed carbon cycling in subsurface environments.

Microbial production of metabolites and associated enzymatic reactions under high pressure.

PubMed

Dong, Yongsheng; Jiang, Hua

2016-11-01

High environmental pressure exerts an external stress on the survival of microorganisms that are commonly found under normal pressure. In response, many growth traits alter, including cell morphology and physiology, cellular structure, metabolism, physical and chemical properties, the reproductive process, and defense mechanisms. The high-pressure technology (HP) has been industrially utilized in pressurized sterilization, synthesis of stress-induced products, and microbial/enzymatic transformation of chemicals. This article reviews current research on pressure-induced production of metabolites in normal-pressure microbes and their enzymatic reactions. Factors that affect the production of such metabolites are summarized, as well as the effect of pressure on the performance of microbial fermentation and the yield of flavoring compounds, different categories of induced enzymatic reactions and their characteristics in the supercritical carbon dioxide fluid, effects on enzyme activity, and the selection of desirable bacterial strains. Technological challenges are discussed, and future research directions are proposed. Information presented here will benefit the research, development, and application of the HP technology to improve microbial fermentation and enzymatic production of biologically active substances, thereby help to meet their increasing demand from the ever-expanding market.

Enzymatic browning and antioxidant activities in harvested litchi fruit as influenced by apple polyphenols.

PubMed

Zhang, Zhengke; Huber, Donald J; Qu, Hongxia; Yun, Ze; Wang, Hui; Huang, Zihui; Huang, Hua; Jiang, Yueming

2015-03-15

'Guiwei' litchi fruit were treated with 5 ga.i. L(-1) apple polyphenols (APP) and then stored at 25°C to investigate the effects on pericarp browning. APP treatment effectively reduced pericarp browning and retarded the loss of red colour. APP-treated fruit exhibited higher levels of anthocyanins and cyanidin-3-rutinoside, which correlated with suppressed anthocyanase activity. APP treatment also maintained membrane integrity and reduced oxidative damage, as indicated by a lower relative leakage rate, malondialdehyde content, and reactive oxygen species (ROS) generation. The data suggest that decompartmentalisation of peroxidase and polyphenoloxidase and respective browning substrates was reduced. In addition, APP treatment enhanced the activities of antioxidant enzymes (superoxide dismutase, catalase, ascorbate peroxidase and glutathione reductase), as well as non-enzymatic antioxidant capacity (DPPH radical-scavenging activity and reducing power), which might be beneficial in scavenging ROS. We propose that APP treatment is a promising safe strategy for controlling postharvest browning of litchi fruit. Copyright © 2014 Elsevier Ltd. All rights reserved.

Quinolone resistance-associated amino acid substitutions affect enzymatic activity of Mycobacterium leprae DNA gyrase.

PubMed

Yamaguchi, Tomoyuki; Yokoyama, Kazumasa; Nakajima, Chie; Suzuki, Yasuhiko

2017-07-01

Quinolones are important antimicrobials for treatment of leprosy, a chronic infectious disease caused by Mycobacterium leprae. Although it is well known that mutations in DNA gyrase are responsible for quinolone resistance, the effect of those mutations on the enzymatic activity is yet to be studied in depth. Hence, we conducted in vitro assays to observe supercoiling reactions of wild type and mutated M. leprae DNA gyrases. DNA gyrase with amino acid substitution Ala91Val possessed the highest activity among the mutants. DNA gyrase with Gly89Cys showed the lowest level of activity despite being found in clinical strains, but it supercoiled DNA like the wild type does if applied at a sufficient concentration. In addition, patterns of time-dependent conversion from relaxed circular DNA into supercoiled DNA by DNA gyrases with clinically unreported Asp95Gly and Asp95Asn were observed to be distinct from those by the other DNA gyrases.

Control of enzymatic browning in potato (Solanum tuberosum L.) by sense and antisense RNA from tomato polyphenol oxidase.

PubMed

Coetzer, C; Corsini, D; Love, S; Pavek, J; Tumer, N

2001-02-01

Polyphenol oxidase (PPO) activity of Russet Burbank potato was inhibited by sense and antisense PPO RNAs expressed from a tomato PPO cDNA under the control of the 35S promoter from the cauliflower mosaic virus. Transgenic Russet Burbank potato plants from 37 different lines were grown in the field. PPO activity and the level of enzymatic browning were measured in the tubers harvested from the field. Of the tubers from 28 transgenic lines that were sampled, tubers from 5 lines exhibited reduced browning. The level of PPO activity correlated with the reduction in enzymatic browning in these lines. These results indicate that expression of tomato PPO RNA in sense or antisense orientation inhibits PPO activity and enzymatic browning in the major commercial potato cultivar. Expression of tomato PPO RNA in sense orientation led to the greatest decrease in PPO activity and enzymatic browning, possibly due to cosuppression. These results suggest that expression of closely related heterologous genes can be used to prevent enzymatic browning in a wide variety of food crops without the application of various food additives.

Contrasting effects of untreated textile wastewater onto the soil available nitrogen-phosphorus and enzymatic activities in aridisol.

PubMed

Arif, Muhammad Saleem; Riaz, Muhammad; Shahzad, Sher Muhammad; Yasmeen, Tahira; Buttler, Alexandre; Garcıa-Gil, Juan Carlos; Roohi, Mahnaz; Rasool, Akhtar

2016-02-01

Water shortage and soil qualitative degradation are significant environmental problems in arid and semi-arid regions of the world. The increasing demand for water in agriculture and industry has resulted in the emergence of wastewater use as an alternative in these areas. Textile wastewater is produced in surplus amounts which poses threat to the environment as well as associated flora and fauna. A 60-day incubation study was performed to assess the effects of untreated textile wastewater at 0, 25, 50, 75, and 100% dilution levels on the physico-chemical and some microbial and enzymatic properties of an aridisol soil. The addition of textile wastewater provoked a significant change in soil pH and electrical conductivity and soil dehydrogenase and urease activities compared to the distilled-water treated control soil. Moreover, compared to the control treatment, soil phosphomonoesterase activity was significantly increased from 25 to 75% application rates, but decreased at 100% textile wastewater application rate. Total and available soil N contents increased significantly in response to application of textile wastewater. Despite significant increases in the soil total P contents after the addition of textile wastewater, soil available P content decreased with increasing concentration of wastewater. Changes in soil nutrient contents and related enzymatic activities suggested a dynamic match between substrate availability and soil N and P contents. Aridisols have high fixation and low P availability, application of textile wastewater to such soils should be considered only after careful assessment.

White grape pomace extracts, obtained by a sequential enzymatic plus ethanol-based extraction, exert antioxidant, anti-tyrosinase and anti-inflammatory activities.

PubMed

Ferri, Maura; Rondini, Greta; Calabretta, Maria Maddalena; Michelini, Elisa; Vallini, Veronica; Fava, Fabio; Roda, Aldo; Minnucci, Giordano; Tassoni, Annalisa

2017-10-25

The present work aimed at optimizing a two-step enzymatic plus solvent-based process for the recovery of bioactive compounds from white grape (Vitis vinifera L., mix of Trebbiano and Verdicchio cultivars) pomace, the winemaking primary by-product. Phenolic compounds solubilised by water enzyme-assisted and ethanol-based extractions of wet (WP) and dried (DP) pomace were characterised for composition and tested for antioxidant, anti-tyrosinase and anti-inflammatory bioactivities. Ethanol treatment led to higher phenol yields than water extraction, while DP samples showed the highest capacity of releasing polyphenols, most probably as a positive consequence of the pomace drying process. Different compositions and bioactivities were observed between water and ethanol extracts and among different treatments and for the first time the anti-tyrosinase activity of V. vinifera pomace extracts, was here reported. Enzymatic treatments did not significantly improve the total amount of solubilised compounds; Celluclast in DP led to the recovery of extracts enriched in specific compounds, when compared to control. The best extracts (enzymatic plus ethanol treatment total levels) were obtained from DP showing significantly higher amounts of polyphenols, flavonoids, flavanols and tannins and exerted higher antioxidant and anti-tyrosinase activities than WP total extracts. Conversely, anti-inflammatory capacity was only detected in water (with and without enzyme) extracts, with WP samples showing on average a higher activity than DP. The present findings demonstrate that white grape pomace constitute a sustainable source for the extraction of phytochemicals that might be exploited as functional ingredients in the food, nutraceutical, pharmaceutical or cosmetic industries. Copyright © 2017 Elsevier B.V. All rights reserved.

Ultrasound assisted enzymatic depolymerization of aqueous guar gum solution.

PubMed

Prajapat, Amrutlal L; Subhedar, Preeti B; Gogate, Parag R

2016-03-01

The present work investigates the effectiveness of application of low intensity ultrasonic irradiation for the intensification of enzymatic depolymerization of aqueous guar gum solution. The extent of depolymerization of guar gum has been analyzed in terms of intrinsic viscosity reduction. The effect of ultrasonic irradiation on the kinetic and thermodynamic parameters related to the enzyme activity as well as the intrinsic viscosity reduction of guar gum using enzymatic approach has been evaluated. The kinetic rate constant has been found to increase with an increase in the temperature and cellulase loading. It has been observed that application of ultrasound not only enhances the extent of depolymerization but also reduces the time of depolymerization as compared to conventional enzymatic degradation technique. In the presence of cellulase enzyme, the maximum extent of depolymerization of guar gum has been observed at 60 W of ultrasonic rated power and ultrasonic treatment time of 30 min. The effect of ultrasound on the kinetic and thermodynamic parameters as well as the molecular structure of cellulase enzyme was evaluated with the help of the chemical reaction kinetics model and fluorescence spectroscopy. Application of ultrasound resulted in a reduction in the thermodynamic parameters of activation energy (Ea), enthalpy (ΔH), entropy (ΔS) and free energy (ΔG) by 47%, 50%, 65% and 1.97%, respectively. The changes in the chemical structure of guar gum treated using ultrasound assisted enzymatic approach in comparison to the native guar gum were also characterized by FTIR. The results revealed that enzymatic depolymerization of guar gum resulted in a polysaccharide with low degree of polymerization, viscosity and consistency index without any change in the core chemical structure which could make it useful for incorporation in food products. Copyright © 2015 Elsevier B.V. All rights reserved.

Enzymatic activity and partial purification of solanapyrone synthase: first enzyme catalyzing Diels-Alder reaction.

PubMed

Katayama, K; Kobayashi, T; Oikawa, H; Honma, M; Ichihara, A

1998-05-19

In cell-free extracts of Alternaria solani, an enzymatic activity converting prosolanapyrone II to solanapyrones A and D via oxidation and subsequent Diels-Alder reaction has been found. Chromatography with DEAE-Sepharose provided two active fractions, pools 1 and 2. The former fraction converted prosolanapyrone II to solanapyrones A and D in a ratio of 2.2:1 with optical purities of 99% and 45% ee, respectively. The latter fraction did so in a ratio of 7.6:1 with 99% and nearly 0% ee, respectively. The enzyme partially purified from pool 2 native molecular weight of 40-62 kD and a pl of 4.25. The high reactivity of prosolanapyrone III in aqueous solution and the chromatographic behavior of the enzyme in pool 2 suggest that a single enzyme catalyzes both the oxidation and Diels-Alder reaction.

Production of xylooligosaccharide from wheat bran by microwave assisted enzymatic hydrolysis.

PubMed

Wang, Tseng-Hsing; Lu, Shin

2013-06-01

The effective production of xylooligosaccharides (XOS) from wheat bran was investigated. Wheat bran contains rich hemicellulose which can be hydrolyzed by enzyme; the XOS were obtained by microwave assisted enzymatic hydrolysis. To improve the productivity of XOS, repeated microwave assisted enzymatic hydrolysis and activated carbon adsorption method was chosen to eliminate macromolecules in the XOS. On the basis of experimental data, an industrial XOS production process consisting of pretreatment, repeated microwave assisted enzymatic treatment and purification was designed. Using the designed process, 3.2g dry of purified XOS was produced from 50 g dry wheat bran powder. Copyright © 2012 Elsevier Ltd. All rights reserved.

Enzymatic generation of galactose-rich oligosaccharides/oligomers from potato rhamnogalacturonan I pectic polysaccharides.

PubMed

Khodaei, Nastaran; Karboune, Salwa

2016-04-15

Potato pulp by-product rich in galactan-rich rhamnogalacturonan I (RG I) was investigated as a new source of oligosaccharides with potential prebiotic properties. The efficiency of selected monocomponent enzymes and multi-enzymatic preparations to generate oligosaccharides/oligomers from potato RG I was evaluated. These overall results of yield were dependent on the activity profile of the multi-enzymatic preparations. Highest oligo-RG I yield of 93.9% was achieved using multi-enzymatic preparation (Depol 670L) with higher hydrolytic activity toward side chains of RG I as compared to its backbone. Main oligo-RG I products were oligosaccharides with DP of 2-12 (79.8-100%), while the oligomers with DP of 13-70 comprised smaller proportion (0.0-20.2%). Galactose (58.9-91.2%, w/w) was the main monosaccharide of oligo-RG I, while arabinose represented 0.0-12.1%. An understanding of the relationship between the activity profile of multi-enzymatic preparations and the yield/DP of oligo-RG I was achieved. This is expected to provide the capability to generate galacto- and galacto(arabino) oligosaccharides and their corresponding oligomers from an abundant by-product. Copyright © 2015 Elsevier Ltd. All rights reserved.

Novel enzymatic method for assaying Lp-PLA2 in serum.

PubMed

Yamaura, Saki; Sakasegawa, Shin-Ichi; Koguma, Emisa; Ueda, Shigeru; Kayamori, Yuzo; Sugimori, Daisuke; Karasawa, Ken

2018-06-01

Measurement of lipoprotein-associated phospholipase A 2 (Lp-PLA 2 ) can be used as an adjunct to traditional cardiovascular risk factors for identifying individuals at higher risk of cardiovascular events. This can be performed by quantification of the protein concentration using an ELISA platform or by measuring Lp-PLA 2 activity using platelet-activating factor (PAF) analog as substrate. Here, an enzymatic Lp-PLA 2 activity assay method using 1-O-Hexadecyl-2-acetyl-rac-glycero-3-phosphocholine (rac C 16 PAF) was developed. The newly revealed substrate specificity of lysoplasmalogen-specific phospholipase D (lysophospholipase D (LysoPLD)) was exploited. Lp-PLA 2 hydrolyzes 1-O-Hexadecyl-2-acetyl-sn-glycero-3-phosphocholine (C 16 PAF) to 1-O-Hexadecyl-2-hydroxy-sn-glycero-3-phosphocholine (LysoPAF). LysoPLD acted on LysoPAF, and the hydrolytically released choline was detected by choline oxidase. Regression analysis of Lp-PLA 2 activity measured by the enzymatic Lp-PLA 2 activity assay vs. two chemical Lp-PLA 2 activity assays, i.e. LpPLA 2 FS and PLAC® test, and ELISA, gave the following correlation coefficients: 0.990, 0.893 and 0.785, respectively (n = 30). Advantages of this enzymatic Lp-PLA 2 activity assay compared with chemical Lp-PLA 2 methods include the following; (i) only requires two reagents enabling a simple two-point linear calibration method with one calibrator (ii) no need for inhibitors of esterase-like activity in serum. Copyright © 2018 Elsevier B.V. All rights reserved.

Impact of the redox-cycling herbicide diquat on transcript expression and antioxidant enzymatic activities of the freshwater snail Lymnaea stagnalis.

PubMed

Bouétard, Anthony; Besnard, Anne-Laure; Vassaux, Danièle; Lagadic, Laurent; Coutellec, Marie-Agnès

2013-01-15

The presence of pesticides in the environment results in potential unwanted effects on non-target species. Freshwater organisms inhabiting water bodies adjacent to agricultural areas, such as ditches, ponds and marshes, are good models to test such effects as various pesticides may reach these habitats through several ways, including aerial drift, run-off, and drainage. Diquat is a non-selective herbicide used for crop protection or for weed control in such water bodies. In this study, we investigated the effects of diquat on a widely spread aquatic invertebrate, the holarctic freshwater snail Lymnaea stagnalis. Due to the known redox-cycling properties of diquat, we studied transcript expression and enzymatic activities relative to oxidative and general stress in the haemolymph and gonado-digestive complex (GDC). As diquat is not persistent, snails were exposed for short times (5, 24, and 48 h) to ecologically relevant concentrations (22.2, 44.4, and 222.2 μg l(-1)) of diquat dibromide. RT-qPCR was used to quantify the transcription of genes encoding catalase (cat), a cytosolic superoxide dismutase (Cu/Zn-sod), a selenium-dependent glutathione peroxidase (gpx), a glutathione reductase (gred), the retinoid X receptor (rxr), two heat shock proteins (hsp40 and hsp70), cortactin (cor) and the two ribosomal genes r18S and r28s. Enzymatic activities of SOD, Gpx, Gred and glutathione S-transferase (GST) were investigated in the GDC using spectrophoto/fluorometric methods. Opposite trends were obtained in the haemolymph depending on the herbicide concentration. At the lowest concentration, effects were mainly observed after 24 h of exposure, with over-transcription of cor, hsp40, rxr, and sod, whereas higher concentrations down-regulated the expression of most of the studied transcripts, especially after 48 h of exposure. In the GDC, earlier responses were observed and the fold-change magnitude was generally much higher: transcription of all target genes increased

[Enzymatic degradation of organophosphorus insecticide chlorpyrifos by fungus WZ-I].

PubMed

Xie, Hui; Zhu, Lu-sheng; Wang, Jun; Wang, Xiu-guo; Liu, Wei; Qian, Bo; Wang, Qian

2005-11-01

Degradation characteristics of chlorpyrifos insecticides was determined by the crude enzyme extracted from the isolated strain WZ-I ( Fusarium LK. ex Fx). The best separating condition and the degrading characteristic of chlorpyrifos were studied. Rate of degradation for chlorpyrifos by its intracellular enzyme, extracellular enzyme and cell fragment was 60.8%, 11.3% and 48%, respectively. The degrading enzyme was extracted after this fungus was incubated for 8 generations in the condition of noninducement, and its enzymic activity lost less, the results show that this enzyme is an intracellular and connatural enzyme. The solubility protein of the crude enzyme was determined with Albumin (bovine serum) as standard protein and the solubility protein of the crude enzyme was 3.36 mg x mL(-1). The pH optimum for crude enzyme was 6.8 for enzymatic degradation of chlorpyrifos, and it had comparatively high activity in the range of pH 6.0 - 9.0. The optimum temperature for enzymatic activity was at 40 degrees C, it still had comparatively high activity in the range of temperature 20-50 degrees C, the activity of enzyme rapidly reduced at 55 degrees C, its activity was 41% of the maximal activity. The crude enzyme showed Km value for chlorpyrifos of 1.049 26 mmol x L(-1), and the maximal enzymatic degradation rate was 0.253 5 micromol x (mg x min)(-1). Additional experimental evidence suggests that the enzyme had the stability of endure for temperature and pH, the crude enzyme of fungus WZ-I could effectively degrade chlorpyrifos.

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

PubMed

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

2015-08-28

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

Enzymatic activities in different strains isolated from healthy and brittle leaf disease affected date palm leaves: study of amylase production conditions.

PubMed

Mouna, Jrad; Imen, Fendri; Choba Ines, Ben; Nourredine, Drira; Adel, Kadri; Néji, Gharsallah

2015-02-01

The present study aimed to investigate and compare the enzymatic production of endophytic bacteria isolated from healthy and brittle leaf disease affected date palm leaves (pectinase, cellulase, lipase, and amylase). The findings revealed that the enzymatic products from the bacterial isolates of healthy date palm leaves were primarily 33% amylolytic enzyme, 33 % cellulase, 25 % pectinase, and 25 % lipase. The isolates from brittle leaf disease date palm leaves, on the other hand, were noted to produce 16 % amylolytic enzyme, 20 % cellulose, 50 % pectinase, and 50 % lipase. The effects of temperature and pH on amylase, pectinase, and cellulose activities were investigated. The Bacillus subtilis JN934392 strain isolated from healthy date palm leaves produced higher levels of amylase activity at pH 7. A Box Behnken Design (BBD) was employed to optimize amylase extraction. Maximal activity was observed at pH and temperature ranges of pH 6-6.5 and 37-39 °C, respectively. Under those conditions, amylase activity was noted to be attained 9.37 U/ml. The results showed that the enzyme was able to maintain more than 50 % of its activity over a temperature range of 50-80 °C, with an optimum at 70 °C. This bacterial amylase showed high activity compared to other bacteria, which provides support for its promising candidacy for future industrial application.

Use of enzymatic tools for biomonitoring inorganic pollution in aquatic sediments: a case study (Bor, Serbia)

PubMed Central

2013-01-01

Background Sediment bacterial communities are key players in biogeochemical cycling of elements in the aquatic environment. Copper mining, smelting, and processing operations located in Bor area (Serbia) are major environmental hot spots in the lower Danube Basin and Western Balkans. In the present study, we evaluate the influence of trace element (TE) concentration in sediments and physico-chemical properties of water on sediment microbial communities in water streams adjacent to the Copper Smelter Complex Bor (RTB Bor, Serbia). The degree to which metabolic activities of bacterial biota inhabiting differently polluted sites is inhibited by inorganic pollution were compared using selected enzymatic bioindicators. Results Cu, Zn, Pb, and As concentrations systematically exceeded the target values for metal loadings in aquatic sediments. Water electrical conductivity (WEC) followed the same pattern of spatial variation, irrespective of season. Interestingly, the most intense enzymatic activity occurred at the reference site although this site showed the greatest TE levels in aquatic sediments. Catalase activity (CA), potential dehydrogenase activity (PDA), actual dehydrogenase activity (ADA), urease activity (UA), and phosphatase activity (PA) in aquatic sediments displayed heterogeneous patterns of spatio-temporal variation. Inorganic pollution greatly affected CA, ADA, and PDA, but much less so UA and PA. Canonical correlation analysis showed that pH and WEC were the strongest determinants of enzymatic activity in bacterial biota, with the latter variable being reversely correlated with the enzymatic indicator of sediment quality (EISQ). The median values of EISQ increased with distance from the major sources of pollution. In addition, it was found that sites with different degrees of inorganic pollution can be appropriately classified by applying cluster analysis to EISQ, TE levels in sediments, and physico-chemical properties of water. Conclusions Because EISQ

Modulation of the pharmacological effects of enzymatically-active PLA2 by BTL-2, an isolectin isolated from the Bryothamnion triquetrum red alga

PubMed Central

Oliveira, Simone CB; Fonseca, Fabiana V; Antunes, Edson; Camargo, Enilton A; Morganti, Rafael P; Aparício, Ricardo; Toyama, Daniela O; Beriam, Luís OS; Nunes, Eudismar V; Cavada, Benildo S; Nagano, Celso S; Sampaio, Alexandre H; Nascimento, Kyria S; Toyama, Marcos H

2008-01-01

Background An interaction between lectins from marine algae and PLA2 from rattlesnake was suggested some years ago. We, herein, studied the effects elicited by a small isolectin (BTL-2), isolated from Bryothamnion triquetrum, on the pharmacological and biological activities of a PLA2 isolated from rattlesnake venom (Crotalus durissus cascavella), to better understand the enzymatic and pharmacological mechanisms of the PLA2 and its complex. Results This PLA2 consisted of 122 amino acids (approximate molecular mass of 14 kDa), its pI was estimated to be 8.3, and its amino acid sequence shared a high degree of similarity with that of other neurotoxic and enzymatically-active PLA2s. BTL-2 had a molecular mass estimated in approximately 9 kDa and was characterized as a basic protein. In addition, BTL-2 did not exhibit any enzymatic activity. The PLA2 and BTL-2 formed a stable heterodimer with a molecular mass of approximately 24–26 kDa, estimated by molecular exclusion HPLC. In the presence of BTL-2, we observed a significant increase in PLA2 activity, 23% higher than that of PLA2 alone. BTL-2 demonstrated an inhibition of 98% in the growth of the Gram-positive bacterial strain, Clavibacter michiganensis michiganensis (Cmm), but only 9.8% inhibition of the Gram-negative bacterial strain, Xanthomonas axonopodis pv passiflorae (Xap). PLA2 decreased bacterial growth by 27.3% and 98.5% for Xap and Cmm, respectively, while incubating these two proteins with PLA2-BTL-2 inhibited their growths by 36.2% for Xap and 98.5% for Cmm. PLA2 significantly induced platelet aggregation in washed platelets, whereas BTL-2 did not induce significant platelet aggregation in any assay. However, BTL-2 significantly inhibited platelet aggregation induced by PLA2. In addition, PLA2 exhibited strong oedematogenic activity, which was decreased in the presence of BTL-2. BTL-2 alone did not induce oedema and did not decrease or abolish the oedema induced by the 48/80 compound. Conclusion The

Effect of salinity tolerant PDH45 transgenic rice on physicochemical properties, enzymatic activities and microbial communities of rhizosphere soils

PubMed Central

Sahoo, Ranjan Kumar; Tuteja, Narendra

2013-01-01

The effect of genetically modified (GM) plants on environment is now major concern worldwide. The plant roots of rhizosphere soil interact with variety of bacteria which could be influenced by the transgene in GM plants. The antibiotic resistance genes in GM plants may be transferred to soil microbes. In this study we have examined the effect of overexpression of salinity tolerant pea DNA helicase 45 (PDH45) gene on microbes and enzymatic activities in the rhizosphere soil of transgenic rice IR64 in presence and absence of salt stress in two different rhizospheric soils (New Delhi and Odisha, India). The diversity of the microbial community and soil enzymes viz., dehydrogenase, alkaline phosphatase, urease and nitrate reductase was assessed. The results revealed that there was no significant effect of transgene expression on rhizosphere soil of the rice plants. The isolated bacteria were phenotyped both in absence and presence of salt and no significant changes were found in their phenotypic characters as well as in their population. Overall, the overexpression of PDH45 in rice did not cause detectable changes in the microbial population, soil enzymatic activities and functional diversity of the rhizosphere soil microbial community. PMID:23733066

Enzymatic- and temperature-sensitive controlled release of ultrasmall superparamagnetic iron oxides (USPIOs)

PubMed Central

2011-01-01

Background Drug and contrast agent delivery systems that achieve controlled release in the presence of enzymatic activity are becoming increasingly important, as enzymatic activity is a hallmark of a wide array of diseases, including cancer and atherosclerosis. Here, we have synthesized clusters of ultrasmall superparamagnetic iron oxides (USPIOs) that sense enzymatic activity for applications in magnetic resonance imaging (MRI). To achieve this goal, we utilize amphiphilic poly(propylene sulfide)-bl-poly(ethylene glycol) (PPS-b-PEG) copolymers, which are known to have excellent properties for smart delivery of drug and siRNA. Results Monodisperse PPS polymers were synthesized by anionic ring opening polymerization of propylene sulfide, and were sequentially reacted with commercially available heterobifunctional PEG reagents and then ssDNA sequences to fashion biofunctional PPS-bl-PEG copolymers. They were then combined with hydrophobic 12 nm USPIO cores in the thin-film hydration method to produce ssDNA-displaying USPIO micelles. Micelle populations displaying complementary ssDNA sequences were mixed to induce crosslinking of the USPIO micelles. By design, these crosslinking sequences contained an EcoRV cleavage site. Treatment of the clusters with EcoRV results in a loss of R2 negative contrast in the system. Further, the USPIO clusters demonstrate temperature sensitivity as evidenced by their reversible dispersion at ~75°C and re-clustering following return to room temperature. Conclusions This work demonstrates proof of concept of an enzymatically-actuatable and thermoresponsive system for dynamic biosensing applications. The platform exhibits controlled release of nanoparticles leading to changes in magnetic relaxation, enabling detection of enzymatic activity. Further, the presented functionalization scheme extends the scope of potential applications for PPS-b-PEG. Combined with previous findings using this polymer platform that demonstrate controlled drug

Enzymatic- and temperature-sensitive controlled release of ultrasmall superparamagnetic iron oxides (USPIOs).

PubMed

Yu, Shann S; Scherer, Randy L; Ortega, Ryan A; Bell, Charleson S; O'Neil, Conlin P; Hubbell, Jeffrey A; Giorgio, Todd D

2011-02-27

Drug and contrast agent delivery systems that achieve controlled release in the presence of enzymatic activity are becoming increasingly important, as enzymatic activity is a hallmark of a wide array of diseases, including cancer and atherosclerosis. Here, we have synthesized clusters of ultrasmall superparamagnetic iron oxides (USPIOs) that sense enzymatic activity for applications in magnetic resonance imaging (MRI). To achieve this goal, we utilize amphiphilic poly(propylene sulfide)-bl-poly(ethylene glycol) (PPS-b-PEG) copolymers, which are known to have excellent properties for smart delivery of drug and siRNA. Monodisperse PPS polymers were synthesized by anionic ring opening polymerization of propylene sulfide, and were sequentially reacted with commercially available heterobifunctional PEG reagents and then ssDNA sequences to fashion biofunctional PPS-bl-PEG copolymers. They were then combined with hydrophobic 12 nm USPIO cores in the thin-film hydration method to produce ssDNA-displaying USPIO micelles. Micelle populations displaying complementary ssDNA sequences were mixed to induce crosslinking of the USPIO micelles. By design, these crosslinking sequences contained an EcoRV cleavage site. Treatment of the clusters with EcoRV results in a loss of R2 negative contrast in the system. Further, the USPIO clusters demonstrate temperature sensitivity as evidenced by their reversible dispersion at ~75°C and re-clustering following return to room temperature. This work demonstrates proof of concept of an enzymatically-actuatable and thermoresponsive system for dynamic biosensing applications. The platform exhibits controlled release of nanoparticles leading to changes in magnetic relaxation, enabling detection of enzymatic activity. Further, the presented functionalization scheme extends the scope of potential applications for PPS-b-PEG. Combined with previous findings using this polymer platform that demonstrate controlled drug release in oxidative

Ultrasonic-assisted enzymatic extraction of phenolics from broccoli (Brassica oleracea L. var. italica) inflorescences and evaluation of antioxidant activity in vitro.

PubMed

Wu, Hao; Zhu, Junxiang; Yang, Long; Wang, Ran; Wang, Chengrong

2015-06-01

An efficient ultrasonic-assisted enzymatic extraction technique was applied to extracting phenolics from broccoli inflorescences without organic solvents. The synergistic model of enzymolysis and ultrasonication simultaneously was selected, and the enzyme combination was optimized by orthogonal test: cellulase 7.5 mg/g FW (fresh weight), pectinase 10 mg/g FW, and papain 1.0 mg/g FW. The operating parameters in ultrasonic-assisted enzymatic extraction were optimized with response surface methodology using Box-Behnken design. The optimal extraction conditions were as follows: ultrasonic power, 440 W; liquid to material ratio, 7.0:1 mL/g; pH value of 6.0 at 54.5 ℃ for 10 min. Under these conditions, the extraction yield of phenolics achieved 1.816 ± 0.0187 mg gallic acid equivalents/gram FW. The free radical scavenging activity of ultrasonic-assisted enzymatic extraction extracts was determined by 1,1-diphenyl-2-picrylhydrazyl·assay with EC50 values of 0.25, and total antioxidant activity was determined by ferric reducing antioxidant power assay with ferric reducing antioxidant power value of 0.998 mmol FeSO4/g compared with the referential ascorbic acid of 1.184 mmol FeSO4/g. © The Author(s) 2014 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav.

Enzymatic and non-enzymatic comparison of two different industrial tomato (Solanum lycopersicum) varieties against drought stress.

PubMed

Çelik, Özge; Ayan, Alp; Atak, Çimen

2017-12-01

The aim of this study is to compare the tolerance mechanisms of two industrial tomato varieties (X5671R and 5MX12956) under drought stress. 14 days-old tomato seedlings were subjected to 7 days-long drought stress by withholding irrigation. The effects of stress were determined by enzymatic and non-enzymatic parameters. The physiological damages were evaluated via lipid peroxidation ratio, total protein content, relative water content, chlorophyll content and proline accumulation. Enzymatic responses were determined by biochemical analysis and electrophoresis of SOD, APX, POX and CAT enzymes. Relative water contents of X5671R and 5MX12956 varieties at 7th day of drought were decreased to 8.4 and 12.2%, respectively. Applied drought decreased all photosynthetic pigments of X5671R and 5MX12956 varieties during the treatment period significantly comparing to the Day 0 as the control. Total protein content, lipid peroxidation and proline accumulation presented increased values in both varieties in accordance with the increasing stress intensity. According to lipid peroxidation analysis, 5MX12956 tomato variety was found more drought sensitive than X5671R variety. Antioxidative enzyme activities showed increases in both varieties as a response to drought stress, although CAT and APX activities presented decrease on the 7th day of applied stress. 7 days long drought stress differentially altered POX, APX and SOD isozyme patterns. Same POX bands were observed in both varieties with different band intensities. However, main isozyme pattern differences were obtained for SOD and APX. APX1, Fe-SOD and Cu/Zn-SOD2 isozyme bands should be evaluated to define their main role in the tolerance mechanism of both tomato varieties.

Mechanistic investigation in ultrasound induced enhancement of enzymatic hydrolysis of invasive biomass species.

PubMed

Borah, Arup Jyoti; Agarwal, Mayank; Poudyal, Manisha; Goyal, Arun; Moholkar, Vijayanand S

2016-08-01

This study has assessed four invasive weeds, viz. Saccharum spontaneum (SS), Mikania micrantha (MM), Lantana camara (LC) and Eichhornia crassipes (EC) for enzymatic hydrolysis prior to bioalcohol fermentation. Enzymatic hydrolysis of pretreated biomasses of weeds has been conducted with mechanical agitation and sonication under constant (non-optimum) conditions. Profiles of total reducible sugar release have been fitted to HCH-1 model of enzymatic hydrolysis using Genetic Algorithm. Trends in parameters of this model reveal physical mechanism of ultrasound-induced enhancement of enzymatic hydrolysis. Sonication accelerates hydrolysis kinetics by ∼10-fold. This effect is contributed by several causes, attributed to intense micro-convection generated during sonication: (1) increase in reaction velocity, (2) increase in enzyme-substrate affinity, (3) reduction in product inhibition, and (4) enhancement of enzyme activity due to conformational changes in its secondary structure. Enhancement effect of sonication is revealed to be independent of conditions of enzymatic hydrolysis - whether optimum or non-optimum. Copyright © 2016 Elsevier Ltd. All rights reserved.

Enzyme-immobilized SiO2-Si electrode: Fast interfacial electron transfer with preserved enzymatic activity

NASA Astrophysics Data System (ADS)

Wang, Gang; Yau, Siu-Tung

2005-12-01

The enzyme, glucose oxidase (GOx), is immobilized using electrostatic interaction on the native oxide of heavily doped n-type silicon. Voltammetric measurement shows that the immobilized GOx gives rise to a very fast enzyme-silicon interfacial electron transfer rate constant of 7.9s-1. The measurement also suggests that the enzyme retains its native conformation when immobilized on the silicon surface. The preserved native conformation of GOx is further confirmed by testing the enzymatic activity of the immobilized GOx using glucose. The GOx-immobilized silicon is shown to behave as a glucose sensor that detects glucose with concentrations as low as 50μM.

cPLA2α Gene Activation by IL-1β is Dependent on an Upstream Kinase pathway, Enzymatic Activation and Downstream 15-lipoxygenase Activity: A Positive Feedback Loop

PubMed Central

Walters, Jewell N.; Bickford, Justin S.; Beachy, Dawn E.; Newsom, Kimberly J.; Herlihy, John-David H.; Peck, Molly V.; Qiu, Xiaolei; Nick, Harry S.

2011-01-01

Cytosolic phospholipase A2α (cPLA2α) is the most widely studied member of the Group IV PLA2 family. The enzyme is Ca2+-dependent with specificity for phospholipid substrates containing arachidonic acid. As the pinnacle of the arachidonic acid pathway, cPLA2α has a primary role in the biosynthesis of a diverse family of eicosanoid metabolites, with potent physiological, inflammatory and pathological consequences. cPLA2α activity is regulated by pro-inflammatory stimuli through pathways involving increased intracellular Ca2+ levels, phosphorylation coupled to increased enzymatic activity and de novo gene transcription. This study addresses the signal transduction pathways for protein phosphorylation and gene induction following IL-1β stimulation in human fetal lung fibroblasts. Our results utilizing both inhibitors and kinase-deficient cells demonstrate that cPLA2α is phosphorylated within 10 min of IL-1β treatment, an event requiring p38 MAPK as well as the upstream kinase, MKK3/MKK6. Inhibition of p38 MAPK also blocks the phosphorylation of a downstream, nuclear kinase, MSK-1. Our results further demonstrate that the activities of both cPLA2α and a downstream lipoxygenase (15-LOX2) are required for IL-1β-dependent induction of cPLA2α mRNA expression. Overall, these data support an MKK3/MKK6→p38 MAPK→MSK-1→cPLA2α→15-LOX2-dependent, positive feedback loop where a protein’s enzymatic activity is required to regulate its own gene induction by a pro-inflammatory stimulus. PMID:21771656

Evaluation of enzymatic and non-enzymatic antioxidants in seminal plasma of men with genitourinary infections, varicocele and idiopathic infertility.

PubMed

Micheli, L; Cerretani, D; Collodel, G; Menchiari, A; Moltoni, L; Fiaschi, A I; Moretti, E

2016-05-01

This study was aimed to assess the antioxidant enzymatic and non-enzymatic compounds in semen of infertile men. Seventy-four infertile patients were grouped according to their clinical diagnosis: genitourinary infection, varicocele, idiopathic infertility. Semen samples of fertile men represent the control. Semen characteristics were evaluated by light and transmission electron microscopy (TEM). TEM data was quantified with a mathematical formula, which provides numerical scores. Spectrophotometric and HPLC methods were used to measure the amount of reduced (GSH), oxidised glutathione (GSSG), ascorbic acid (AA) and malondialdehyde (MDA, marker of lipid peroxidation) and the activity of glutathione reductase, catalase (CAT), glutathione peroxidase. Infertile groups showed significantly decreased values of sperm parameters vs. In infection and varicocele groups, the seminal MDA levels were significantly increased when compared to controls (p < 0.001), indicating an alteration of oxidative status and a peroxidative damage. In infection and varicocele groups, AA levels were reduced (p < 0.05) vs. control; in the varicocele group, the GSH levels were also decreased (p < 0.05). Significantly higher CAT activity was observed in infection and varicocele groups vs. fertile men (p < 0.001 and p < 0.05 respectively). The GSH/GSSG ratio was significantly decreased in varicocele and idiopathic infertility groups vs. control (p < 0.01). The study of the alteration of a single parameter of oxidative stress or of the antioxidant system may not have a relevant clinical value to estimate male fertilising potential and the background of infertility causes, since complex and multifactorial mechanisms are involved in different pathologies. In our study, each pathology is characterised by a definite pattern of markers such as MDA and enzymatic and non-enzymatic antioxidant compounds. In the different pathologies related to infertility, the identification of the complex of

Impacts of dissolved organic matter on aqueous behavior of nano/micron-titanium nitride and their induced enzymatic/non-enzymatic antioxidant activities in Scenedesmus obliquus.

PubMed

Zhang, Xin; Wang, Zhuang; Wang, Se; Fang, Hao; Zhang, Fan; Wang, De-Gao

2017-01-02

Freshwater dispersion stability and ecotoxicological effects of titanium nitride (TiN) with particle size of 20 nm, 50 nm, and 2-10 μm in the presence of dissolved organic matter (DOM) at various concentrations were studied. The TiN particles that had a more negative zeta potential and smaller hydrodynamic size showed more stable dispersion in an aqueous medium when DOM was present than when DOM was absent. Biochemical assays indicated that relative to the control, the TiN particles in the presence of DOM alleviated to some extent the antioxidative stress enzyme activity in Scenedesmus obliquus. In addition, it was found that the TiN with a primary size of 50 nm at a high concentration presented a significant impact on non-enzymatic antioxidant defense in algal cells.

THE ENZYMATIC RESPONSE OF ASTROCYTES TO VARIOUS IONS IN VITRO

PubMed Central

Friede, Reinhard L.

1964-01-01

The effect of environmental ion concentration on the enzyme activity of astrocytes was investigated in tissue cultures of rat cerebral cortex. It was found that the oxidative enzymatic activity (succinic dehydrogenase, DPN-diaphorase, and several other enzymes) of astrocytes depended on the concentration of NaCl in the environment. This response was not specific for NaCl, but was also elicited by MgCl2 and LiCl; the response was less consistent, and often questionable for KCl. However, only NaCl could elicit enzymatic changes in astrocytes at concentrations known to be present in a living organism. Astrocytes were the only cells which responded this way; it appeared that the foot-plates were particularly involved in the response since increase of enzyme activity occurred earlier in the foot-plates than in the perikarya. It was concluded that astrocytes are metabolically involved in the maintenance of the ionic and osmotic environment of the central nervous system, particularly in regard to the active transport of sodium. PMID:14105217

Allergenic Properties of Enzymatically Hydrolyzed Peanut Flour Extracts

USDA-ARS?s Scientific Manuscript database

Peanut flour is a high protein, low oil, powdered material prepared from roasted 21 peanut seed. In addition to being a well-established food ingredient, peanut flour is also the 22 active ingredient in peanut oral immunotherapy trials. Enzymatic hydrolysis was evaluated as a 23 processing strategy ...

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

PubMed Central

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

2015-01-01

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

Cloning and characterization of microbial activated Aedes aegypti MEK4 (AaMEK4): influences of noncatalytic domains on enzymatic activity.

PubMed

Wu, R C-C; Cho, W-L

2014-10-01

Protein kinases are known to be involved in a number of signal transduction cascades. Both the stress-activated Jun N-terminal kinase (JNK) and mitogen-activated protein kinase (MAPK) p38 pathways have been shown to correlate with the insect immune response to microbial infection. MAP kinase kinase 4 (MEK4) is an upstream kinase of JNK and p38 kinase. The cDNA of AaMEK4 was cloned and characterized. AaMEK4 was activated by microbial lysates of Gram-positive, Gram-negative bacteria and yeast. The conserved lysine (K112 ) and the putative phosphorylation sites (S238 and T242 ) were shown to be important for kinase activity by site-directed mutagenesis. A common MAPK docking site (MAPK_dsA) was found and in addition, a new nearby docking site, MAPK_dsB, was identified in the N-terminal noncatalytic domain of AaMEK4. MAPK_dsB was shown to be a unique element in the MEK4 family. In this study, both MAPK_dsA and _dsB were demonstrated to be important to AaMEK4 enzymatic activity for the downstream protein kinase, Aap38. © 2014 The Royal Entomological Society.

Structural basis of the lack of endo-glucanase inhibitory activity of Lupinus albus γ-conglutin.

PubMed

Scarafoni, Alessio; Consonni, Alessandro; Pessina, Stefano; Balzaretti, Silvia; Capraro, Jessica; Galanti, Elisabetta; Duranti, Marcello

2016-02-01

Lupin γ-conglutin and soybean BG7S are two legume seed proteins strongly similar to plant endo-β-glucanases inhibitors acting against fungal GH11 and GH12 glycoside hydrolase. However these proteins lack inhibitory activity. Here we describe the conversion of lupin γ-conglutin to an active inhibitor of endo-β-glucanases belonging to GH11 family. A set of γ-conglutin mutants was designed and expressed in Pichia pastoris, along with the wild-type protein. Unexpectedly, this latter was able to inhibit a GH11 enzyme, but not GH12, whereas the mutants were able to modulate the inhibition capacity. In lupin, γ-conglutin is naturally cleaved in two subunits, whereas in P. pastoris it is not. The lack of proteolytic cleavage is one of the reasons at the basis of the inhibitory activity of recombinant γ-conglutin. The results provide new insights about structural features at the basis of the lack of inhibitory activity of wild-type γ-conglutin and its legume homologues. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

Antagonists' impact on enzymatic response in wilt infected cotton plants

USDA-ARS?s Scientific Manuscript database

A number of PR-proteins possess enzymatic activity. As such, these proteins maybe indicators of defensive response of plants. Thus, we have conducted a comparative analysis of beta-1,3-glucanase, peroxidase and xylanase activity in cotton plants to determine how these enzymes are affected by the pat...

Continuous enzymatic hydrolysis of lignocellulosic biomass with simultaneous detoxification and enzyme recovery.

PubMed

Gurram, Raghu N; Menkhaus, Todd J

2014-07-01

Recovering hydrolysis enzymes and/or alternative enzyme addition strategies are two potential mechanisms for reducing the cost during the biochemical conversion of lignocellulosic materials into renewable biofuels and biochemicals. Here, we show that enzymatic hydrolysis of acid-pretreated pine wood with continuous and/or fed-batch enzyme addition improved sugar conversion efficiencies by over sixfold. In addition, specific activity of the hydrolysis enzymes (cellulases, hemicellulases, etc.) increased as a result of continuously washing the residual solids with removal of glucose (avoiding the end product inhibition) and other enzymatic inhibitory compounds (e.g., furfural, hydroxymethyl furfural, organic acids, and phenolics). As part of the continuous hydrolysis, anion exchange resin was tested for its dual application of simultaneous enzyme recovery and removal of potential enzymatic and fermentation inhibitors. Amberlite IRA-96 showed favorable adsorption profiles of inhibitors, especially furfural, hydroxymethyl furfural, and acetic acid with low affinity toward sugars. Affinity of hydrolysis enzymes to adsorb onto the resin allowed for up to 92 % of the enzymatic activity to be recovered using a relatively low-molar NaCl wash solution. Integration of an ion exchange column with enzyme recovery into the proposed fed-batch hydrolysis process can improve the overall biorefinery efficiency and can greatly reduce the production costs of lignocellulosic biorenewable products.

Effects of 24-epibrassinolide on enzymatic browning and antioxidant activity of fresh-cut lotus root slices.

PubMed

Gao, Hui; Chai, HongKang; Cheng, Ni; Cao, Wei

2017-02-15

Fresh-cut lotus root slices were treated with 80nM 24-epibrassinolide (EBR) and then stored at 4°C for 8days to investigate the effects on cut surface browning. The results showed that EBR treatment reduced cut surface browning in lotus root slices and alleviated membrane lipid peroxidation as reflected by low malondialdehyde content and lipoxygenase activity. EBR treatment inhibited the activity of phenylalanine ammonia lyase and polyphenol oxidase, and subsequently decreased phenolics accumulation and soluble quniones formation. The treatment also stimulated the activity of peroxidase, catalase and ascorbate peroxidase and delayed the loss of ascorbic acid, which would help prevent membrane lipid peroxidation, as a consequence, reducing decompartmentation of enzymes and substrates causing enzymatic browning. These results indicate that EBR treatment is a promising attempt to control browning at cut surface of fresh-cut lotus root slices. Copyright © 2016 Elsevier Ltd. All rights reserved.

Enzymatic reactions in confined environments

NASA Astrophysics Data System (ADS)

Küchler, Andreas; Yoshimoto, Makoto; Luginbühl, Sandra; Mavelli, Fabio; Walde, Peter

2016-05-01

Within each biological cell, surface- and volume-confined enzymes control a highly complex network of chemical reactions. These reactions are efficient, timely, and spatially defined. Efforts to transfer such appealing features to in vitro systems have led to several successful examples of chemical reactions catalysed by isolated and immobilized enzymes. In most cases, these enzymes are either bound or adsorbed to an insoluble support, physically trapped in a macromolecular network, or encapsulated within compartments. Advanced applications of enzymatic cascade reactions with immobilized enzymes include enzymatic fuel cells and enzymatic nanoreactors, both for in vitro and possible in vivo applications. In this Review, we discuss some of the general principles of enzymatic reactions confined on surfaces, at interfaces, and inside small volumes. We also highlight the similarities and differences between the in vivo and in vitro cases and attempt to critically evaluate some of the necessary future steps to improve our fundamental understanding of these systems.

Enzymatic activity of a subtilisin homolog, Tk-SP, from Thermococcus kodakarensis in detergents and its ability to degrade the abnormal prion protein

PubMed Central

2013-01-01

Background Tk-SP is a member of subtilisin-like serine proteases from a hyperthermophilic archaeon Thermococcus kodakarensis. It has been known that the hyper-stable protease, Tk-SP, could exhibit enzymatic activity even at high temperature and in the presence of chemical denaturants. In this work, the enzymatic activity of Tk-SP was measured in the presence of detergents and EDTA. In addition, we focused to demonstrate that Tk-SP could degrade the abnormal prion protein (PrPSc), a protease-resistant isoform of normal prion protein (PrPC). Results Tk-SP was observed to maintain its proteolytic activity with nonionic surfactants and EDTA at 80°C. We optimized the condition in which Tk-SP functions efficiently, and demonstrated that the enzyme is highly stable in the presence of 0.05% (w/v) nonionic surfactants and 0.01% (w/v) EDTA, retaining up to 80% of its activity. Additionally, we also found that Tk-SP can degrade PrPSc to a level undetectable by western-blot analysis. Conclusions Our results indicate that Tk-SP has a great potential for technological applications, such as thermo-stable detergent additives. In addition, it is also suggested that Tk-SP-containing detergents can be developed to decrease the secondary infection risks of transmissible spongiform encephalopathies (TSE). PMID:23448268

Bio-conversion of apple pomace into ethanol and acetic acid: Enzymatic hydrolysis and fermentation.

PubMed

Parmar, Indu; Rupasinghe, H P Vasantha

2013-02-01

Enzymatic hydrolysis of cellulose present in apple pomace was investigated using process variables such as enzyme activity of commercial cellulase, pectinase and β-glucosidase, temperature, pH, time, pre-treatments and end product separation. The interaction of enzyme activity, temperature, pH and time had a significant effect (P<0.05) on release of glucose. Optimal conditions of enzymatic saccharification were: enzyme activity of cellulase, 43units; pectinase, 183units; β-glucosidase, 41units/g dry matter (DM); temperature, 40°C; pH 4.0 and time, 24h. The sugars were fermented using Saccharomyces cerevisae yielding 19.0g ethanol/100g DM. Further bio-conversion using Acetobacter aceti resulted in the production of acetic acid at a concentration of 61.4g/100g DM. The present study demonstrates an improved process of enzymatic hydrolysis of apple pomace to yield sugars and concomitant bioconversion to produce ethanol and acetic acid. Copyright © 2012 Elsevier Ltd. All rights reserved.

Drosophila immunity: analysis of PGRP-SB1 expression, enzymatic activity and function.

PubMed

Zaidman-Rémy, Anna; Poidevin, Mickael; Hervé, Mireille; Welchman, David P; Paredes, Juan C; Fahlander, Carina; Steiner, Hakan; Mengin-Lecreulx, Dominique; Lemaitre, Bruno

2011-02-18

Peptidoglycan is an essential and specific component of the bacterial cell wall and therefore is an ideal recognition signature for the immune system. Peptidoglycan recognition proteins (PGRPs) are conserved from insects to mammals and able to bind PGN (non-catalytic PGRPs) and, in some cases, to efficiently degrade it (catalytic PGRPs). In Drosophila, several non-catalytic PGRPs function as selective peptidoglycan receptors upstream of the Toll and Imd pathways, the two major signalling cascades regulating the systemic production of antimicrobial peptides. Recognition PGRPs specifically activate the Toll pathway in response to Lys-type peptidoglycan found in most Gram-positive bacteria and the Imd pathway in response to DAP-type peptidoglycan encountered in Gram-positive bacilli-type bacteria and in Gram-negative bacteria. Catalytic PGRPs on the other hand can potentially reduce the level of immune activation by scavenging peptidoglycan. In accordance with this, PGRP-LB and PGRP-SC1A/B/2 have been shown to act as negative regulators of the Imd pathway. In this study, we report a biochemical and genetic analysis of PGRP-SB1, a catalytic PGRP. Our data show that PGRP-SB1 is abundantly secreted into the hemolymph following Imd pathway activation in the fat body, and exhibits an enzymatic activity towards DAP-type polymeric peptidoglycan. We have generated a PGRP-SB1/2 null mutant by homologous recombination, but its thorough phenotypic analysis did not reveal any immune function, suggesting a subtle role or redundancy of PGRP-SB1/2 with other molecules. Possible immune functions of PGRP-SB1 are discussed.

Enzymatic activity of albumin shown by coelenterazine chemiluminescence.

PubMed

Vassel, N; Cox, C D; Naseem, R; Morse, V; Evans, R T; Power, R L; Brancale, A; Wann, K T; Campbell, A K

2012-01-01

Bioluminescence, the emission of light from live organisms, occurs in 18 phyla and is the major communication system in the deep sea. It has appeared independently many times during evolution but its origins remain unknown. Coelenterazine bioluminescence discovered in luminous jellyfish is the most common chemistry causing bioluminescence in the sea, occurring in seven phyla. Sequence similarities between coelenterazine luciferases and photoproteins from different phyla are poor (often < 5%). The aim of this study was to examine albumin that binds organic substances as a coelenterazine luciferase to test the hypothesis that the evolutionary origin of a bioluminescent protein was the result of the formation of a solvent cage containing just a few key amino acids. The results show for the first time that bovine and human albumin catalysed coelenterazine chemiluminescence consistent with a mono-oxygenase, whereas gelatin and haemoglobin, an oxygen carrier, had very weak activity. Insulin also catalysed coelenterazine chemiluminescence and was increased by Zn(2+). Albumin chemiluminescence was heat denaturable, exhibited saturable substrate characteristics and was inhibited by cations that bound these proteins and by drugs that bind to human albumin drug site I. Molecular modelling confirmed the coelenterazine binding site and identified four basic amino acids: lys195, arg222, his242 and arg257, potentially important in binding and catalysis similar to naturally occurring coelenterazine bioluminescent proteins. These results support the 'solvent cage' hypothesis for the evolutionary origin of enzymatic coelenterazine bioluminescent proteins. They also have important consequences in diseases such as diabetes, gut disorders and food intolerance where a mono-oxygenase could affect cell surface proteins. Copyright © 2012 John Wiley & Sons, Ltd.

Bacterial Production and Enzymatic Activities in Deep-Sea Sediments of the Pacific Ocean: Biogeochemical Implications of Different Temperature Constraints

NASA Astrophysics Data System (ADS)

Danovaro, R.; Corinaldesi, C.; dell'Anno, A.

2002-12-01

The deep-sea bed, acting as the ultimate sink for organic material derived from the upper oceans primary production, is now assumed to play a key role in biogeochemical cycling of organic matter on global scale. Early diagenesis of organic matter in marine sediments is dependent upon biological processes (largely mediated by bacterial activity) and by molecular diffusion. Organic matter reaching the sea floor by sedimentation is subjected to complex biogeochemical transformations that make organic matter largely unsuitable for direct utilization by benthic heterotrophs. Extracellular enzymatic activities in the sediment is generally recognized as the key step in the degradation and utilization of organic polymers by bacteria and a key role in biopolymeric carbon mobilization is played by aminopeptidase, alkaline phosphatase and glucosidase activities. In the present study we investigated bacterial density, bacterial C production and exo-enzymatic activities (aminopeptidase, glucosidase and phosphatase activity) in deep-sea sediments of the Pacific Ocean in relation with the biochemical composition of sediment organic matter (proteins, carbohydrates and lipids), in order to gather information on organic matter cycling and diagenesis. Benthic viral abundance was also measured to investigate the potential role of viruses on microbial loop functioning. Sediment samples were collected at eight stations (depth ranging from 2070-3100 m) along two transects located at the opposite side (north and south) of ocean seismic ridge Juan Fernandez (along latitudes 33° 20' - 33° 40'), constituted by the submerged vulcanoes, which connects the Chilean coasts to Rapa Nui Island. Since the northern and southern sides of this ridge apparently displayed small but significant differences in deep-sea temperature (related to the general ocean circulation), this sampling strategy allowed also investigating the role of different temperature constraints on bacterial activity and

Comparative study of enzymatic activities of new KatG mutants from low- and high-level isoniazid-resistant clinical isolates of Mycobacterium tuberculosis.

PubMed

Brossier, Florence; Boudinet, Marlène; Jarlier, Vincent; Petrella, Stéphanie; Sougakoff, Wladimir

2016-09-01

Resistance to isoniazid (INH-R) in Mycobacterium tuberculosis is mainly due to mutations at position 315 (S315T) of the catalase-peroxidase KatG. We identified 16 mutations (including 13 biochemically uncharacterized mutations) in KatG from INH-R clinical isolates of M. tuberculosis showing mutations other than S315T. The KatG enzymatic activities (catalase, peroxidase, free radical production and isonicotinoyl-NAD formation) of wild-type KatG and the 16 mutants were determined and correlated to their spatial location in a KatG model structure. Of all mutations studied, H270R, which conferred a high level of INH-R and results in the disruption of a coordination bond with the heme, caused complete loss of all enzymatic KatG activities. The mutants generally associated with a very high level of INH-R were all characterized by a drastic reduction in catalase activity and a marked decrease in INH activation activities. One mutant, A162E, displayed a behavior similar to S315T, i.e. a moderate decrease in catalase activity and a drastic decrease in the formation of the radical form of INH. Finally, the mutants associated with a low level of INH-R showed a moderate reduction in the four catalytic activities, likely stemming from an overall alteration of the folding and/or stability of the KatG protein. Copyright © 2016 Elsevier Ltd. All rights reserved.

Isoprene Production on Enzymatic Hydrolysate of Peanut Hull Using Different Pretreatment Methods.

PubMed

Wang, Sumeng; Li, Ruichao; Yi, Xiaohua; Fang, Tigao; Yang, Jianming; Bae, Hyeun-Jong

2016-01-01

The present study is about the use of peanut hull for isoprene production. In this study, two pretreatment methods, hydrogen peroxide-acetic acid (HPAC) and popping, were employed prior to enzymatic hydrolysis, which could destroy the lignocellulosic structure and accordingly improve the efficiency of enzymatic hydrolysis. It is proven that the isoprene production on enzymatic hydrolysate with HPAC pretreatment is about 1.9-fold higher than that of popping pretreatment. Moreover, through High Performance Liquid Chromatography (HPLC) analysis, the amount and category of inhibitors such as formic acid, acetic acid, and HMF were assayed and were varied in different enzymatic hydrolysates, which may be the reason leading to a decrease in isoprene production during fermentation. To further increase the isoprene yield, the enzymatic hydrolysate of HPAC was detoxified by activated carbon. As a result, using the detoxified enzymatic hydrolysate as the carbon source, the engineered strain YJM21 could accumulate 297.5 mg/L isoprene, which accounted for about 90% of isoprene production by YJM21 fermented on pure glucose (338.6 mg/L). This work is thought to be the first attempt on isoprene production by E. coli using peanut hull as the feedstock. More importantly, it also shows the prospect of peanut hull to be considered as an alternative feedstock for bio-based chemicals or biofuels production due to its easy access and high polysaccharide content.

Macrophage Migration Inhibitory Factor Enzymatic Activity, Lung Inflammation, and Cystic Fibrosis

PubMed Central

Adamali, Huzaifa; Armstrong, Michelle E.; McLaughlin, Anne Marie; Cooke, Gordon; McKone, Edward; Costello, Christine M.; Gallagher, Charles G.; Leng, Lin; Baugh, John A.; Fingerle-Rowson, Günter; Bucala, Richard J.; McLoughlin, Paul

2012-01-01

Rationale: Macrophage migration inhibitory factor (MIF) is a proinflammatory mediator with unique tautomerase enzymatic activity; the precise function has not been clearly defined. We previously demonstrated that individual patients with cystic fibrosis (CF) who are genetically predisposed to be high MIF producers develop accelerated end-organ injury. Objectives: To characterize the effects of the MIF-CATT polymorphism in patients with CF ex vivo. To investigate the role of MIF’s tautomerase activity in a murine model of Pseudomonas aeruginosa infection. Methods: MIF and tumor necrosis factor (TNF)-α protein levels were assessed in plasma or peripheral blood mononuclear cell (PBMC) supernatants by ELISA. A murine pulmonary model of chronic Pseudomonas infection was used in MIF wild-type mice (mif+/+) and in tautomerase-null, MIF gene knockin mice (mif P1G/P1G). Measurements and Main Results: MIF protein was measured in plasma and PBMCs from 5- and 6-CATT patients with CF; LPS-induced TNF-α production from PBMCs was also assessed. The effect of a specific inhibitor of MIF-tautomerase activity, ISO-1, was investigated in PBMCs. In the murine infection model, total weight loss, differential cell counts, bacterial load, and intraacinar airspace/tissue volume were measured. MIF and TNF-α levels were increased in 6-CATT compared with 5-CATT patients with CF. LPS-induced TNF-α production from PBMCs was attenuated in the presence of ISO-1. In a murine model of Pseudomonas infection, significantly less pulmonary inflammation and bacterial load was observed in mifP1G/P1G compared with mif+/+ mice. Conclusions: MIF-tautomerase activity may provide a novel therapeutic target in patients with chronic inflammatory diseases such as CF, particularly those patients who are genetically predisposed to produce increased levels of this cytokine. PMID:22592805

Substitution-inert trinuclear platinum complexes efficiently condense/aggregate nucleic acids and inhibit enzymatic activity.

PubMed

Malina, Jaroslav; Farrell, Nicholas P; Brabec, Viktor

2014-11-17

The trinuclear platinum complexes (TriplatinNC-A [{Pt(NH3 )3 }2 -μ-{trans-Pt(NH3 )2 (NH2 (CH2 )6 NH2 )2 }](6+) , and TriplatinNC [{trans-Pt(NH3 )2 (NH2 (CH2 )6 NH3 (+) )}2 -μ-{trans-Pt(NH3 )2 (NH2 (CH2 )6 NH2 )2 }](8+) ) are biologically active agents that bind to DNA through noncovalent (hydrogen bonding, electrostatic) interactions. Herein, we show that TriplatinNC condenses DNA with a much higher potency than conventional DNA condensing agents. Both complexes induce aggregation of small transfer RNA molecules, and TriplatinNC in particular completely inhibits DNA transcription at lower concentrations than naturally occurring spermine. Topoisomerase I-mediated relaxation of supercoiled DNA was inhibited by TriplatinNC-A and TriplatinNC at concentrations which were 60 times and 250 times lower than that of spermine. The mechanisms for the biological activity of TriplatinNC-A and TriplatinNC may be associated with their ability to condense/aggregate nucleic acids with consequent inhibitory effects on crucial enzymatic activities. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Determination of the Activation Energy of the Enzymatic Biodegradation Process in Microfabricated Polyhydroxyalkanoate Thin Films Using In-Situ, Real Time Atomic Force Microscopy

NASA Astrophysics Data System (ADS)

Morse, Clinton; Latuga, Brian M.; Delfaus, Stephen; Devore, Thomas C.; Augustine, Brian H.; Hughes, W. Christopher; Warne, Paul G.

2003-11-01

Using the liquid cell capability of the atomic force microscope (AFM), we report the determination of the activation energy of the biodegradation process of the enzymatic biodegradation of poly 3-hydroxybutyrate / poly 3-hydroxyvalerate [P(3HB-HV)] thin films. We have prepared P(3HB-3HV) copolymer microstructures by the selective dewetting of soft lithographically patterned gold substrates with features sizes down to 10 mm. These have been then used as an internal height standard to measure the volume of material as a function of biodegradation time. Biodegradation is measured in-situ and real time using contact mode AFM in an enzymatic solution produced from Streptomyces sp. bacteria. The temperature dependent biodegradation has been measured over a temperature range from 23oC to 40oC. We will discuss the calculation of the activation energy of this process as well as a physical model to describe three distinct regions in the biodegradation process that have been observed.

Hepatitis B Virus Lacks Immune Activating Capacity, but Actively Inhibits Plasmacytoid Dendritic Cell Function

PubMed Central

Woltman, Andrea M.; Shi, Cui C.; Janssen, Harry L. A.

2011-01-01

Chronic hepatitis B virus (HBV) infection is caused by inadequate anti-viral immunity. Activation of plasmacytoid dendritic cells (pDC) leading to IFNα production is important for effective anti-viral immunity. Hepatitis B virus (HBV) infection lacks IFNα induction in animal models and patients and chronic HBV patients display impaired IFNα production by pDC. Therefore, HBV and HBV-derived proteins were examined for their effect on human pDC in vitro. In addition, the in vitro findings were compared to the function of pDC derived from chronic HBV patients ex vivo. In contrast to other viruses, HBV did not activate pDC. Moreover, HBV and HBsAg abrogated CpG-A/TLR9-induced, but not Loxoribine/TLR7-induced, mTOR-mediated S6 phosphorylation, subsequent IRF7 phosphorylation and IFNα gene transcription. HBV/HBsAg also diminished upregulation of co-stimulatory molecules, production of TNFα, IP-10 and IL-6 and pDC-induced NK cell function, whereas TLR7-induced pDC function was hardly affected. In line, HBsAg preferentially bound to TLR9-triggered pDC demonstrating that once pDC are able to bind HBV/HBsAg, the virus exerts its immune regulatory effect. HBV not only directly interfered with pDC function, but also indirectly by interfering with monocyte-pDC interaction. Also HBeAg diminished pDC function to a certain extent, but via another unknown mechanism. Interestingly, patients with HBeAg-positive chronic hepatitis B displayed impaired CpG-induced IFNα production by pDC without significant alterations in Loxoribine-induced pDC function compared to HBeAg-negative patients and healthy controls. The lack of activation and the active inhibition of pDC by HBV may both contribute to HBV persistence. The finding that the interaction between pDC and HBV may change upon activation may aid in the identification of a scavenging receptor supporting immunosuppressive effects of HBV and also in the design of novel treatment strategies for chronic HBV. PMID:21246041

Recent Research Trends on the Enzymatic Synthesis of Structured Lipids.

PubMed

Kim, Byung Hee; Akoh, Casimir C

2015-08-01

Structured lipids (SLs) are lipids that have been chemically or enzymatically modified from their natural biosynthetic form. Because SLs are made to possess desired nutritional, physicochemical, or textural properties for various applications in the food industry, many research activities have been aimed at their commercialization. The production of SLs by enzymatic procedures has a great potential in the future market because of the specificity of lipases and phospholipases used as the biocatalysts. The aim of this review is to provide concise information on the recent research trends on the enzymatic synthesis of SLs of commercial interest, such as medium- and long-chain triacylglycerols, human milk fat substitutes, cocoa butter equivalents, trans-free or low-trans plastic fats (such as margarines and shortenings), low-calorie fats/oils, health-beneficial fatty acid-rich fats/oils, mono- or diacylglycerols, and structurally modified phospholipids. This limited review covers 108 research articles published between 2010 and 2014 which were searched in Web of Science. © 2015 Institute of Food Technologists®

A singular enzymatic megacomplex from Bacillus subtilis.

PubMed

Straight, Paul D; Fischbach, Michael A; Walsh, Christopher T; Rudner, David Z; Kolter, Roberto

2007-01-02

Nonribosomal peptide synthetases (NRPS), polyketide synthases (PKS), and hybrid NRPS/PKS are of particular interest, because they produce numerous therapeutic agents, have great potential for engineering novel compounds, and are the largest enzymes known. The predicted masses of known enzymatic assembly lines can reach almost 5 megadaltons, dwarfing even the ribosome (approximately 2.6 megadaltons). Despite their uniqueness and importance, little is known about the organization of these enzymes within the native producer cells. Here we report that an 80-kb gene cluster, which occupies approximately 2% of the Bacillus subtilis genome, encodes the subunits of approximately 2.5 megadalton active hybrid NRPS/PKS. Many copies of the NRPS/PKS assemble into a single organelle-like membrane-associated complex of tens to hundreds of megadaltons. Such an enzymatic megacomplex is unprecedented in bacterial subcellular organization and has important implications for engineering novel NRPS/PKSs.

Ink-native electrophoresis: an alternative to blue-native electrophoresis more suitable for in-gel detection of enzymatic activity.

PubMed

Kaneko, Keisuke; Sueyoshi, Noriyuki; Kameshita, Isamu; Ishida, Atsuhiko

2013-09-15

Blue-native electrophoresis (BNE) is a useful technique for analyzing protein complexes, but the Coomassie brilliant blue (CBB) dye used in BNE often hampers in-gel detection of enzymatic activity. Here we report an improved method, termed ink-native electrophoresis (INE), in which Pelikan 4001 fountain pen ink is used as a charge-shifting agent instead of CBB. INE is more suitable than BNE for in-gel detection of protein kinase activity after polyacrylamide gel electrophoresis (PAGE), and its performance in protein complex separation is comparable to that of conventional BNE. INE may provide a powerful tool to isolate and analyze various protein complexes. Copyright © 2013 Elsevier Inc. All rights reserved.

Iron(II) supramolecular helicates condense plasmid DNA and inhibit vital DNA-related enzymatic activities.

PubMed

Malina, Jaroslav; Hannon, Michael J; Brabec, Viktor

2015-07-27

The dinuclear iron(II) supramolecular helicates [Fe2 L3 ]Cl4 (L=C25 H20 N4 ) bind to DNA through noncovalent (i.e., hydrogen-bonding, electrostatic) interactions and exhibit antimicrobial and anticancer effects. In this study, we show that the helicates condense plasmid DNA with a much higher potency than conventional DNA-condensing agents. Notably, molecules of DNA in the presence of the M enantiomer of [Fe2 L3 ]Cl4 do not form intermolecular aggregates typically formed by other condensing agents, such as spermidine or spermine. The helicates inhibit the activity of several DNA-processing enzymes, such as RNA polymerase, DNA topoisomerase I, deoxyribonuclease I, and site-specific restriction endonucleases. However, the results also indicate that the DNA condensation induced by the helicates does not play a crucial role in these inhibition reactions. The mechanisms for the inhibitory effects of [Fe2 L3 ]Cl4 helicates on DNA-related enzymatic activities have been proposed. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Isoprene Production on Enzymatic Hydrolysate of Peanut Hull Using Different Pretreatment Methods

PubMed Central

Wang, Sumeng; Li, Ruichao; Yi, Xiaohua; Fang, Tigao

2016-01-01

The present study is about the use of peanut hull for isoprene production. In this study, two pretreatment methods, hydrogen peroxide-acetic acid (HPAC) and popping, were employed prior to enzymatic hydrolysis, which could destroy the lignocellulosic structure and accordingly improve the efficiency of enzymatic hydrolysis. It is proven that the isoprene production on enzymatic hydrolysate with HPAC pretreatment is about 1.9-fold higher than that of popping pretreatment. Moreover, through High Performance Liquid Chromatography (HPLC) analysis, the amount and category of inhibitors such as formic acid, acetic acid, and HMF were assayed and were varied in different enzymatic hydrolysates, which may be the reason leading to a decrease in isoprene production during fermentation. To further increase the isoprene yield, the enzymatic hydrolysate of HPAC was detoxified by activated carbon. As a result, using the detoxified enzymatic hydrolysate as the carbon source, the engineered strain YJM21 could accumulate 297.5 mg/L isoprene, which accounted for about 90% of isoprene production by YJM21 fermented on pure glucose (338.6 mg/L). This work is thought to be the first attempt on isoprene production by E. coli using peanut hull as the feedstock. More importantly, it also shows the prospect of peanut hull to be considered as an alternative feedstock for bio-based chemicals or biofuels production due to its easy access and high polysaccharide content. PMID:27847814

Bacterial communities and enzymatic activities in the vegetation-activated sludge process (V-ASP) and related advantages by comparison with conventional constructed wetland.

PubMed

Yuan, Jiajia; Dong, Wenyi; Sun, Feiyun; Zhao, Ke; Du, Changhang; Shao, Yunxian

2016-11-01

A new-developed vegetation-activated sludge process (V-ASP) was implemented for decentralized domestic wastewater treatment, and studied in lab-scale and full-scale. The main purpose of this work was the investigation of biomass activities and microbial communities in V-ASP by comparison with conventional constructed wetland (CW), to unveil the causations of its consistently higher pollutants removal efficiencies. Compared with CWs, V-ASP has greater vegetation nitrogen and phosphorus uptake rates, higher biomass and enzymatic activities, and more bacteria community diversity. The microbial community structure was comprehensively analyzed by using high-throughput sequencing. It was observed that Proteobacteria was dominated in both CWs and V-ASPs, while their subdivisions distribution was rather different. V-ASPs contained a higher nitrite-oxidizing bacteria (Nitrospira) abundances that resulted in a consistently better nitrogen removal efficiency. Hence, a long-term experiment of full-scale V-ASP displayed stably excellent capability in resistance of influent loading shocks and seasonal temperature effect. Copyright © 2016 Elsevier Ltd. All rights reserved.

[Enzymatic analysis of the quality of foodstuffs].

PubMed

Kolesnov, A Iu

1997-01-01

Enzymatic analysis is an independent and separate branch of enzymology and analytical chemistry. It has become one of the most important methodologies used in food analysis. Enzymatic analysis allows the quick, reliable determination of many food ingredients. Often these contents cannot be determined by conventional methods, or if methods are available, they are determined only with limited accuracy. Today, methods of enzymatic analysis are being increasingly used in the investigation of foodstuffs. Enzymatic measurement techniques are used in industry, scientific and food inspection laboratories for quality analysis. This article describes the requirements of an optimal analytical method: specificity, sample preparation, assay performance, precision, sensitivity, time requirement, analysis cost, safety of reagents.

Enzymatic Detoxication, Conformational Selection, and the Role of Molten Globule Active Sites*

PubMed Central

Honaker, Matthew T.; Acchione, Mauro; Zhang, Wei; Mannervik, Bengt; Atkins, William M.

2013-01-01

The role of conformational ensembles in enzymatic reactions remains unclear. Discussion concerning “induced fit” versus “conformational selection” has, however, ignored detoxication enzymes, which exhibit catalytic promiscuity. These enzymes dominate drug metabolism and determine drug-drug interactions. The detoxication enzyme glutathione transferase A1–1 (GSTA1–1), exploits a molten globule-like active site to achieve remarkable catalytic promiscuity wherein the substrate-free conformational ensemble is broad with barrierless transitions between states. A quantitative index of catalytic promiscuity is used to compare engineered variants of GSTA1–1 and the catalytic promiscuity correlates strongly with characteristics of the thermodynamic partition function, for the substrate-free enzymes. Access to chemically disparate transition states is encoded by the substrate-free conformational ensemble. Pre-steady state catalytic data confirm an extension of the conformational selection model, wherein different substrates select different starting conformations. The kinetic liability of the conformational breadth is minimized by a smooth landscape. We propose that “local” molten globule behavior optimizes detoxication enzymes. PMID:23649628

Enzymatic biofilm digestion in soil aggregates facilitates the release of particulate organic matter by sonication

NASA Astrophysics Data System (ADS)

Büks, Frederick; Kaupenjohann, Martin

2016-10-01

The stability of soil aggregates against shearing and compressive forces as well as water-caused dispersion is an integral marker of soil quality. High stability results in less compaction and erosion and has been linked to enhanced water retention, dynamic water transport and aeration regimes, increased rooting depth, and protection of soil organic matter (SOM) against microbial degradation. In turn, particulate organic matter is supposed to support soil aggregate stabilization. For decades the importance of biofilm extracellular polymeric substances (EPSs) regarding particulate organic matter (POM) occlusion and aggregate stability has been canonical because of its distribution, geometric structure and ability to link primary particles. However, experimental proof is still missing. This lack is mainly due to methodological reasons. Thus, the objective of this work is to develop a method of enzymatic biofilm detachment for studying the effects of EPSs on POM occlusion. The method combines an enzymatic pre-treatment with different activities of α-glucosidase, β-galactosidase, DNAse and lipase with a subsequent sequential ultrasonic treatment for disaggregation and density fractionation of soils. POM releases of treated samples were compared to an enzyme-free control. To test the efficacy of biofilm detachment the ratio of bacterial DNA from suspended cells and the remaining biofilm after enzymatic treatment were measured by quantitative real-time PCR. Although the enzyme treatment was not sufficient for total biofilm removal, our results indicate that EPSs may attach POM within soil aggregates. The tendency to additional POM release with increased application of enzymes was attributed to a slight loss in aggregate stability. This suggests that an effect of agricultural practices on soil microbial populations could influence POM occlusion/aggregate stability and thereby carbon cycle/soil quality.

Enzymatic regeneration of adenosine triphosphate cofactor

NASA Technical Reports Server (NTRS)

Marshall, D. L.

1974-01-01

Regenerating adenosine triphosphate (ATP) from adenosine diphosphate (ADP) by enzymatic process which utilizes carbamyl phosphate as phosphoryl donor is technique used to regenerate expensive cofactors. Process allows complex enzymatic reactions to be considered as candidates for large-scale continuous processes.

Enzymatic Depilation of Animal Hide: Identification of Elastase (LasB) from Pseudomonas aeruginosa MCM B-327 as a Depilating Protease

PubMed Central

Pandeeti, Emmanuel Vijay Paul; Pitchika, Gopi Krishna; Jotshi, Jyotsna; Nilegaonkar, Smita S.; Kanekar, Pradnya P.; Siddavattam, Dayananda

2011-01-01

Conventional leather processing involving depilation of animal hide by lime and sulphide treatment generates considerable amounts of chemical waste causing severe environmental pollution. Enzymatic depilation is an environmentally friendly process and has been considered to be a viable alternative to the chemical depilation process. We isolated an extracellular protease from Pseudomonas aeruginosa strain MCM B-327 with high depilation activity using buffalo hide as a substrate. This 33 kDa protease generated a peptide mass fingerprint and de novo sequence that matched perfectly with LasB (elastase), of Pseudomonas aeruginosa. In support of this data a lasB mutant of MCM B-327 strain lacked depilatory activity and failed to produce LasB. LasB heterologously over-produced and purified from Escherichia coli also exhibited high depilating activity. Moreover, reintroduction of the lasB gene to the P. aeruginosa lasB mutant via a knock-in strategy also successfully restored depilation activity thus confirming the role of LasB as the depilating enzyme. PMID:21347249

D-Amino acid oxidase bio-functionalized platforms: Toward an enhanced enzymatic bio-activity

NASA Astrophysics Data System (ADS)

Herrera, Elisa; Valdez Taubas, Javier; Giacomelli, Carla E.

2015-11-01

The purpose of this work is to study the adsorption process and surface bio-activity of His-tagged D-amino acid oxidase (DAAO) from Rhodotorula gracilis (His6-RgDAAO) as the first step for the development of an electrochemical bio-functionalized platform. With such a purpose this work comprises: (a) the His6-RgDAAO bio-activity in solution determined by amperometry, (b) the adsorption mechanism of His6-RgDAAO on bare gold and carboxylated modified substrates in the absence (substrate/COO-) and presence of Ni(II) (substrate/COO- + Ni(II)) determined by reflectometry, and (c) the bio-activity of the His6-RgDAAO bio-functionalized platforms determined by amperometry. Comparing the adsorption behavior and bio-activity of His6-RgDAAO on these different solid substrates allows understanding the contribution of the diverse interactions responsible for the platform performance. His6-RgDAAO enzymatic performance in solution is highly improved when compared to the previously used pig kidney (pk) DAAO. His6-RgDAAO exhibits an amperometrically detectable bio-activity at concentrations as low as those expected on a bio-functional platform; hence, it is a viable bio-recognition element of D-amino acids to be coupled to electrochemical platforms. Moreover, His6-RgDAAO bio-functionalized platforms exhibit a higher surface activity than pkDAAO physically adsorbed on gold. The platform built on Ni(II) modified substrates present enhanced bio-activity because the surface complexes histidine-Ni(II) provide with site-oriented, native-like enzymes. The adsorption mechanism responsible of the excellent performance of the bio-functionalized platform takes place in two steps involving electrostatic and bio-affinity interactions whose prevalence depends on the degree of surface coverage.

Interaction with Single-stranded DNA-binding Protein Stimulates Escherichia coli Ribonuclease HI Enzymatic Activity*

PubMed Central

Petzold, Christine; Marceau, Aimee H.; Miller, Katherine H.; Marqusee, Susan; Keck, James L.

2015-01-01

Single-stranded (ss) DNA-binding proteins (SSBs) bind and protect ssDNA intermediates formed during replication, recombination, and repair reactions. SSBs also directly interact with many different genome maintenance proteins to stimulate their enzymatic activities and/or mediate their proper cellular localization. We have identified an interaction formed between Escherichia coli SSB and ribonuclease HI (RNase HI), an enzyme that hydrolyzes RNA in RNA/DNA hybrids. The RNase HI·SSB complex forms by RNase HI binding the intrinsically disordered C terminus of SSB (SSB-Ct), a mode of interaction that is shared among all SSB interaction partners examined to date. Residues that comprise the SSB-Ct binding site are conserved among bacterial RNase HI enzymes, suggesting that RNase HI·SSB complexes are present in many bacterial species and that retaining the interaction is important for its cellular function. A steady-state kinetic analysis shows that interaction with SSB stimulates RNase HI activity by lowering the reaction Km. SSB or RNase HI protein variants that disrupt complex formation nullify this effect. Collectively our findings identify a direct RNase HI/SSB interaction that could play a role in targeting RNase HI activity to RNA/DNA hybrid substrates within the genome. PMID:25903123

Interaction with Single-stranded DNA-binding Protein Stimulates Escherichia coli Ribonuclease HI Enzymatic Activity

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

Petzold, Christine; Marceau, Aimee H.; Miller, Katherine H.

Single-stranded (ss) DNA-binding proteins (SSBs) bind and protect ssDNA intermediates formed during replication, recombination, and repair reactions. SSBs also directly interact with many different genome maintenance proteins to stimulate their enzymatic activities and/or mediate their proper cellular localization. We have identified an interaction formed between Escherichia coli SSB and ribonuclease HI (RNase HI), an enzyme that hydrolyzes RNA in RNA/DNA hybrids. The RNase HI·SSB complex forms by RNase HI binding the intrinsically disordered C terminus of SSB (SSB-Ct), a mode of interaction that is shared among all SSB interaction partners examined to date. Residues that comprise the SSB-Ct binding sitemore » are conserved among bacterial RNase HI enzymes, suggesting that RNase HI·SSB complexes are present in many bacterial species and that retaining the interaction is important for its cellular function. A steady-state kinetic analysis shows that interaction with SSB stimulates RNase HI activity by lowering the reaction Km. SSB or RNase HI protein variants that disrupt complex formation nullify this effect. Collectively our findings identify a direct RNase HI/SSB interaction that could play a role in targeting RNase HI activity to RNA/DNA hybrid substrates within the genome.« less

Interaction with Single-stranded DNA-binding Protein Stimulates Escherichia coli Ribonuclease HI Enzymatic Activity.

PubMed

Petzold, Christine; Marceau, Aimee H; Miller, Katherine H; Marqusee, Susan; Keck, James L

2015-06-05

Single-stranded (ss) DNA-binding proteins (SSBs) bind and protect ssDNA intermediates formed during replication, recombination, and repair reactions. SSBs also directly interact with many different genome maintenance proteins to stimulate their enzymatic activities and/or mediate their proper cellular localization. We have identified an interaction formed between Escherichia coli SSB and ribonuclease HI (RNase HI), an enzyme that hydrolyzes RNA in RNA/DNA hybrids. The RNase HI·SSB complex forms by RNase HI binding the intrinsically disordered C terminus of SSB (SSB-Ct), a mode of interaction that is shared among all SSB interaction partners examined to date. Residues that comprise the SSB-Ct binding site are conserved among bacterial RNase HI enzymes, suggesting that RNase HI·SSB complexes are present in many bacterial species and that retaining the interaction is important for its cellular function. A steady-state kinetic analysis shows that interaction with SSB stimulates RNase HI activity by lowering the reaction Km. SSB or RNase HI protein variants that disrupt complex formation nullify this effect. Collectively our findings identify a direct RNase HI/SSB interaction that could play a role in targeting RNase HI activity to RNA/DNA hybrid substrates within the genome. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Clinical manifestations and enzymatic activities of mitochondrial respiratory chain complexes in Pearson marrow-pancreas syndrome with 3-methylglutaconic aciduria: a case report and literature review.

PubMed

Sato, Takeshi; Muroya, Koji; Hanakawa, Junko; Iwano, Reiko; Asakura, Yumi; Tanaka, Yukichi; Murayama, Kei; Ohtake, Akira; Hasegawa, Tomonobu; Adachi, Masanori

2015-12-01

Pearson marrow-pancreas syndrome (PS) is a rare mitochondrial disorder. Impaired mitochondrial respiratory chain complexes (MRCC) differ among individuals and organs, which accounts for variable clinical pictures. A subset of PS patients develop 3-methylglutaconic aciduria (3-MGA-uria), but the characteristic symptoms and impaired MRCC remain unknown. Our patient, a girl, developed pancytopenia, hyperlactatemia, steatorrhea, insulin-dependent diabetes mellitus, liver dysfunction, Fanconi syndrome, and 3-MGA-uria. She died from cerebral hemorrhage at 3 years of age. We identified a novel 5.4-kbp deletion of mitochondrial DNA. The enzymatic activities of MRCC I and IV were markedly reduced in the liver and muscle and mildly reduced in skin fibroblasts and the heart. To date, urine organic acid analysis has been performed on 29 PS patients, including our case. Eight patients had 3-MGA-uria, while only one patient did not. The remaining 20 patients were not reported to have 3-MGA-uria. In this paper, we included these 20 patients as PS patients without 3-MGA-uria. PS patients with and without 3-MGA-uria have similar manifestations. Only a few studies have examined the enzymatic activities of MRCC. No clinical characteristics distinguish between PS patients with and without 3-MGA-uria. The correlation between 3-MGA-uria and the enzymatic activities of MRCC remains to be elucidated. • The clinical characteristics of patients with Pearson marrow-pancreas syndrome and 3-methylglutaconic aciduria remain unknown. • No clinical characteristics distinguish between Pearson marrow-pancreas syndrome patients with and without 3-methylglutaconic aciduria.

How Metal Substitution Affects the Enzymatic Activity of Catechol-O-Methyltransferase

PubMed Central

Sparta, Manuel; Alexandrova, Anastassia N.

2012-01-01

Catechol-O-methyltransferase (COMT) degrades catecholamines, such as dopamine and epinephrine, by methylating them in the presence of a divalent metal cation (usually Mg(II)), and S-adenosyl-L-methionine. The enzymatic activity of COMT is known to be vitally dependent on the nature of the bound metal: replacement of Mg(II) with Ca(II) leads to a complete deactivation of COMT; Fe(II) is slightly less than potent Mg(II), and Fe(III) is again an inhibitor. Considering the fairly modest role that the metal plays in the catalyzed reaction, this dependence is puzzling, and to date remains an enigma. Using a quantum mechanical / molecular mechanical dynamics method for extensive sampling of protein structure, and first principle quantum mechanical calculations for the subsequent mechanistic study, we explicate the effect of metal substitution on the rate determining step in the catalytic cycle of COMT, the methyl transfer. In full accord with experimental data, Mg(II) bound to COMT is the most potent of the studied cations and it is closely followed by Fe(II), whereas Fe(III) is unable to promote catalysis. In the case of Ca(II), a repacking of the protein binding site is observed, leading to a significant increase in the activation barrier and higher energy of reaction. Importantly, the origin of the effect of metal substitution is different for different metals: for Fe(III) it is the electronic effect, whereas in the case of Ca(II) it is instead the effect of suboptimal protein structure. PMID:23056605

Ultrasonic accelerates asparagine-glucose non-enzymatic browning reaction without acrylamide formation.

PubMed

Gao, Zhiqiang; Zheng, Junfeng; Chen, Lian

2017-01-01

Ultrasonic accelerated the asparagine-glucose non-enzymatic browning reaction with significant decrease of glucose and asparagine concentrations, and marked increase of intermediate products (UV-absorbance value at 294nm, Abs 294 ), melanoidins (UV-absorbance value at 420nm, Abs 420 ) and in vitro antioxidant activity (DPPH free radical scavenging activity). As the ultrasonic intensity was 17.83W/cm 2 , the asparagine-glucose solution's Abs 294 , Abs 420 and antioxidant activity increased from 0 to 1.26, 0.88 and 21.56%, respectively, and the glucose and asparagine concentrations of the asparagine-glucose solution reduced 58.97 and 12.57%, respectively. The high performance liquid chromatography (HPLC)-Diode Array Detector (DAD) analyses showed that no acrylamide was detected after 50-min ultrasonic reaction. This study suggested that ultrasonic at higher intensity was a potential method to accelerate the non-enzymatic browning reaction in the asparagine-glucose solution without acrylamide production. Copyright © 2016 Elsevier B.V. All rights reserved.

Maltodextrin-powered enzymatic fuel cell through a non-natural enzymatic pathway

NASA Astrophysics Data System (ADS)

Zhu, Zhiguang; Wang, Yiran; Minteer, Shelley D.; Percival Zhang, Y.-H.

Enzymatic fuel cells (EFCs) use a variety of fuels to generate electricity through oxidoreductase enzymes, such as oxidases or dehydrogenases, as catalysts on electrodes. We have developed a novel synthetic enzymatic pathway containing two free enzymes (maltodextrin phosphorylase and phosphoglucomutase) and one immobilized glucose-6-phosphate dehydrogenase that can utilize an oligomeric substrate maltodextrin for producing electrons mediated via a diaphorase and vitamin K 3 electron shuttle system. Three different enzyme immobilization approaches were compared based on electrostatic force entrapment, chemical cross-linking, and cross-linking with the aid of carbon nanotubes. At 10 mM glucose-6-phosphate (G6P) as a substrate concentration, the maximum power density of 0.06 mW cm -2 and retaining 42% of power output after 11 days were obtained through the method of chemical cross-linking with carbon nanotubes, approximately 6-fold and 3.5-fold better than those of the electrostatic force-based method, respectively. When changed to maltodextrin (degree of polymerization = 19) as the substrate, the EFC achieved a maximum power density of 0.085 mW cm -2. With the advantages of stable, low cost, high energy density, non-inhibitor to enzymes, and environmental friendly, maltodextrin is suggested to be an ideal fuel to power enzymatic fuel cells.

The effect of ultraviolet treatment on enzymatic activity and total phenolic content of minimally processed potato slices.

PubMed

Teoh, Li Shing; Lasekan, Ola; Adzahan, Noranizan Mohd; Hashim, Norhashila

2016-07-01

In this work, potato slices were exposed to different doses of UV-C irradiation (i.e. 2.28, 6.84, 11.41, and 13.68 kJ m -2 ) with or without pretreatment [i.e. ascorbic acid and calcium chloride (AACCl) dip] and stored at 4 ± 1 °C. Changes in enzymatic activities of polyphenol oxidase (PPO), peroxidase (POD) and phenylalanine ammonia lyase (PAL), as well as total phenolic content (TPC) were investigated after 0, 3, 7 and 10 days of storage. Results showed that untreated and UV-C treated potato slices at 13.68 kJ m -2 dosage level showed significantly higher PPO, POD and PAL activities. Conversely, untreated potato slices showed the lowest TPC during storage period. Potato slices subjected to AACCl dip plus UV-C at 6.84 kJ m -2 produced lower PPO, POD and PAL activities, as well as maintained a high TPC during storage.

Proteomic analysis of tylosin-resistant Mycoplasma gallisepticum reveals enzymatic activities associated with resistance.

PubMed

Xia, Xi; Wu, Congming; Cui, Yaowen; Kang, Mengjiao; Li, Xiaowei; Ding, Shuangyang; Shen, Jianzhong

2015-11-20

Mycoplasma gallisepticum is a significant pathogenic bacterium that infects poultry, causing chronic respiratory disease and sinusitis in chickens and turkeys, respectively. M. gallisepticum infection poses a substantial economic threat to the poultry industry, and this threat is made worse by the emergence of antibiotic-resistant strains. The mechanisms of resistance are often difficult to determine; for example, little is known about antibiotic resistance of M. gallisepticum at the proteome level. In this study, we performed comparative proteomic analyses of an antibiotic (tylosin)-resistant M. gallisepticum mutant and a susceptible parent strain using a combination of two-dimensional differential gel electrophoresis and nano-liquid chromatography-quadrupole-time of flight mass spectrometry. Thirteen proteins were identified as differentially expressed in the resistant strain compared to the susceptible strain. Most of these proteins were related to catalytic activity, including catalysis that promotes the formylation of initiator tRNA and energy production. Elongation factors Tu and G were over-expressed in the resistant strains, and this could promote the binding of tRNA to ribosomes and catalyze ribosomal translocation, the coordinated movement of tRNA, and conformational changes in the ribosome. Taken together, our results indicate that M. gallisepticum develops resistance to tylosin by regulating associated enzymatic activities.

[Isolation of wood-decaying fungi and evaluation of their enzymatic activity (Quindío, Colombia)].

PubMed

Chaparro, Deisy Fernanda; Rosas, Diana Carolina; Varela, Amanda

2009-12-31

White rot fungi (Ascomycota and Basidiomycota) were collected on fallen trunks with different decay stages, in a subandean forest (La Montaña del Ocaso nature reserve), and it was evaluated their ligninolitic activity. They were cultured on malt extract agar. Then it was performed semiquantitative tests for laccase and cellobiose dehydrogenase (CDH) activity using ABTS and DCPIP as enzymatic inducers. Based on the results of these tests, the fungi with higher activities from trunks with different decay stages were selected: Cookeina sulcipes (for stage 1), a fungus from the family Corticiaceae (for stage 2), Xylaria polymorpha (for stage 3) and Earliella sp. (for stage 4). A fermentation was performed at 28 degrees C, during 11 days, in a rotatory shaker at 150 rpm. Biomass, glucose, proteins and enzyme activities measurements were performed daily. The fungi that were in the trunks with decay states from 1 to 3, showed higher laccase activity as the state of decay increased. A higher DCH activity was also associated with a higher. Also, there was a positive relationship between both enzymes' activities. Erliella was the fungus which presented the highest biomass production (1140,19 g/l), laccase activity (157 UL(-1)) and CDH activity (43,50 UL(-1)). This work is the first report of laccase and CDH activity for Cookeina sulcipes and Earliella sp. Moreover, it gives basis for the use of these native fungi in biotechnological applications and the acknowledgment of their function in the wood decay process in native forest.

Optimization of Process Parameters and Kinetic Model of Enzymatic Extraction of Polyphenols from Lonicerae Flos

PubMed Central

Kong, Fansheng; Yu, Shujuan; Bi, Yongguang; Huang, Xiaojun; Huang, Mengqian

2016-01-01

Objective: To optimize and verify the cellulase extraction of polyphenols from honeysuckle and provide a reference for enzymatic extracting polyphenols from honeysuckle. Materials and Methods: The uniform design was used According to Fick's first law and kinetic model, fitting analysis of the dynamic process of enzymatic extracting polyphenols was conducted. Results: The optimum enzymatic extraction parameters for polyphenols from honeysuckle are found to be 80% (v/v) of alcohol, 35:1 (mL/g) of liquid-solid ratio, 80°C of extraction temperature, 8.5 of pH, 6.0 mg of enzyme levels, and 130 min of extraction time. Under the optimal conditions, the extraction rate of polyphenols was 3.03%. The kinetic experiments indicated kinetic equation had a good linear relationship with t even under the conditions of different levels of enzyme and temperature, which means fitting curve tallies well with the experimental values. Conclusion: The results of quantification showed that the results provide a reference for enzymatic extracting polyphenols from honeysuckle. SUMMARY Lonicerae flos (Lonicera japonica Thunb.) is a material of traditional Chinese medicine and healthy drinks, of which active compounds mainly is polyphenols. At present, plant polyphenols are the hotspots centents of food, cosmetic and medicine, because it has strong bioactivity. Several traditional methods are available for the extraction of plant polyphenols including impregnation, solvent extraction, ultrasonic extraction, hot-water extraction, alkaline dilute alcohol or alkaline water extraction, microwave extraction and Supercritical CO2 extraction. But now, an increasing number of research on using cellulase to extract active ingredients from plants. Enzymatic method is widely used for enzyme have excellent properties of high reaction efficiency and specificity, moderate reaction conditions, shorter extraction time and easier to control, less damage to the active ingredient. At present, the enzymatic

Inhibition of enzymatic browning of chlorogenic acid by sulfur-containing compounds.

PubMed

Kuijpers, Tomas F M; Narváez-Cuenca, Carlos-Eduardo; Vincken, Jean-Paul; Verloop, Annewieke J W; van Berkel, Willem J H; Gruppen, Harry

2012-04-04

The antibrowning activity of sodium hydrogen sulfite (NaHSO(3)) was compared to that of other sulfur-containing compounds. Inhibition of enzymatic browning was investigated using a model browning system consisting of mushroom tyrosinase and chlorogenic acid (5-CQA). Development of brown color (spectral analysis), oxygen consumption, and reaction product formation (RP-UHPLC-PDA-MS) were monitored in time. It was found that the compounds showing antibrowning activity either prevented browning by forming colorless addition products with o-quinones of 5-CQA (NaHSO(3), cysteine, and glutathione) or inhibiting the enzymatic activity of tyrosinase (NaHSO(3) and dithiothreitol). NaHSO(3) was different from the other sulfur-containing compounds investigated, because it showed a dual inhibitory effect on browning. Initial browning was prevented by trapping the o-quinones formed in colorless addition products (sulfochlorogenic acid), while at the same time, tyrosinase activity was inhibited in a time-dependent way, as shown by pre-incubation experiments of tyrosinase with NaHSO(3). Furthermore, it was demonstrated that sulfochlorogenic and cysteinylchlorogenic acids were not inhibitors of mushroom tyrosinase.

Modulation of the Activity of Mycobacterium tuberculosis LipY by Its PE Domain

PubMed Central

Garrett, Christopher K.; Broadwell, Lindsey J.; Hayne, Cassandra K.; Neher, Saskia B.

2015-01-01

Mycobacterium tuberculosis harbors over 160 genes encoding PE/PPE proteins, several of which have roles in the pathogen’s virulence. A number of PE/PPE proteins are secreted via Type VII secretion systems known as the ESX secretion systems. One PE protein, LipY, has a triglyceride lipase domain in addition to its PE domain. LipY can regulate intracellular triglyceride levels and is also exported to the cell wall by one of the ESX family members, ESX-5. Upon export, LipY’s PE domain is removed by proteolytic cleavage. Studies using cells and crude extracts suggest that LipY’s PE domain not only directs its secretion by ESX-5, but also functions to inhibit its enzymatic activity. Here, we attempt to further elucidate the role of LipY’s PE domain in the regulation of its enzymatic activity. First, we established an improved purification method for several LipY variants using detergent micelles. We then used enzymatic assays to confirm that the PE domain down-regulates LipY activity. The PE domain must be attached to LipY in order to effectively inhibit it. Finally, we determined that full length LipY and the mature lipase lacking the PE domain (LipYΔPE) have similar melting temperatures. Based on our improved purification strategy and activity-based approach, we concluded that LipY’s PE domain down-regulates its enzymatic activity but does not impact the thermal stability of the enzyme. PMID:26270534

Enzymatic treatment to improve the quality of black tea extracts.

PubMed

Chandini, S K; Rao, L Jaganmohan; Gowthaman, M K; Haware, D J; Subramanian, R

2011-08-01

Enzymatic extraction was investigated to improve the quality of black tea extracts with pretreatment of pectinase and tannase independently, successively and simultaneously. Pectinase improved the extractable-solids-yield (ESY) up to 11.5%, without much of an improvement in polyphenols recovery, while tannase pre-treatment showed a significant improvement in polyphenols recovery (14.3%) along with an 11.1% improvement in ESY. Among the four treatments, tannase-alone treatment showed the maximum improvement in tea quality, with higher polyphenols-in-extracted solids. Treatments involving tannase resulted in the significant release of gallic acid, due to its hydrolytic activity, leading to greater solubility besides favourably improving TF/TR ratio. The results suggested that employing a single enzyme, tannase, for the pre-treatment of black tea is desirable. Enzymatic extraction may be preferred over enzymatic clarification as it not only displayed reduction in tea cream and turbidity but also improved the recovery of polyphenols and ESY in the extract, as well as maintaining a good balance of tea quality. Copyright © 2011 Elsevier Ltd. All rights reserved.

What’s New in Enzymatic Halogenations

PubMed Central

Fujimori, Danica Galoniæ; Walsh, Christopher T.

2007-01-01

Summary The halogenation of thousands of natural products occurs during biosynthesis and often confers important functional properties. While haloperoxidases had been the default paradigm for enzymatic incorporation of halogens, via X+ equivalents into organic scaffolds, a combination of microbial genome sequencing, enzymatic studies and structural biology have provided deep new insights into enzymatic transfer of halide equivalents in three oxidation states. These are: (1) the halide ions (X−) abundant in nature, (2) halogen atoms (X•), and (3) the X+ equivalents. The mechanism of halogen incorporation is tailored to the electronic demands of specific substrates and involves enzymes with distinct redox coenzyme requirements. PMID:17881282

Modification of chemical properties, Cu fractionation and enzymatic activities in an acid vineyard soil amended with winery wastes: A field study.

PubMed

Rodríguez-Salgado, Isabel; Pérez-Rodríguez, Paula; Gómez-Armesto, Antía; Díaz-Raviña, Montserrat; Nóvoa-Muñoz, Juan Carlos; Arias-Estévez, Manuel; Fernández-Calviño, David

2017-11-01

The effects of adding two winery wastes, perlite waste (PW) and bentonite waste (BW), to an acid vineyard soil were assessed using some chemical and biological soil properties in a field study that lasted 18 months. The addition of PW (up to 81 Mg ha -1 ) had neither significant nor permanent effects on soil characteristics such as the pH, organic matter content or nutrient concentrations, the amounts of copper or zinc, or the electrical conductivity. Moreover, no persistent negative effects were found on the enzymatic activities after PW application. In contrast, soil that was amended with up to 71 Mg BW ha -1 showed increases in its soil pH values, exchangeable potassium and water soluble potassium and phosphorus contents. In addition, it caused significant increases in the electrical conductivity and water-soluble Cu. In addition, the phosphomonoesterase enzymatic activity decreased significantly (up to 28%) in response to the amendment with 71 Mg BW ha -1 . These results showed that adding BW and PW to the soil may be a good agronomic practice for recycling these types of wastes. However, in the case of PW, its use as a soil amendment must be performed with caution to control its possible harmful effects. Copyright © 2017 Elsevier Ltd. All rights reserved.

Extraction and quantitation of coumarin from cinnamon and its effect on enzymatic browning in fresh apple juice: a bioinformatics approach to illuminate its antibrowning activity.

PubMed

Thada, Rajarajeshwari; Chockalingam, Shivashri; Dhandapani, Ramesh Kumar; Panchamoorthy, Rajasekar

2013-06-05

Enzymatic browning by polyphenoloxidase (PPO) affects food quality and taste in fruits and vegetables. Thus, the study was designed to reduce browning in apple juice by coumarin. The ethanolic extract of cinnamon was prepared and its coumarin content was quantitated by HPLC, using authentic coumarin (AC) as standard. The effect of cinnamon extract (CE) and AC on enzymatic browning, its time dependent effects, and the specific activity of PPO and peroxidase (POD) were studied in apple juice. The docking of coumarin with PPO and POD was also performed to elucidate its antibrowning mechanism. The CE (73%) and AC (82%) showed better reduction in browning, maintained its antibrowning effect at all time points, and significantly (p < 0.05) reduced the specific activity of PPO and POD when compared with controls. Coumarin showed strong interaction with binding pockets of PPO and POD, suggesting its potential use as inhibitor to enzyme mediated browning in apple juice.

Algal extracellular release in river-floodplain dissolved organic matter: response of extracellular enzymatic activity during a post-flood period

PubMed Central

Sieczko, Anna; Maschek, Maria; Peduzzi, Peter

2015-01-01

River-floodplain systems are susceptible to rapid hydrological events. Changing hydrological connectivity of the floodplain generates a broad range of conditions, from lentic to lotic. This creates a mixture of allochthonously and autochthonously derived dissolved organic matter (DOM). Autochthonous DOM, including photosynthetic extracellular release (PER), is an important source supporting bacterial secondary production (BSP). Nonetheless, no details are available regarding microbial extracellular enzymatic activity (EEA) as a response to PER under variable hydrological settings in river-floodplain systems. To investigate the relationship between bacterial and phytoplankton components, we therefore used EEA as a tool to track the microbial response to non-chromophoric, but reactive and ecologically important DOM. The study was conducted in three floodplain subsystems with distinct hydrological regimes (Danube Floodplain National Park, Austria). The focus was on the post-flood period. Enhanced %PER (up to 48% of primary production) in a hydrologically isolated subsystem was strongly correlated with β-glucosidase, which was related to BSP. This shows that—in disconnected floodplain backwaters with high terrestrial input—BSP can also be driven by autochthonous carbon sources (PER). In a semi-isolated section, in the presence of fresh labile material from primary producers, enhanced activity of phenol oxidase was observed. In frequently flooded river-floodplain systems, BSP was mainly driven by enzymatic degradation of particulate primary production. Our research demonstrates that EEA measurements are an excellent tool to describe the coupling between bacteria and phytoplankton, which cannot be deciphered when focusing solely on chromophoric DOM. PMID:25741326

Dual role of the carboxyl-terminal region of pig liver L-kynurenine 3-monooxygenase: mitochondrial-targeting signal and enzymatic activity.

PubMed

Hirai, Kumiko; Kuroyanagi, Hidehito; Tatebayashi, Yoshitaka; Hayashi, Yoshitaka; Hirabayashi-Takahashi, Kanako; Saito, Kuniaki; Haga, Seiich; Uemura, Tomihiko; Izumi, Susumu

2010-12-01

l-kynurenine 3-monooxygenase (KMO) is an NAD(P)H-dependent flavin monooxygenase that catalyses the hydroxylation of l-kynurenine to 3-hydroxykynurenine, and is localized as an oligomer in the mitochondrial outer membrane. In the human brain, KMO may play an important role in the formation of two neurotoxins, 3-hydroxykynurenine and quinolinic acid, both of which provoke severe neurodegenerative diseases. In mosquitos, it plays a role in the formation both of eye pigment and of an exflagellation-inducing factor (xanthurenic acid). Here, we present evidence that the C-terminal region of pig liver KMO plays a dual role. First, it is required for the enzymatic activity. Second, it functions as a mitochondrial targeting signal as seen in monoamine oxidase B (MAO B) or outer membrane cytochrome b(5). The first role was shown by the comparison of the enzymatic activity of two mutants (C-terminally FLAG-tagged KMO and carboxyl-terminal truncation form, KMOΔC50) with that of the wild-type enzyme expressed in COS-7 cells. The second role was demonstrated with fluorescence microscopy by the comparison of the intracellular localization of the wild-type, three carboxyl-terminal truncated forms (ΔC20, ΔC30 and ΔC50), C-terminally FLAG-tagged wild-type and a mutant KMO, where two arginine residues, Arg461-Arg462, were replaced with Ser residues.

Similar potential ATP-P production and enzymatic activities in the microplankton community off Concepción (Chile) under oxic and suboxic conditions

NASA Astrophysics Data System (ADS)

González, Rodrigo R.; Gutiérrez, Marcelo H.; Quiñones, Renato A.

2007-11-01

The effects of the oxygen minimum zone on the metabolism of the heterotrophic microplankton community (0.22-100 μm) in the Humboldt Current System, as well as the factors controlling its biomass production, remain unknown. Here we compare the effect of four sources of dissolved organic carbon (glucose, oxaloacetate, glycine, leucine) on microbial biomass production (such as ATP-P) and the potential enzymatic activities involved in catabolic pathways under oxic and suboxic conditions. Our results show significant differences ( p < 0.05) in the ATP-P production when induced by the different substrates that are used as dissolved organic carbon herein. The induction of ATP-P production is enhanced from glucose < oxaloacetate < glycine < leucine. Nevertheless, for individual substrates, no significant differences were found between incubation under oxic and suboxic conditions except in the case of leucine. For this amino acid, the induction of ATP-P synthesis was higher under suboxic than oxic conditions. The data sets of all the substrates used showed greater potential ATP-P production under suboxic than oxic conditions. The results of the potential enzymatic activities suggest that malate dehydrogenase has the highest signal of NADH oxidization activity in the microbial assemblage. Furthermore, for all experiments, the malate dehydrogenase activity data set had a significant relationship with ATP-P production. These findings suggest that the microbial community inhabiting the oxygen minimum zone has the same or greater potential growth than the community inhabiting more oxygenated strata of the water column and that malate dehydrogenase is the activity that best represents the metabolic potential of the community.

Mechanism of lignin inhibition of enzymatic biomass deconstruction

DOE PAGES

Vermaas, Josh V.; Petridis, Loukas; Qi, Xianghong; ...

2015-12-01

The conversion of plant biomass to ethanol via enzymatic cellulose hydrolysis offers a potentially sustainable route to biofuel production. However, the inhibition of enzymatic activity in pretreated biomass by lignin severely limits the efficiency of this process. By performing atomic-detail molecular dynamics simulation of a biomass model containing cellulose, lignin, and cellulases (TrCel7A), we elucidate detailed lignin inhibition mechanisms. We find that lignin binds preferentially both to the elements of cellulose to which the cellulases also preferentially bind (the hydrophobic faces) and also to the specific residues on the cellulose-binding module of the cellulase that are critical for cellulose bindingmore » of TrCel7A (Y466, Y492, and Y493). In conclusion, lignin thus binds exactly where for industrial purposes it is least desired, providing a simple explanation of why hydrolysis yields increase with lignin removal.« less

Changes on lipid peroxidation,enzymatic activities and gene expression in planarian (Dugesia japonica) following exposure to perfluorooctanoic acid.

PubMed

Yuan, Zuoqing; Miao, Zili; Gong, Xiaoning; Zhao, Baoying; Zhang, Yuanyuan; Ma, Hongdou; Zhang, Jianyong; Zhao, Bosheng

2017-11-01

We investigated perfluorooctanoic acid (PFOA)-induced stress response in planarians. We administered different concentrations of PFOA to planarians for up to 10 d. PFOA exposure resulted in significant concentration-dependent elevations in lipid peroxidation, glutathione S-transferase and caspase-3 protease activities, and a significant decline in glutathione peroxidase activities compared with control groups. Exposure to PFOA significantly up-regulated the heat shock proteins hsp70 and hsp90, and p53, and down-regulated hsp40 compared with controls. PFOA exposure also increased HSP70 protein levels, as demonstrated by western blot analysis. These alterations indicated that PFOA exposure induced a stress response and affected the regulation of oxidative stress, enzymatic activities and gene expression. These results suggest that these sensitive parameters, together with other biomarkers, could be used for evaluating toxicity, for ecological risk assessment of PFOA in freshwaters. Copyright © 2017 Elsevier Inc. All rights reserved.

Enzymatic reaction paths as determined by transition path sampling

NASA Astrophysics Data System (ADS)

Masterson, Jean Emily

Enzymes are biological catalysts capable of enhancing the rates of chemical reactions by many orders of magnitude as compared to solution chemistry. Since the catalytic power of enzymes routinely exceeds that of the best artificial catalysts available, there is much interest in understanding the complete nature of chemical barrier crossing in enzymatic reactions. Two specific questions pertaining to the source of enzymatic rate enhancements are investigated in this work. The first is the issue of how fast protein motions of an enzyme contribute to chemical barrier crossing. Our group has previously identified sub-picosecond protein motions, termed promoting vibrations (PVs), that dynamically modulate chemical transformation in several enzymes. In the case of human heart lactate dehydrogenase (hhLDH), prior studies have shown that a specific axis of residues undergoes a compressional fluctuation towards the active site, decreasing a hydride and a proton donor--acceptor distance on a sub-picosecond timescale to promote particle transfer. To more thoroughly understand the contribution of this dynamic motion to the enzymatic reaction coordinate of hhLDH, we conducted transition path sampling (TPS) using four versions of the enzymatic system: a wild type enzyme with natural isotopic abundance; a heavy enzyme where all the carbons, nitrogens, and non-exchangeable hydrogens were replaced with heavy isotopes; and two versions of the enzyme with mutations in the axis of PV residues. We generated four separate ensembles of reaction paths and analyzed each in terms of the reaction mechanism, time of barrier crossing, dynamics of the PV, and residues involved in the enzymatic reaction coordinate. We found that heavy isotopic substitution of hhLDH altered the sub-picosecond dynamics of the PV, changed the favored reaction mechanism, dramatically increased the time of barrier crossing, but did not have an effect on the specific residues involved in the PV. In the mutant systems

Substitution-inert trinuclear platinum complexes efficiently condense/aggregate nucleic acids and inhibit enzymatic activity**

PubMed Central

Malina, Jaroslav; Farrell, Nicholas P.; Brabec, Viktor

2015-01-01

The trinuclear platinum complexes ([{Pt(NH3)3}2-μ-{trans-Pt(NH3)2(NH2(CH2)6NH2)2}]6+, TriplatinNC‐A; [{trans-Pt(NH3)2(NH2(CH2)6NH3+)}2-μ-{trans-Pt(NH3)2(NH2(CH2)6NH2)2}]8+, TriplatinNC) belong to a class of biologically active agents that bind to DNA via nonbonding noncovalent (hydrogen bonding, electrostatic) interactions. Charge delocalization (6+ to 8+) in these linear trinuclear platinum complexes results in a high cellular uptake and promising cytotoxic activity in several carcinoma cell lines. We show in the present work with the aid of the methods of biophysical chemistry that in particular TriplatinNC condenses DNA with unprecedented potency which is much higher than that of conventional DNA condensing agents. In addition, in contrast to other DNA condensing agents, both platinum complexes induce aggregation of small transfer RNA molecules. We also demonstrate for the first time that TriplatinNC-A and TriplatinNC in particular completely inhibit DNA transcriptional activity at markedly lower concentration than naturally occurring spermine. Notably, the topoisomerase I-mediated relaxation of supercoiled DNA was inhibited by TriplatinNC-A and TriplatinNC at ~60-fold and ~250-fold lower concentration than that of spermine, respectively. We suggest that the general mechanisms of biological activity of TriplatinNC-A and TriplatinNC may be associated with their unique ability to condense/aggregate nucleic acids with consequent inhibitory effect on crucial enzymatic activities. PMID:25256921

Engineering of vault nanocapsules with enzymatic and fluorescent properties

PubMed Central

Kickhoefer, Valerie A.; Garcia, Yvette; Mikyas, Yeshi; Johansson, Erik; Zhou, Jing C.; Raval-Fernandes, Sujna; Minoofar, Payam; Zink, Jeffrey I.; Dunn, Bruce; Stewart, Phoebe L.; Rome, Leonard H.

2005-01-01

One of the central issues facing the emerging field of nanotechnology is cellular compatibility. Nanoparticles have been proposed for diagnostic and therapeutic applications, including drug delivery, gene therapy, biological sensors, and controlled catalysis. Viruses, liposomes, peptides, and synthetic and natural polymers have been engineered for these applications, yet significant limitations continue to prevent their use. Avoidance of the body's natural immune system, lack of targeting specificity, and the inability to control packaging and release are remaining obstacles. We have explored the use of a naturally occurring cellular nanoparticle known as the vault, which is named for its morphology with multiple arches reminiscent of cathedral ceilings. Vaults are 13-MDa ribonucleoprotein particles with an internal cavity large enough to sequester hundreds of proteins. Here, we report a strategy to target and sequester biologically active materials within the vault cavity. Attachment of a vault-targeting peptide to two proteins, luciferase and a variant of GFP, resulted in their sequestration within the vault cavity. The targeted proteins confer enzymatic and fluorescent properties on the recombinant vaults, both of which can be detected by their emission of light. The modified vaults are compatible with living cells. The ability to engineer vault particles with designed properties and functionalities represents an important step toward development of a biocompatible nanocapsule. PMID:15753293

Engineering of vault nanocapsules with enzymatic and fluorescent properties.

PubMed

Kickhoefer, Valerie A; Garcia, Yvette; Mikyas, Yeshi; Johansson, Erik; Zhou, Jing C; Raval-Fernandes, Sujna; Minoofar, Payam; Zink, Jeffrey I; Dunn, Bruce; Stewart, Phoebe L; Rome, Leonard H

2005-03-22

One of the central issues facing the emerging field of nanotechnology is cellular compatibility. Nanoparticles have been proposed for diagnostic and therapeutic applications, including drug delivery, gene therapy, biological sensors, and controlled catalysis. Viruses, liposomes, peptides, and synthetic and natural polymers have been engineered for these applications, yet significant limitations continue to prevent their use. Avoidance of the body's natural immune system, lack of targeting specificity, and the inability to control packaging and release are remaining obstacles. We have explored the use of a naturally occurring cellular nanoparticle known as the vault, which is named for its morphology with multiple arches reminiscent of cathedral ceilings. Vaults are 13-MDa ribonucleoprotein particles with an internal cavity large enough to sequester hundreds of proteins. Here, we report a strategy to target and sequester biologically active materials within the vault cavity. Attachment of a vault-targeting peptide to two proteins, luciferase and a variant of GFP, resulted in their sequestration within the vault cavity. The targeted proteins confer enzymatic and fluorescent properties on the recombinant vaults, both of which can be detected by their emission of light. The modified vaults are compatible with living cells. The ability to engineer vault particles with designed properties and functionalities represents an important step toward development of a biocompatible nanocapsule.

Influence of crop rotation, intermediate crops, and organic fertilizers on the soil enzymatic activity and humus content in organic farming systems

NASA Astrophysics Data System (ADS)

Marcinkeviciene, A.; Boguzas, V.; Balnyte, S.; Pupaliene, R.; Velicka, R.

2013-02-01

The influence of crop rotation systems with different portions of nitrogen-fixing crops, intermediate crops, and organic fertilizers on the enzymatic activity and humus content of soils in organic farming was studied. The highest activity of the urease and invertase enzymes was determined in the soil under the crop rotation with 43% nitrogen-fixing crops and with perennial grasses applied twice per rotation. The application of manure and the growing of intermediate crops for green fertilizers did not provide any significant increase in the content of humus. The activity of urease slightly correlated with the humus content ( r = 0.30 at the significance level of 0.05 and r = 0.39 at the significance level of 0.01).

Enzymatic approaches to rare sugar production.

PubMed

Zhang, Wenli; Zhang, Tao; Jiang, Bo; Mu, Wanmeng

Rare sugars have recently attracted much attention because of their potential applications in the food, nutraceutical, and pharmaceutical industries. A systematic strategy for enzymatic production of rare sugars, named Izumoring, was developed >10years ago. The strategy consists of aldose-ketose isomerization, ketose C-3 epimerization, and monosaccharide oxidation-reduction. Recent development of the Izumoring strategy is reviewed herein, especially the genetic approaches to the improvement of rare sugar-producing enzymes and the applications of target-oriented bioconversion. In addition, novel non-Izumoring enzymatic approaches are also summarized, including enzymatic condensation, phosphorylation-dephosphorylation cascade reaction, aldose epimerization, ulosonic acid decarboxylation, and biosynthesis of rare disaccharides. Copyright © 2017 Elsevier Inc. All rights reserved.

Altered enzymatic activity and allele frequency of OMI/HTRA2 in Alzheimer's disease

PubMed Central

Westerlund, Marie; Behbahani, Homira; Gellhaar, Sandra; Forsell, Charlotte; Belin, Andrea Carmine; Anvret, Anna; Zettergren, Anna; Nissbrandt, Hans; Lind, Charlotta; Sydow, Olof; Graff, Caroline; Olson, Lars; Ankarcrona, Maria; Galter, Dagmar

2011-01-01

The serine-protease OMI/HTRA2, required for several cellular processes, including mitochondrial function, autophagy, chaperone activity, and apoptosis, has been implicated in the pathogenesis of both Alzheimer's disease (AD) and Parkinson's disease (PD). Western blot quantification of OMI/HTRA2 in frontal cortex of patients with AD (n=10) and control subjects (n=10) in two separate materials indicated reduced processed (active, 35 kDa) OMI/HTRA2 levels, whereas unprocessed (50 kDa) enzyme levels were not significantly different between the groups. Interestingly, the specific protease activity of OMI/HTRA2 was found to be significantly increased in patients with AD (n=10) compared to matched control subjects (n=10) in frontal cortex in two separate materials. Comparison of OMI/HTRA2 mRNA levels in frontal cortex and hippocampus, two brain areas particularly affected by AD, indicated similar levels in patients with AD (n=10) and matched control subjects (n=10). In addition, we analyzed the occurrence of the OMI/HTRA2 variants A141S and G399S in Swedish case-control materials for AD and PD and found a weak association of A141S with AD, but not with PD. In conclusion, our genetic, histological, and biochemical findings give further support to an involvement of OMI/HTRA2 in the pathology of AD; however, further studies are needed to clarify the role of this gene in neurodegeneration.—Westerlund, M., Behbahani, H., Gellhaar, S., Forsell, C., Carmine Belin, A., Anvret, A., Zettergren, A., Nissbrandt, H., Lind, C., Sydow, O., Graff, C., Olson, L., Ankarcrona, M., Galter, D. Altered enzymatic activity and allele frequency of OMI/HTRA2 in Alzheimer's disease. PMID:21163861

Glycated cholecystokinin-8 has an enhanced satiating activity and is protected against enzymatic degradation.

PubMed

O'Harte, F P; Mooney, M H; Kelly, C M; Flatt, P R

1998-10-01

Monoglycated cholecystokinin octapeptide (CCK-8) (glucitol-Asp1 adduct) modified at the NH2-terminus was prepared under hyperglycemic conditions, purified by high-performance liquid chromatography, and characterized by mass spectrometry (Mr 1228.4 Da) and peptide sequencing. CCK-8 (100 nmol/kg, i.p.) significantly (P < 0.001) reduced voluntary food intake of fasted mice for up to 30 min after its administration, compared with saline-administered controls. Glycated CCK-8 reduced food intake at 30-120 min (P < 0.01 to P < 0.001) and significantly reduced feeding compared with CCK-8 from 60 to 120 min (P < 0.01). In vitro plasma degradation studies indicated that glycated CCK-8 was resistant to the normal rapid enzymatic conversion to CCK fragments. This study demonstrated that CCK-8 is a potent short-term inhibitor of food intake, and that structural modification of this peptide by amino-terminal glycation leads to enhanced satiating activity, partially due to increased resistance to serum aminopeptidase degradation.

Topical formulations with superoxide dismutase: influence of formulation composition on physical stability and enzymatic activity.

PubMed

Di Mambro, Valéria M; Borin, Maria F; Fonseca, Maria J V

2003-04-24

Three different topical formulations were supplemented with superoxide dismutase (SOD) and evaluated concerning physical and chemical stabilities in order to determine the most stable formulation that would maintain SOD activity. Physical stability was evaluated by storing the formulation at room temperature, and at 37 and 45 degrees C for 28 days. Samples were collected at 7-day intervals for assessment of rheological behavior. Chemical stability was evaluated by the measurement of enzymatic activity in formulations stored at room temperature and at 45 degrees C for 75 days. The formulations showed a pseudoplastic behavior, with a flow index of less than 1. There was no significant difference in the initial values of flow index, hysteresis loop or minimum apparent viscosity. The simple emulsion and the one stabilized with hydroxyethylcellulose showed decreased viscosity by the 21st day and with higher temperature, but no significant changes concerning the presence of SOD. Although there were no significant changes concerning storage time or temperature, the formulation stabilized with hydroxyethylcellulose showed a marked loss of SOD activity. The addition of SOD to the formulations studied did not affect their physical stability. Simple emulsions or emulsions stabilized with carboxypolymethylene seem to be better bases for enzyme addition than emulsion stabilized with hydroxyethylcellulose.

Enzymatic hydrolysis of biomimetic bacterial cellulose-hemicellulose composites.

PubMed

Penttilä, Paavo A; Imai, Tomoya; Hemming, Jarl; Willför, Stefan; Sugiyama, Junji

2018-06-15

The production of biofuels and other chemicals from lignocellulosic biomass is limited by the inefficiency of enzymatic hydrolysis. Here a biomimetic composite material consisting of bacterial cellulose and wood-based hemicelluloses was used to study the effects of hemicelluloses on the enzymatic hydrolysis with a commercial cellulase mixture. Bacterial cellulose synthesized in the presence of hemicelluloses, especially xylan, was found to be more susceptible to enzymatic hydrolysis than hemicellulose-free bacterial cellulose. The reason for the easier hydrolysis could be related to the nanoscale structure of the substrate, particularly the packing of cellulose microfibrils into ribbons or bundles. In addition, small-angle X-ray scattering was used to show that the average nanoscale morphology of bacterial cellulose remained unchanged during the enzymatic hydrolysis. The reported easier enzymatic hydrolysis of bacterial cellulose produced in the presence of wood-based xylan offers new insights to overcome biomass recalcitrance through genetic engineering. Copyright © 2018 Elsevier Ltd. All rights reserved.

Enzymatic Decontamination of Environmental Organophosphorus Compounds

DTIC Science & Technology

2006-12-04

ABSTRACT (Maximum 200 words) The abstract is below since many authors do not follow the 200 word limit 14. SUBJECT TERMS organophosphorus compounds ...5404 Enzymatic decontamination of environmental organophosphorus compounds REPORT DOCUMENTATION PAGE 18. SECURITY CLASSIFICATION ON THIS PAGE...239-18 298-102 15. NUMBER OF PAGES 20. LIMITATION OF ABSTRACT UL - 4-Dec-2006 Enzymatic decontamination of environmental organophosphorus compounds

Enzymatic production of biodiesel from microalgal oil using ethyl acetate as an acyl acceptor.

PubMed

Alavijeh, Razieh Shafiee; Tabandeh, Fatemeh; Tavakoli, Omid; Karkhane, Aliasghar; Shariati, Parvin

2015-01-01

Microalgae have become an important source of biomass for biodiesel production. In enzymatic transesterification reaction, the enzyme activity is decreased in presence of alcohols. The use of different acyl acceptors such as methyl/ethyl acetate is suggested as an alternative and effective way to overcome this problem. In this study, ethyl acetate was used for the first time in the enzymatic production of biodiesel by using microalga, Chlorella vulgaris, as a triglyceride source. Enzymatic conversion of such fatty acids to biodiesel was catalyzed by Novozym 435 as an efficient immobilized lipase which is extensively used in biodiesel production. The best conversion yield of 66.71% was obtained at the ethyl acetate to oil molar ratio of 13:1 and Novozym 435 concentration of 40%, based on the amount of oil, and a time period of 72 h at 40℃. The results showed that ethyl acetate have no adverse effect on lipase activity and the biodiesel amount was not decreased even after seven transesterification cycles, so ethyl acetate has a great potential to be substituted for short-chain alcohols in transesterification reaction.

Enzymatic extraction of star gooseberry (Phyllanthus acidus) juice with high antioxidant level

NASA Astrophysics Data System (ADS)

Loan, Do Thi Thanh; Tra, Tran Thi Thu; Nguyet, Ton Nu Minh; Man, Le Van Viet

2017-09-01

Ascorbic acid and phenolic compounds are main antioxidants in star gooseberry (Phyllanthus acidus) fruit. In this study, Pectinex Ultra SP-L preparation with pectinase activity was used in the extraction of star gooseberry juice. The effects of pectinase concentration and biocatalytic time on the content of ascorbic acid, phenolic compounds and antioxidant activity of the fruit juice were firstly investigated. Response surface methodology was then used to optimize the conditions of enzymatic extraction for maximizing the antioxidant activity of the star gooseberry juice. The optimal pectinase concentration and biocatalytic time were 19 polygalacturonase units per 100g pulp dry weight and 67 min, respectively under which the maximal antioxidant activity achieved 5595±6 µmol Trolox equivalent per 100g juice dry weight. On the basis of kinetic model of second-order extraction, the extraction rate constant of ascorbic acid and phenolic compounds in the enzymatic extraction increased approximately 21% and 157%, respectively in comparison with that in the conventional extraction. Application of pectinase preparation to the fruit juice extraction was therefore potential for improvement in antioxidant level of the product.

Enhanced enzymatic hydrolysis of kenaf core using irradiation and dilute acid

NASA Astrophysics Data System (ADS)

Lee, Byoung-Min; Jeun, Joon-Pyo; Kang, Phil-Hyun

2017-01-01

This study was performed to determine the effect of electron beam dose and enzymatic hydrolysis time for production of sugar such as glucose and xylose. After kenaf core was exposed to an irradiation dose that ranged from 0 to 500 kGy, the irradiated kenaf core was treated with a 3% (v/v) sulfuric acid solution using an autoclave for 5 h at 120 °C. The pretreated kenaf core was subsequently subjected to enzymatic hydrolysis at 50 °C in a shaking water bath at 150 rpm for 12, 24, 48, and 72 h. The determined enzyme activity rates were 70 FPU (Celluclast 1.5 L) and 40 CBU (Novozyme-188). The crystallinity index decreased from 50.6% in a non-pretreated kenaf core to 27.7% in kenaf core that was subjected to the two-stage pretreatment at dose of 500 kGy. The sugar yield of the two-stage pretreated kenaf core increased with an increase in irradiation dose. The sugar yield after 72 h of enzymatic hydrolysis was 73.6% at its highest with an irradiation dose of 500 kGy. The enhancement of enzymatic hydrolysis by two-stage pretreatment was more effective than non- and single pretreatment (36.9%, 40.6% and 44.0% in non-pretreatment, electron beam and dilute acid, respectively).

Oxygen isotope ratios of PO4: An inorganic indicator of enzymatic activity and P metabolism and a new biomarker in the search for life

PubMed Central

Blake, Ruth E.; Alt, Jeffrey C.; Martini, Anna M.

2001-01-01

The distinctive relations between biological activity and isotopic effect recorded in biomarkers (e.g., carbon and sulfur isotope ratios) have allowed scientists to suggest that life originated on this planet nearly 3.8 billion years ago. The existence of life on other planets may be similarly identified by geochemical biomarkers, including the oxygen isotope ratio of phosphate (δ18Op) presented here. At low near-surface temperatures, the exchange of oxygen isotopes between phosphate and water requires enzymatic catalysis. Because enzymes are indicative of cellular activity, the demonstration of enzyme-catalyzed PO4–H2O exchange is indicative of the presence of life. Results of laboratory experiments are presented that clearly show that δ18OP values of inorganic phosphate can be used to detect enzymatic activity and microbial metabolism of phosphate. Applications of δ18Op as a biomarker are presented for two Earth environments relevant to the search for extraterrestrial life: a shallow groundwater reservoir and a marine hydrothermal vent system. With the development of in situ analytical techniques and future planned sample return strategies, δ18Op may provide an important biosignature of the presence of life in extraterrestrial systems such as that on Mars. PMID:11226207

Oxygen isotope ratios of PO4: an inorganic indicator of enzymatic activity and P metabolism and a new biomarker in the search for life.

PubMed

Blake, R E; Alt, J C; Martini, A M

2001-02-27

The distinctive relations between biological activity and isotopic effect recorded in biomarkers (e.g., carbon and sulfur isotope ratios) have allowed scientists to suggest that life originated on this planet nearly 3.8 billion years ago. The existence of life on other planets may be similarly identified by geochemical biomarkers, including the oxygen isotope ratio of phosphate (delta(18)O(p)) presented here. At low near-surface temperatures, the exchange of oxygen isotopes between phosphate and water requires enzymatic catalysis. Because enzymes are indicative of cellular activity, the demonstration of enzyme-catalyzed PO(4)-H(2)O exchange is indicative of the presence of life. Results of laboratory experiments are presented that clearly show that delta(18)O(P) values of inorganic phosphate can be used to detect enzymatic activity and microbial metabolism of phosphate. Applications of delta(18)O(p) as a biomarker are presented for two Earth environments relevant to the search for extraterrestrial life: a shallow groundwater reservoir and a marine hydrothermal vent system. With the development of in situ analytical techniques and future planned sample return strategies, delta(18)O(p) may provide an important biosignature of the presence of life in extraterrestrial systems such as that on Mars.

Oxidant and enzymatic antioxidant status (gene expression and activity) in the brain of chickens with cold-induced pulmonary hypertension

NASA Astrophysics Data System (ADS)

Hassanpour, Hossein; Khalaji-Pirbalouty, Valiallah; Nasiri, Leila; Mohebbi, Abdonnaser; Bahadoran, Shahab

2015-11-01

To evaluate oxidant and antioxidant status of the brain (hindbrain, midbrain, and forebrain) in chickens with cold-induced pulmonary hypertension, the measurements of lipid peroxidation, protein oxidation, antioxidant capacity, enzymatic activity, and gene expression (for catalase, glutathione peroxidase, and superoxide dismutases) were done. There were high lipid peroxidation/protein oxidation and low antioxidant capacity in the hindbrain of cold-induced pulmonary hypertensive chickens compared to control ( P < 0.05). In the hypertensive chickens, superoxide dismutase activity was decreased (forebrain, midbrain, and hindbrain), while catalase activity was increased (forebrain and midbrain) ( P < 0.05). Glutathione peroxidase activity did not change. Relative gene expression of catalase and superoxide dismutases (1 and 2) was downregulated, while glutathione peroxidase was upregulated in the brain of the cold-induced pulmonary hypertensive chickens. Probably, these situations in the oxidant and antioxidant status of the brain especially hindbrain may change its function at cardiovascular center and sympathetic nervous system to exacerbate pulmonary hypertension.

Characterization of enzymatic micromachining for construction of variable cross-section microchannel topologies

PubMed Central

Ruggles, Molly E.; Jayaraman, Arul; Ugaz, Victor M.

2016-01-01

The ability to harness enzymatic activity as an etchant to precisely machine biodegradable substrates introduces new possibilities for microfabrication. This flow-based etching is straightforward to implement, enabling patterning of microchannels with topologies that incorporate variable depth along the cross-sectional dimension. Additionally, unlike conventional small-molecule formulations, the macromolecular nature of enzymatic etchants enables features to be precisely positioned. Here, we introduce a kinetic model to characterize the enzymatic machining process and its localization by co-injection of a macromolecular inhibitor species. Our model captures the interaction between enzyme, inhibitor, and substrate under laminar flow, enabling rational prediction of etched microchannel profiles so that cross-sectional topologies incorporating complex lateral variations in depth can be constructed. We also apply this approach to achieve simultaneous widening of an entire network of microchannels produced in the biodegradable polymeric substrate poly(lactic acid), laying a foundation to construct systems incorporating a broad range of internal cross-sectional dimensions by manipulating the process conditions. PMID:27190566

A Single Glycan at the 99-Loop of Human Kallikrein-related Peptidase 2 Regulates Activation and Enzymatic Activity*

PubMed Central

Guo, Shihui; Skala, Wolfgang; Magdolen, Viktor; Briza, Peter; Biniossek, Martin L.; Schilling, Oliver; Kellermann, Josef; Brandstetter, Hans; Goettig, Peter

2016-01-01

Human kallikrein-related peptidase 2 (KLK2) is a key serine protease in semen liquefaction and prostate cancer together with KLK3/prostate-specific antigen. In order to decipher the function of its potential N-glycosylation site, we produced pro-KLK2 in Leishmania tarentolae cells and compared it with its non-glycosylated counterpart from Escherichia coli expression. Mass spectrometry revealed that Asn-95 carries a core glycan, consisting of two GlcNAc and three hexoses. Autocatalytic activation was retarded in glyco-pro-KLK2, whereas the activated glyco-form exhibited an increased proteolytic resistance. The specificity patterns obtained by the PICS (proteomic identification of protease cleavage sites) method are similar for both KLK2 variants, with a major preference for P1-Arg. However, glycosylation changes the enzymatic activity of KLK2 in a drastically substrate-dependent manner. Although glyco-KLK2 has a considerably lower catalytic efficiency than glycan-free KLK2 toward peptidic substrates with P2-Phe, the situation was reverted toward protein substrates, such as glyco-pro-KLK2 itself. These findings can be rationalized by the glycan-carrying 99-loop that prefers to cover the active site like a lid. By contrast, the non-glycosylated 99-loop seems to favor a wide open conformation, which mostly increases the apparent affinity for the substrates (i.e. by a reduction of Km). Also, the cleavage pattern and kinetics in autolytic inactivation of both KLK2 variants can be explained by a shift of the target sites due to the presence of the glycan. These striking effects of glycosylation pave the way to a deeper understanding of kallikrein-related peptidase biology and pathology. PMID:26582203

JMJD6 and U2AF65 co-regulate alternative splicing in both JMJD6 enzymatic activity dependent and independent manner

PubMed Central

Yi, Jia; Shen, Hai-Feng; Qiu, Jin-Song; Huang, Ming-Feng; Zhang, Wen-Juan; Ding, Jian-Cheng; Zhu, Xiao-Yan; Zhou, Yu

2017-01-01

Abstract JMJD6, a jumonji C (Jmj C) domain-containing protein demethylase and hydroxylase, has been implicated in an array of biological processes. It has been shown that JMJD6 interacts with and hydroxylates multiple serine/arginine-rich (SR) proteins and SR related proteins, including U2AF65, all of which are known to function in alternative splicing regulation. However, whether JMJD6 is widely involved in alternative splicing and the molecular mechanism underlying JMJD6-regulated alternative splicing have remained incompletely understood. Here, by using RASL-Seq, we investigated the functional impact of RNA-dependent interaction between JMJD6 and U2AF65, revealing that JMJD6 and U2AF65 co-regulated a large number of alternative splicing events. We further demonstrated the JMJD6 function in alternative splicing in jmjd6 knockout mice. Mechanistically, we showed that the enzymatic activity of JMJD6 was required for a subset of JMJD6-regulated splicing, and JMJD6-mediated lysine hydroxylation of U2AF65 could account for, at least partially, their co-regulated alternative splicing events, suggesting both JMJD6 enzymatic activity-dependent and independent control of alternative splicing. These findings reveal an intimate link between JMJD6 and U2AF65 in alternative splicing regulation, which has important implications in development and disease processes. PMID:27899633

Two enzymatic reaction pathways in the formation of pyropheophorbide a.

PubMed

Suzuki, Yasuyo; Doi, Michio; Shioi, Yuzo

2002-01-01

The demethoxycarbonyl reaction of pheophorbide a in plants and algae was investigated. Two types of enzyme that catalyze alternative reactions in the formation of pyropheophorbide a were found. One enzyme, designated 'pheophorbidase (Phedase)', was purified nearly to homogeneity from cotyledons of radish (Raphanus sativus). This enzyme catalyzes the conversion of pheophorbide a to a precursor of pyropheophorbide a, C-13(2)-carboxylpyropheophorbide a, by demethylation, and then the precursor is decarboxylated non-enzymatically to yield pyropheophorbide a. The activity of Phedase was inhibited by the reaction product, methanol. The other enzyme, termed 'pheophorbide demethoxycarbonylase (PDC)', was highly purified from the Chl b-less mutant NL-105 of Chlamydomonas reinhardtii. This enzyme had produced no intermediate as shown in the Phedase reaction, indicating that it converts pheophorbide a directly into pyropheophorbide a, probably by nucleophilic reaction. Phedase and PDC consisted of both senescence-induced and constitutive enzymes. The molecular weight of both Phedases was 113 000 and of senescence-induced PDC was 170 000. The K (m) values against pheophorbide a for both Phedases were 14-15 muM and 283 muM for senescence-induced PDC. The activity of both Phedases was inhibited by the reaction product, methanol, whereas methanol had no specific effect on senescence-induced PDC. Phenylmethylsulfonic fluoride and N-ethylmaleimide inhibited the senescence-induced Phedase and PDC, respectively. Among the 23 species from 15 different families tested, Phedase activity was found in 10 species from three families. PDC activity was not detected in plants lacking Phedase activity, except for Chlamydomonas. Based on these findings, a likely conclusion is that at least two alternative pathways that are catalyzed by two different enzymes, Phedase and PDC, exist for the formation of pyropheophorbide a.

Conformational Changes in a Hyperthermostable Glycoside Hydrolase: Enzymatic Activity Is a Consequence of the Loop Dynamics and Protonation Balance

PubMed Central

de Oliveira, Leandro C.; da Silva, Viviam M.; Colussi, Francieli; Cabral, Aline D.; de Oliveira Neto, Mario; Squina, Fabio M.; Garcia, Wanius

2015-01-01

Endo-β-1, 4-mannanase from Thermotoga petrophila (TpMan) is a modular hyperthermostable enzyme involved in the degradation of mannan-containing polysaccharides. The degradation of these polysaccharides represents a key step for several industrial applications. Here, as part of a continuing investigation of TpMan, the region corresponding to the GH5 domain (TpManGH5) was characterized as a function of pH and temperature. The results indicated that the enzymatic activity of the TpManGH5 is pH-dependent, with its optimum activity occurring at pH 6. At pH 8, the studies demonstrated that TpManGH5 is a molecule with a nearly spherical tightly packed core displaying negligible flexibility in solution, and with size and shape very similar to crystal structure. However, TpManGH5 experiences an increase in radius of gyration in acidic conditions suggesting expansion of the molecule. Furthermore, at acidic pH values, TpManGH5 showed a less globular shape, probably due to a loop region slightly more expanded and flexible in solution (residues Y88 to A105). In addition, molecular dynamics simulations indicated that conformational changes caused by pH variation did not change the core of the TpManGH5, which means that only the above mentioned loop region presents high degree of fluctuations. The results also suggested that conformational changes of the loop region may facilitate polysaccharide and enzyme interaction. Finally, at pH 6 the results indicated that TpManGH5 is slightly more flexible at 65°C when compared to the same enzyme at 20°C. The biophysical characterization presented here is well correlated with the enzymatic activity and provide new insight into the structural basis for the temperature and pH-dependent activity of the TpManGH5. Also, the data suggest a loop region that provides a starting point for a rational design of biotechnological desired features. PMID:25723179

Label-free electrochemical detection of botulinum neurotoxin type E based on its enzymatic activity using interdigitated electrodes

NASA Astrophysics Data System (ADS)

Hyun, Sang Hwa; Park, Dae Keun; Kang, Aeyeon; Kim, Soohyun; Kim, Daehee; Shin, Yu Mi; Song, Ji-Joon; Yun, Wan Soo

2016-02-01

We report a simple label-free electrochemical method of detecting low concentrations of botulinum neurotoxin type E light chain (BoNT/E LC) based on its peptide cleavage activity. Dual-mode cyclic voltammetry was employed to observe changes in the redox signal of ferri-/ferro-cyanide on interdigitated microelectrodes, whose surfaces were covered by peptides designed from synaptosomal-associated protein 25 to be cleaved by BoNT/E LC. With the introduction of BoNT/E LC, the redox signal showed a time-dependent increase due to cleavage of the immobilized peptide molecules. In addition to the increased redox signal intensity, its time-dependence can be considered as a strong evidence of BoNT/E sensing, since the time-dependent increase can only result from the enzymatic activity of BoNT/E LC. Using this method, BoNT/E LC, at concentrations as low as 5 pg/ml, was readily measurable with only an hour of incubation.

Bioelectrocatalytic NAD+/NADH inter-conversion: transformation of an enzymatic fuel cell into an enzymatic redox flow battery.

PubMed

Quah, Timothy; Milton, Ross D; Abdellaoui, Sofiene; Minteer, Shelley D

2017-07-25

Diaphorase and a benzylpropylviologen redox polymer were combined to create a bioelectrode that can both oxidize NADH and reduce NAD + . We demonstrate how bioelectrocatalytic NAD + /NADH inter-conversion can transform a glucose/O 2 enzymatic fuel cell (EFC) with an open circuit potential (OCP) of 1.1 V into an enzymatic redox flow battery (ERFB), which can be rapidly recharged by operation as an EFC.

Extracellular enzymatic activity of two hydrolases in wastewater treatment for biological nutrient removal.

PubMed

Berrio-Restrepo, Jorge Mario; Saldarriaga, Julio César; Correa, Mauricio Andrés; Aguirre, Néstor Jaime

2017-10-01

Due to the complex nature of the wastewater (both domestic and non-domestic) composition, biological processes are widely used to remove nutrients, such as carbon (C), nitrogen (N), and phosphorous (P), which cause instability and hence contribute to the damage of water bodies. Systems with different configurations have been developed (including anaerobic, anoxic, and aerobic conditions) for the joint removal of carbon, nitrogen, and phosphorus. The goal of this research is to evaluate the extracellular activity of β-glucosidase and phosphatase enzymes in a University of Cape Town (UCT) system fed with two synthetic wastewaters of different molecular complexity. Both types of waters have medium strength characteristics similar to those of domestic wastewater with a mean C/N/P ratio of 100:13:1. The operation parameters were hydraulic retention time (HRT) of 10 h, solid retention time (SRT) of 12 days, mean concentration of the influent in terms of chemical oxygen demand (COD), total Kjeldahl nitrogen (TKN), and total phosphorus (TP) of 600, 80, and 6 mg/L, respectively. According to the results obtained, statistically significant differences have been found in the extracellular enzyme activities with the evaluated wastewaters and in the units comprising the treatment system in some of the cases. An analysis of principal components showed that the extracellular enzymatic activity has been correlated to nutrient concentration in wastewater, biomass concentration in the system, and metabolic conditions of treatment phases. Additionally, this research has allowed determining an inverse relationship between wastewater biodegradability and the extracellular enzyme activity of β-glucosidase and phosphatase. These results highlight the importance of including the analysis of biomass biochemical characteristics as control methods in wastewater treatment systems for the nutrient removal.

PSA-alpha-2-macroglobulin complex is enzymatically active in the serum of patients with advanced prostate cancer and can degrade circulating peptide hormones.

PubMed

Kostova, Maya B; Brennen, William Nathaniel; Lopez, David; Anthony, Lizamma; Wang, Hao; Platz, Elizabeth; Denmeade, Samuel R

2018-08-01

Prostate cancer cells produce high levels of the serine protease Prostate-Specific Antigen (PSA). PSA is enzymatically active in the tumor microenvironment but is presumed to be enzymatically inactive in the blood due to complex formation with serum protease inhibitors α-1-antichymotrypsin and α-2-macroglobulin (A2M). PSA-A2M complexes cannot be measured by standard ELISA assays and are also rapidly cleared from the circulation. Thus the exact magnitude of PSA production by prostate cancer cells is not easily measured. The PSA complexed to A2M is unable to cleave proteins but maintains the ability to cleave small peptide substrates. Thus, in advanced prostate cancer, sufficient PSA-A2M may be in circulation to effect total A2M levels, levels of cytokines bound to A2M and hydrolyze small circulating peptide hormones. Total A2M levels in men with advanced prostate cancer and PSA levels above 1000 ng/mL were measured by ELISA and compared to controls. Additional ELISA assays were used to measure levels of IL-6 and TGF-beta which can bind to A2M. The ability of PSA-A2M complexes to hydrolyze protein and peptide substrates was analyzed ± PSA inhibitor. Enzymatic activity of PSA-A2M in serum of men with high PSA levels was also assayed. Serum A2M levels are inversely correlated with PSA levels in men with advanced prostate cancer. Il-6 Levels are significantly elevated in men with PSA >1000 ng/mL compared to controls with PSA <0.1 ng/mL. PSA-A2M complex in serum of men with PSA levels >1000 ng/mL can hydrolyze small fluorescently labeled peptide substrates but not large proteins that are PSA substrates. PSA can hydrolyze small peptide hormones like PTHrP and osteocalcin. PSA complexed to A2M retains the ability to degrade PTHrP. In advanced prostate cancer with PSA levels >1000 ng/mL, sufficient PSA-A2M is present in circulation to produce enzymatic activity against circulating small peptide hormones. Sufficient PSA is produced in advanced prostate

Lack of facilities rather than sociocultural factors as the primary barrier to physical activity among female Saudi university students.

PubMed

Samara, Anastasia; Nistrup, Anne; Al-Rammah, Tamader Y; Aro, Arja R

2015-01-01

The Kingdom of Saudi Arabia is experiencing a dramatic increase in physical inactivity, with women having higher levels of inactivity than men among all age groups. It is assumed that factors such as dress codes, restrictions on going outdoors, and conservative norms are the main reasons for women's low physical activity. Our aim was to explore the different parameters related to physical activity, including self-efficacy, as well as the perceived barriers to and benefits of physical activity in young Saudi females. Ninety-four first-year female Saudi university students in Riyadh, Kingdom of Saudi Arabia, participated in the present study in 2014. The students were from eight bachelor's programs in health and well-being, and each completed a questionnaire with questions divided into five parts as follows: 1) socioeconomic status, 2) physical activity, 3) self-efficacy 4) social factors, and 5) barriers and facilitators related to physical activity. The students exercised at home and alone, and there was low self-efficacy for physical activity (mean score =42±14). Among social factors, attending university was the only factor that hindered physical activity (32%). Physical activity was positively perceived overall (mean score =131±10). Students showed awareness of the benefits of physical activity for health and well-being. The most important barrier was the lack of designated areas available for physical activity. Students disagreed that family or the Islamic community were barriers to physical activity. The lack of facilities and lack of encouragement from the university, but not a lack of knowledge (a high level of knowledge is to be expected given their health and well-being studies backgrounds) and/or restrictions from families and society, seem to hinder female students' physical activity, at least young Saudi students.

Single-Molecule Probing the Energy Landscape of Enzymatic Reaction and Non-Covalent Interactions

NASA Astrophysics Data System (ADS)

Lu, H. Peter; Hu, Dehong; Chen, Yu; Vorpagel, Erich R.

2002-03-01

We have applied single-molecule spectroscopy under physiological conditions to study the mechanisms and dynamics of T4 lysozyme enzymatic reactions, characterizing mode-specific protein conformational dynamics. Enzymatic reaction turnovers and the associated structure changes of individual protein molecules were observed simultaneously in real-time. The overall reaction rates were found to vary widely from molecule-to-molecule, and the initial non-specific binding of the enzyme to the substrate was seen to dominate this inhomogeneity. The reaction steps subsequent to the initial binding were found to have homogeneous rates. Molecular dynamics simulation has been applied to elucidate the mechanism and intermediate states of the single-molecule enzymatic reaction. Combining the analysis of single-molecule experimental trajectories, MD simulation trajectories, and statistical modeling, we have revealed the nature of multiple intermediate states involved in the active enzyme-substrate complex formation and the associated conformational change mechanism and dynamics.

Rational design of functional and tunable oscillating enzymatic networks

NASA Astrophysics Data System (ADS)

Semenov, Sergey N.; Wong, Albert S. Y.; van der Made, R. Martijn; Postma, Sjoerd G. J.; Groen, Joost; van Roekel, Hendrik W. H.; de Greef, Tom F. A.; Huck, Wilhelm T. S.

2015-02-01

Life is sustained by complex systems operating far from equilibrium and consisting of a multitude of enzymatic reaction networks. The operating principles of biology's regulatory networks are known, but the in vitro assembly of out-of-equilibrium enzymatic reaction networks has proved challenging, limiting the development of synthetic systems showing autonomous behaviour. Here, we present a strategy for the rational design of programmable functional reaction networks that exhibit dynamic behaviour. We demonstrate that a network built around autoactivation and delayed negative feedback of the enzyme trypsin is capable of producing sustained oscillating concentrations of active trypsin for over 65 h. Other functions, such as amplification, analog-to-digital conversion and periodic control over equilibrium systems, are obtained by linking multiple network modules in microfluidic flow reactors. The methodology developed here provides a general framework to construct dissipative, tunable and robust (bio)chemical reaction networks.

Methods for improving enzymatic trans-glycosylation for synthesis of human milk oligosaccharide biomimetics.

PubMed

Zeuner, Birgitte; Jers, Carsten; Mikkelsen, Jørn Dalgaard; Meyer, Anne S

2014-10-08

Recently, significant progress has been made within enzymatic synthesis of biomimetic, functional glycans, including, for example, human milk oligosaccharides. These compounds are mainly composed of N-acetylglucosamine, fucose, sialic acid, galactose, and glucose, and their controlled enzymatic synthesis is a novel field of research in advanced food ingredient chemistry, involving the use of rare enzymes, which have until now mainly been studied for their biochemical significance, not for targeted biosynthesis applications. For the enzymatic synthesis of biofunctional glycans reaction parameter optimization to promote "reverse" catalysis with glycosidases is currently preferred over the use of glycosyl transferases. Numerous methods exist for minimizing the undesirable glycosidase-catalyzed hydrolysis and for improving the trans-glycosylation yields. This review provides an overview of the approaches and data available concerning optimization of enzymatic trans-glycosylation for novel synthesis of complex bioactive carbohydrates using sialidases, α-l-fucosidases, and β-galactosidases as examples. The use of an adequately high acceptor/donor ratio, reaction time control, continuous product removal, enzyme recycling, and/or the use of cosolvents may significantly improve trans-glycosylation and biocatalytic productivity of the enzymatic reactions. Protein engineering is also a promising technique for obtaining high trans-glycosylation yields, and proof-of-concept for reversing sialidase activity to trans-sialidase action has been established. However, the protein engineering route currently requires significant research efforts in each case because the structure-function relationship of the enzymes is presently poorly understood.

A comparison of the distribution of enzymatically and non-enzymatically produced lead phosphate in insect flight muscle.

PubMed

Tice, L W

1969-01-01

Lead phosphate precipitates were produced in indirect flight muscles of Phormia regina by sequential incubation in solutions containing lead and inorganic phosphate and their distribution was compared with those produced by ATP hydrolysis in the presence of lead. Enzymatically produced precipitates were associated almost exclusively with thick filaments. Non-enzymatically produced precipitates were associated with thick filaments but were also found associated with thin filaments in significant numbers.

Enzymatic DNA molecules

NASA Technical Reports Server (NTRS)

Joyce, Gerald F. (Inventor); Breaker, Ronald R. (Inventor)

1998-01-01

The present invention discloses deoxyribonucleic acid enzymes--catalytic or enzymatic DNA molecules--capable of cleaving nucleic acid sequences or molecules, particularly RNA, in a site-specific manner, as well as compositions including same. Methods of making and using the disclosed enzymes and compositions are also disclosed.

Nature and position of functional group on thiopurine substrates influence activity of xanthine oxidase--enzymatic reaction pathways of 6-mercaptopurine and 2-mercaptopurine are different.

PubMed

Tamta, Hemlata; Kalra, Sukirti; Thilagavathi, Ramasamy; Chakraborti, Asit K; Mukhopadhyay, Anup K

2007-02-01

Xanthine oxidase-catalyzed hydroxylation reactions of the anticancer drug 6-mercaptopurine (6-MP) and its analog 2-mercaptopurine (2-MP) as well as 6-thioxanthine (6-TX) and 2-thioxanthine (2-TX) have been studied using UV-spectroscopy, high pressure liquid chromatography, photodiode array, and liquid chromatography-based mass spectral analysis. It is shown that 6-MP and 2-MP are oxidatively hydroxylated through different pathways. Enzymatic hydroxylation of 6-MP forms 6-thiouric acid in two steps involving 6-TX as the intermediate, whereas 2-MP is converted to 8-hydroxy-2-mercaptopurine as the expected end product in one step. Surprisingly, in contrast to the other thiopurines, enzymatic hydroxylation of 2-MP showed a unique hyperchromic effect at 264 nm as the reaction proceeded. However, when 2-TX is used as the substrate, it is hydroxylated to 2-thiouric acid. The enzymatic hydroxylation of 2-MP is considerably faster than that of 6-MP, while 6-TX and 2-TX show similar rates under identical reaction conditions. The reason why 2-MP is a better substrate than 6-MP and how the chemical nature and position of the functional groups present on the thiopurine substrates influence xanthine oxidase activity are discussed.

Structure, enzymatic transformation, anticancer activity of fucoidan and sulphated fucooligosaccharides from Sargassum horneri.

PubMed

Silchenko, Artem S; Rasin, Anton B; Kusaykin, Mikhail I; Kalinovsky, Anatoly I; Miansong, Zhang; Changheng, Liu; Malyarenko, Olesya; Zueva, Anastasiya O; Zvyagintseva, Tatyana N; Ermakova, Svetlana P

2017-11-01

Structure and anticancer activity of fucoidan from Sargassum horneri and from products of its enzymatic transformation were investigated. A gene that encodes fucoidanase ffa1 in the marine bacteria F. algae was identified, cloned and the protein (FFA1) was produced in Escherichia coli. The mass of the gene product FFA1 is 111kDa. Sequence analysis has revealed that fucoidanase FFA1 belongs to the GH107 (CAZy) family. Recombinant fucoidanase FFA1 was used to produce fucooligosaccharides. Structure of 5 sulphated oligosaccharides with polymerization degree 4-10 was established by NMR-spectroscopy. The fucoidan extracted from S. horneri is almost pure fucan. The main chain of the fucoidan is established to consist mostly of the repeating →3-α-l-Fucp(2SO 3 - )-1→4-α-l-Fucp(2,3SO 3 - )-1→ fragment, with insertions of →3-α-l-Fucp(2,4SO 3 - )-1→ fragment. Unsulphated side chains with the α-l-Fucp-1→2-α-l-Fucp-1→ structure connect to the main one at the C4 of monosaccharide residue. Copyright © 2017 Elsevier Ltd. All rights reserved.

Particle size distribution of rice flour affecting the starch enzymatic hydrolysis and hydration properties.

PubMed

de la Hera, Esther; Gomez, Manuel; Rosell, Cristina M

2013-10-15

Rice flour is becoming very attractive as raw material, but there is lack of information about the influence of particle size on its functional properties and starch digestibility. This study evaluates the degree of dependence of the rice flour functional properties, mainly derived from starch behavior, with the particle size distribution. Hydration properties of flours and gels and starch enzymatic hydrolysis of individual fractions were assessed. Particle size heterogeneity on rice flour significantly affected functional properties and starch features, at room temperature and also after gelatinization; and the extent of that effect was grain type dependent. Particle size heterogeneity on rice flour induces different pattern in starch enzymatic hydrolysis, with the long grain having slower hydrolysis as indicated the rate constant (k). No correlation between starch digestibility and hydration properties or the protein content was observed. It seems that in intact granules interactions with other grain components must be taken into account. Overall, particle size fractionation of rice flour might be advisable for selecting specific physico-chemical properties. Copyright © 2013. Published by Elsevier Ltd.

Volatile Compound, Physicochemical, and Antioxidant Properties of Beany Flavor-Removed Soy Protein Isolate Hydrolyzates Obtained from Combined High Temperature Pre-Treatment and Enzymatic Hydrolysis.

PubMed

Yoo, Sang-Hun; Chang, Yoon Hyuk

2016-12-01

The present study investigated the volatile compound, physicochemical, and antioxidant properties of beany flavor-removed soy protein isolate (SPI) hydrolyzates produced by combined high temperature pre-treatment and enzymatic hydrolysis. Without remarkable changes in amino acid composition, reductions of residual lipoxygenase activity and beany flavor-causing volatile compounds such as hexanol, hexanal, and pentanol in SPI were observed after combined heating and enzymatic treatments. The degree of hydrolysis, emulsion capacity and stability, 2,2-diphenyl-1-picrylhydrazyl radical scavenging activity, and superoxide radical scavenging activity of SPI were significantly increased, but the magnitudes of apparent viscosity, consistency index, and dynamic moduli (G', G″) of SPI were significantly decreased after the combined heating and enzymatic treatments. Based on these results, it was suggested that the enzymatic hydrolysis in combination with high temperature pre-treatment may allow for the production of beany flavor-removed SPI hydrolyzates with superior emulsifying and antioxidant functionalities.

Comparison of enzymatic activities in different Candida species isolated from women with vulvovaginitis.

PubMed

Fatahinia, M; Halvaeezadeh, M; Rezaei-Matehkolaei, A

2017-06-01

Comparing the activities of secreted enzymes in different fungal species can improve our understanding of their pathogenic role. Secretion of various enzymes by Candida species has been considered for determination of their virulence in different Candida infections including vulvovaginitis. The aim of this study was to determine and compare the activity of secreted enzymes in Candidia strains isolated from women suspected to vulvovaginal candidiasis (VVC) and referred to some health centers in Khuzestan, Southwestern Iran. The vaginal secretion samples were taken by swap from 250 suspected women with symptoms of vulvovaginal candidiasis and cultured on CHROMagar Candida medium. Identification of the isolated Candida from culture positive samples performed by the color of colonies and some standard mycological procedures. Activities of phospholipase, hemolysin-α, hemolysin-β, esterase and proteinase were measured in vitro by standard laboratory protocols. The enzymatic activity index (EAI) was calculated for each enzyme in accordance to relevant protocols. Totally in eighty cases (32%), a Candida strain was isolated which found to be as 52 (65%) Candida albicans; 12 (15%) C. glabrata; 10 (12.5%) C. dubliniensis; 4 (5%) C. krusei, C. tropicalis and C. parapsilosis species (each=1; 1.3%). Among C. albicans strains, 89.1% produced all studied enzymes, while 86% of C. glabrata strains failed to produce proteinase and phospholipase. The EAIs in decreasing order were as hemolysin-β=0.2895, hemolysin-α=0.5420, esterase=0.5753, proteinase=0.7413, and phospholipase=0.7446, respectively. Activity of phospholipase, esterase and proteinase secreted by C. albicans and C. dubliniensis were significantly more than those released by C. glabrata and C. krusei, while 86% of C. glabrata strains did not show esterase activity. On the other hand, the activity rates of hemolysin α and β among all studied isolates were almost similar. In the present study, the prevalence

JMJD6 and U2AF65 co-regulate alternative splicing in both JMJD6 enzymatic activity dependent and independent manner.

PubMed

Yi, Jia; Shen, Hai-Feng; Qiu, Jin-Song; Huang, Ming-Feng; Zhang, Wen-Juan; Ding, Jian-Cheng; Zhu, Xiao-Yan; Zhou, Yu; Fu, Xiang-Dong; Liu, Wen

2017-04-07

JMJD6, a jumonji C (Jmj C) domain-containing protein demethylase and hydroxylase, has been implicated in an array of biological processes. It has been shown that JMJD6 interacts with and hydroxylates multiple serine/arginine-rich (SR) proteins and SR related proteins, including U2AF65, all of which are known to function in alternative splicing regulation. However, whether JMJD6 is widely involved in alternative splicing and the molecular mechanism underlying JMJD6-regulated alternative splicing have remained incompletely understood. Here, by using RASL-Seq, we investigated the functional impact of RNA-dependent interaction between JMJD6 and U2AF65, revealing that JMJD6 and U2AF65 co-regulated a large number of alternative splicing events. We further demonstrated the JMJD6 function in alternative splicing in jmjd6 knockout mice. Mechanistically, we showed that the enzymatic activity of JMJD6 was required for a subset of JMJD6-regulated splicing, and JMJD6-mediated lysine hydroxylation of U2AF65 could account for, at least partially, their co-regulated alternative splicing events, suggesting both JMJD6 enzymatic activity-dependent and independent control of alternative splicing. These findings reveal an intimate link between JMJD6 and U2AF65 in alternative splicing regulation, which has important implications in development and disease processes. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

Enzymatic modification of egg lecithin to improve properties.

PubMed

Asomaning, Justice; Curtis, Jonathan M

2017-04-01

This research studied the enzymatic modification of egg yolk phospholipids and its effect on physicochemical properties. Egg yolk lipids were extracted with food grade ethanol and egg phospholipids (ePL) produced by deoiling with acetone. Vegetable oils were used to interesterify ePL utilizing Lipozyme®: sn-1,3 specific lipase. The enzymatic interesterification resulted in a single phase liquid product, whereas simple blending of the ePL and vegetable oil resulted in a product with two phases. In addition solid fat content decreased by 50% at -10°C and 94% at 35°C when compared with egg yolk lipids extract. A decrease in melting temperature resulted from the interesterification process. Interesterification improved emulsion stability index when used as an emulsifier in oil-in-water emulsion and compared to the native and soy lecithin. Enzyme reusability test showed retention of 63% activity after 10 cycles. Overall, the properties of native egg phospholipids were significantly enhanced in a potentially useful manner through interesterification. Copyright © 2016 Elsevier Ltd. All rights reserved.

Enzymatic and chemical synthesis of new anticoagulant peptides.

PubMed

Origone, Anabella; Bersi, Grisel; Illanes, Andrés; Sturniolo, Héctor; Liggieri, Constanza; Guzmán, Fanny; Barberis, Sonia

2018-06-08

In this study we report the enzymatic synthesis of N-α-[Carbobenzyloxy]-Tyr-Gln-Gln (Z-YQQ), a new anticoagulant tripeptide. It was obtained using phytoproteases from the stems and petioles of Asclepias curassavica L. as catalyst in an aqueous-organic biphasic system formed by 50% (v/v) ethyl acetate and 0.1 M Tris - HCl buffer pH 8. The resulting peptide was compared with the analogous peptide Tyr-Gln-Gln (YQQ) produced by solid-phase chemical synthesis. The in vitro anticoagulant activity of the above mentioned peptides was determined using Wiener Lab Test (Wiener, Argentina). The toxicological activity of the peptides was also determined. The enzymatically synthesized Z-YQQ peptide acted on the extrinsic pathway of the coagulation cascade, delaying the conversion time of prothrombin to thrombin and fibrinogen to fibrin by 136% and 50%, respectively, with respect to the controls. The chemically synthesized YQQ peptide acted specifically on the intrinsic pathway of the coagulation cascade, affecting factors VIII, IX, XI and XII from such cascade, and increasing the coagulation time by 105% with respect to the control. The results suggest that two new anticoagulant peptides (Z-YQQ and YQQ) can be useful for safe pharmaceutical applications. Nevertheless, some aspects related to peptide production should be optimized. This article is protected by copyright. All rights reserved. © 2018 American Institute of Chemical Engineers.

Effect of alkali lignins with different molecular weights from alkali pretreated rice straw hydrolyzate on enzymatic hydrolysis.

PubMed

Li, Yun; Qi, Benkun; Luo, Jianquan; Wan, Yinhua

2016-01-01

This study investigated the effect of alkali lignins with different molecular weights on enzymatic hydrolysis of lignocellulose. Different alkali lignins fractions, which were obtained from cascade ultrafiltration, were added into the dilute acid pretreated (DAP) and alkali pretreated (AP) rice straws respectively during enzymatic hydrolysis. The results showed that the addition of alkali lignins enhanced the hydrolysis and the enhancement for hydrolysis increased with increasing molecular weights of alkali lignins, with maximum enhancement being 28.69% for DAP and 20.05% for AP, respectively. The enhancement was partly attributed to the improved cellulase activity, and filter paper activity increased by 18.03% when adding lignin with highest molecular weight. It was found that the enhancement of enzymatic hydrolysis was correlated with the adsorption affinity of cellulase on alkali lignins, and the difference in surface charge and hydrophobicity of alkali lignins were responsible for the difference in affinity between cellulase and lignins. Copyright © 2015 Elsevier Ltd. All rights reserved.

Scale-up and integration of alkaline hydrogen peroxide pretreatment, enzymatic hydrolysis, and ethanolic fermentation.

PubMed

Banerjee, Goutami; Car, Suzana; Liu, Tongjun; Williams, Daniel L; Meza, Sarynna López; Walton, Jonathan D; Hodge, David B

2012-04-01

Alkaline hydrogen peroxide (AHP) has several attractive features as a pretreatment in the lignocellulosic biomass-to-ethanol pipeline. Here, the feasibility of scaling-up the AHP process and integrating it with enzymatic hydrolysis and fermentation was studied. Corn stover (1 kg) was subjected to AHP pretreatment, hydrolyzed enzymatically, and the resulting sugars fermented to ethanol. The AHP pretreatment was performed at 0.125 g H(2) O(2) /g biomass, 22°C, and atmospheric pressure for 48 h with periodic pH readjustment. The enzymatic hydrolysis was performed in the same reactor following pH neutralization of the biomass slurry and without washing. After 48 h, glucose and xylose yields were 75% and 71% of the theoretical maximum. Sterility was maintained during pretreatment and enzymatic hydrolysis without the use of antibiotics. During fermentation using a glucose- and xylose-utilizing strain of Saccharomyces cerevisiae, all of the Glc and 67% of the Xyl were consumed in 120 h. The final ethanol titer was 13.7 g/L. Treatment of the enzymatic hydrolysate with activated carbon prior to fermentation had little effect on Glc fermentation but markedly improved utilization of Xyl, presumably due to the removal of soluble aromatic inhibitors. The results indicate that AHP is readily scalable and can be integrated with enzyme hydrolysis and fermentation. Compared to other leading pretreatments for lignocellulosic biomass, AHP has potential advantages with regard to capital costs, process simplicity, feedstock handling, and compatibility with enzymatic deconstruction and fermentation. Biotechnol. Bioeng. 2012; 109:922-931. © 2011 Wiley Periodicals, Inc. Copyright © 2011 Wiley Periodicals, Inc.

Enzymatic Inverse Opal Hydrogel Particles for Biocatalyst.

PubMed

Wang, Huan; Gu, Hongcheng; Chen, Zhuoyue; Shang, Luoran; Zhao, Ze; Gu, Zhongze; Zhao, Yuanjin

2017-04-19

Enzymatic carriers have a demonstrated value for chemical reactions and industrial applications. Here, we present a novel kind of inverse opal hydrogel particles as the enzymatic carriers. The particles were negatively replicated from spherical colloidal crystal templates by using magnetic nanoparticles tagged acrylamide hydrogel. Thus, they were endowed with the features of monodispersity, small volume, complete penetrating structure, and controllable motion, which are all beneficial for improving the efficiency of biocatalysis. In addition, due to the ordered porous nanostructure, the inverse opal hydrogel particles were imparted with unique photonic band gaps (PBGs) and vivid structural colors for encoding varieties of immobilized enzymes and for constructing a multienzymes biocatalysis system. These features of the inverse opal hydrogel particles indicate that they are ideal enzymatic carriers for biocatalysis.

Enzymatic saccharification of biologically pre-treated wheat straw with white-rot fungi.

PubMed

Dias, Albino A; Freitas, Gil S; Marques, Guilhermina S M; Sampaio, Ana; Fraga, Irene S; Rodrigues, Miguel A M; Evtuguin, Dmitry V; Bezerra, Rui M F

2010-08-01

Wheat straw was submitted to a pre-treatment by the basidiomycetous fungi Euc-1 and Irpex lacteus, aiming to improve the accessibility of cellulose towards enzymatic hydrolysis via previous selective bio-delignification. This allowed the increase of substrate saccharification nearly four and three times while applying the basidiomycetes Euc-1 and I. lacteus, respectively. The cellulose/lignin ratio increased from 2.7 in the untreated wheat straw to 5.9 and 4.6 after the bio-treatment by the basidiomycetes Euc-1 and I. lacteus, respectively, thus evidencing the highly selective lignin biodegradation. The enzymatic profile of both fungi upon bio-treatment of wheat straw have been assessed including laccase, manganese-dependent peroxidase, lignin peroxidase, carboxymethylcellulase, xylanase, avicelase and feruloyl esterase activities. The difference in efficiency and selectivity of delignification within the two fungi treatments was interpreted in terms of specific lignolytic enzyme profiles and moderate xylanase and cellulolytic activities. (c) 2010 Elsevier Ltd. All rights reserved.

Printing enzymatic reactions.

PubMed

Tian, Junfei; Shen, Wei

2011-02-07

We used relief and planographic printing methods to print the catalytic effect of an enzyme, but not the enzyme molecules, onto paper. Printing enzymatic reactions have applications in bioactive papers, low-cost diagnostics, anti-counterfeiting devices and advanced packaging materials. These methods can create novel printing effects on commodity surfaces for advanced applications.

Genome-Scale Architecture of Small Molecule Regulatory Networks and the Fundamental Trade-Off between Regulation and Enzymatic Activity

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

Reznik, Ed; Christodoulou, Dimitris; Goldford, Joshua E.

Metabolic flux is in part regulated by endogenous small molecules that modulate the catalytic activity of an enzyme, e.g., allosteric inhibition. In contrast to transcriptional regulation of enzymes, technical limitations have hindered the production of a genome-scale atlas of small molecule-enzyme regulatory interactions. Here, we develop a framework leveraging the vast, but fragmented, biochemical literature to reconstruct and analyze the small molecule regulatory network (SMRN) of the model organism Escherichia coli, including the primary metabolite regulators and enzyme targets. Using metabolic control analysis, we prove a fundamental trade-off between regulation and enzymatic activity, and we combine it with metabolomic measurementsmore » and the SMRN to make inferences on the sensitivity of enzymes to their regulators. By generalizing the analysis to other organisms, we identify highly conserved regulatory interactions across evolutionarily divergent species, further emphasizing a critical role for small molecule interactions in the maintenance of metabolic homeostasis.« less

Genome-Scale Architecture of Small Molecule Regulatory Networks and the Fundamental Trade-Off between Regulation and Enzymatic Activity

DOE PAGES

Reznik, Ed; Christodoulou, Dimitris; Goldford, Joshua E.; ...

2017-09-12

Metabolic flux is in part regulated by endogenous small molecules that modulate the catalytic activity of an enzyme, e.g., allosteric inhibition. In contrast to transcriptional regulation of enzymes, technical limitations have hindered the production of a genome-scale atlas of small molecule-enzyme regulatory interactions. Here, we develop a framework leveraging the vast, but fragmented, biochemical literature to reconstruct and analyze the small molecule regulatory network (SMRN) of the model organism Escherichia coli, including the primary metabolite regulators and enzyme targets. Using metabolic control analysis, we prove a fundamental trade-off between regulation and enzymatic activity, and we combine it with metabolomic measurementsmore » and the SMRN to make inferences on the sensitivity of enzymes to their regulators. By generalizing the analysis to other organisms, we identify highly conserved regulatory interactions across evolutionarily divergent species, further emphasizing a critical role for small molecule interactions in the maintenance of metabolic homeostasis.« less

Imbalanced nutrient recycling in a warmer ocean driven by differential response of extracellular enzymatic activities.

PubMed

Ayo, Begoña; Abad, Naiara; Artolozaga, Itxaso; Azua, Iñigo; Baña, Zuriñe; Unanue, Marian; Gasol, Josep M; Duarte, Carlos M; Iriberri, Juan

2017-10-01

Ocean oligotrophication concurrent with warming weakens the capacity of marine primary producers to support marine food webs and act as a CO 2 sink, and is believed to result from reduced nutrient inputs associated to the stabilization of the thermocline. However, nutrient supply in the oligotrophic ocean is largely dependent on the recycling of organic matter. This involves hydrolytic processes catalyzed by extracellular enzymes released by bacteria, which temperature dependence has not yet been evaluated. Here, we report a global assessment of the temperature-sensitivity, as represented by the activation energies (E a ), of extracellular β-glucosidase (βG), leucine aminopeptidase (LAP) and alkaline phosphatase (AP) enzymatic activities, which enable the uptake by bacteria of substrates rich in carbon, nitrogen, and phosphorus, respectively. These E a were calculated from two different approaches, temperature experimental manipulations and a space-for-time substitution approach, which generated congruent results. The three activities showed contrasting E a in the subtropical and tropical ocean, with βG increasing the fastest with warming, followed by LAP, while AP showed the smallest increase. The estimated activation energies predict that the hydrolysis products under projected warming scenarios will have higher C:N, C:P and N:P molar ratios than those currently generated, and suggest that the warming of oceanic surface waters leads to a decline in the nutrient supply to the microbial heterotrophic community relative to that of carbon, particularly so for phosphorus, slowing down nutrient recycling and contributing to further ocean oligotrophication. © 2017 John Wiley & Sons Ltd.

Volatile Compound, Physicochemical, and Antioxidant Properties of Beany Flavor-Removed Soy Protein Isolate Hydrolyzates Obtained from Combined High Temperature Pre-Treatment and Enzymatic Hydrolysis

PubMed Central

Yoo, Sang-Hun; Chang, Yoon Hyuk

2016-01-01

The present study investigated the volatile compound, physicochemical, and antioxidant properties of beany flavor-removed soy protein isolate (SPI) hydrolyzates produced by combined high temperature pre-treatment and enzymatic hydrolysis. Without remarkable changes in amino acid composition, reductions of residual lipoxygenase activity and beany flavor-causing volatile compounds such as hexanol, hexanal, and pentanol in SPI were observed after combined heating and enzymatic treatments. The degree of hydrolysis, emulsion capacity and stability, 2,2-diphenyl-1-picrylhydrazyl radical scavenging activity, and superoxide radical scavenging activity of SPI were significantly increased, but the magnitudes of apparent viscosity, consistency index, and dynamic moduli (G′, G″) of SPI were significantly decreased after the combined heating and enzymatic treatments. Based on these results, it was suggested that the enzymatic hydrolysis in combination with high temperature pre-treatment may allow for the production of beany flavor-removed SPI hydrolyzates with superior emulsifying and antioxidant functionalities. PMID:28078256

Enzymatic Conversion of CO2 to Bicarbonate in Functionalized Mesoporous Silica

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

Yu, Yuehua; Chen, Baowei; Qi, Wen N.

2012-05-01

We report here that carbonic anhydrase (CA), the fastest enzyme that can covert carbon dioxide to bicarbonate, can be spontaneously entrapped in functionalized mesoporous silica (FMS) with super-high loading density (up to 0.5 mg of protein/mg of FMS) due to the dominant electrostatic interaction. The binding of CA to HOOC-FMS can result in the protein’s conformational change comparing to the enzyme free in solution, but can be overcome with increased protein loading density. The higher the protein loading density, the less conformational change, hence the higher enzymatic activity and the higher enzyme immobilization efficiency. The electrostatically bound CA can bemore » released by changing pH. The released enzyme still displayed the native conformational structure and the same high enzymatic activity as that prior to the enzyme entrapment. This work opens up a new approach converting carbon dioxide to biocarbonate in a biomimetic nanoconfiguration that can be integrated with the other part of biosynthesis process for the assimilation of carbon dioxide.« less

The lack of autophagy triggers precocious activation of Notch signaling during Drosophila oogenesis.

PubMed

Barth, Julia M I; Hafen, Ernst; Köhler, Katja

2012-12-05

The proper balance of autophagy, a lysosome-mediated degradation process, is indispensable for oogenesis in Drosophila. We recently demonstrated that egg development depends on autophagy in the somatic follicle cells (FC), but not in the germline cells (GCs). However, the lack of autophagy only affects oogenesis when FCs are autophagy-deficient but GCs are wild type, indicating that a dysfunctional signaling between soma and germline may be responsible for the oogenesis defects. Thus, autophagy could play an essential role in modulating signal transduction pathways during egg development. Here, we provide further evidence for the necessity of autophagy during oogenesis and demonstrate that autophagy is especially required in subsets of FCs. Generation of autophagy-deficient FCs leads to a wide range of phenotypes that are similar to mutants with defects in the classical cell-cell signaling pathways in the ovary. Interestingly, we observe that loss of autophagy leads to a precocious activation of the Notch pathway in the FCs as monitored by the expression of Cut and Hindsight, two downstream effectors of Notch signaling. Our findings point to an unexpected function for autophagy in the modulation of the Notch signaling pathway during Drosophila oogenesis and suggest a function for autophagy in proper receptor activation. Egg development is affected by an imbalance of autophagy between signal sending (germline) and signal receiving cell (FC), thus the lack of autophagy in the germline is likely to decrease the amount of active ligand and accordingly compensates for increased signaling in autophagy-defective follicle cells.

Structural Perspective on Enzymatic Halogenation

PubMed Central

2008-01-01

Simple halogen substituents frequently afford key structural features that account for the potency and selectivity of natural products, including antibiotics and hormones. For example, when a single chlorine atom on the antibiotic vancomycin is replaced by hydrogen, the resulting antibacterial activity decreases by up to 70% (HarrisC. M.; KannanR.; KopeckaH.; HarrisT. M.J. Am. Chem. Soc.1985, 107, 6652−6658). This Account analyzes how structure underlies mechanism in halogenases, the molecular machines designed by nature to incorporate halogens into diverse substrates. Traditional synthetic methods of integrating halogens into complex molecules are often complicated by a lack of specificity and regioselectivity. Nature, however, has developed a variety of elegant mechanisms for halogenating specific substrates with both regio- and stereoselectivity. An improved understanding of the biological routes toward halogenation could lead to the development of novel synthetic methods for the creation of new compounds with enhanced functions. Already, researchers have co-opted a fluorinase from the microorganism Streptomyces cattleya to produce 18F-labeled molecules for use in positron emission tomography (PET) (DengH.; CobbS. L.; GeeA. D.; LockhartA.; MartarelloL.; McGlincheyR. P.; O’HaganD.; OnegaM.Chem. Commun.2006, 652−654). Therefore, the discovery and characterization of naturally occurring enzymatic halogenation mechanisms has become an active area of research. The catalogue of known halogenating enzymes has expanded from the familiar haloperoxidases to include oxygen-dependent enzymes and fluorinases. Recently, the discovery of a nucleophilic halogenase that catalyzes chlorinations has expanded the repertoire of biological halogenation chemistry (DongC.; HuangF.; DengH.; SchaffrathC.; SpencerJ. B.; O’HaganD.; NaismithJ. H.Nature2004, 427, 561−56514765200). Structural characterization has provided a basis toward a mechanistic understanding of the specificity

Co-composting of organic fraction of municipal solid waste mixed with different bulking waste: characterization of physicochemical parameters and microbial enzymatic dynamic.

PubMed

Awasthi, Mukesh Kumar; Pandey, Akhilesh Kumar; Bundela, Pushpendra Singh; Khan, Jamaluddin

2015-04-01

The effect of various bulking waste such as wood shaving, agricultural and yard trimming waste combined with organic fraction of municipal solid waste (OFMSW) composting was investigated through assessing their influence on microbial enzymatic activities and quality of finished compost. All three piles of OFMSW with different bulking waste were inoculated with microbial consortium. The results revealed that OFMSW combined with wood shaving and microbial consortium (Phanerochaete chrysosporium, Trichoderma viride and Pseudomonas aeruginosa) were helpful tool to facilitate the enzymatic activity and shortened composting period within 4 weeks. Maximum enzymatic activity were observed in pile 1 and 3 during the first 3 weeks, while in pile 2 relatively very low. But phosphatase activity was relatively higher in all piles until the end of the process. Maturity parameters of compost quality also favored the pile 1 as the best formulation for OFMSW composting. Copyright © 2015 Elsevier Ltd. All rights reserved.

Orphan Kinesin NOD Lacks Motile Properties But Does Possess a Microtubule-stimulated ATPase Activity

PubMed Central

Matthies, Heinrich J.G.; Baskin, Ronald J.; Hawley, R. Scott

2001-01-01

NOD is a Drosophila chromosome-associated kinesin-like protein that does not fall into the chromokinesin subfamily. Although NOD lacks residues known to be critical for kinesin function, we show that microtubules activate the ATPase activity of NOD >2000-fold. Biochemical and genetic analysis of two genetically identified mutations of NOD (NODDTW and NOD“DR2”) demonstrates that this allosteric activation is critical for the function of NOD in vivo. However, several lines of evidence indicate that this ATPase activity is not coupled to vectorial transport, including 1) NOD does not produce microtubule gliding; and 2) the substitution of a single amino acid in the Drosophila kinesin heavy chain with the analogous amino acid in NOD results in a drastic inhibition of motility. We suggest that the microtubule-activated ATPase activity of NOD provides transient attachments of chromosomes to microtubules rather than producing vectorial transport. PMID:11739796

Physicochemical structural changes of cellulosic substrates during enzymatic saccharification

DOE PAGES

Meng, Xianzhi; Yoo, Chang Geun; Li, Mi; ...

2016-12-30

Enzymatic hydrolysis represents one of the major steps and barriers in the commercialization process of converting cellulosic substrates into biofuels and other value added products. It is usually achieved by a synergistic action of enzyme mixture typically consisting of multiple enzymes such as glucanase, cellobiohydrolase and β-glucosidase with different mode of actions. Due to the innate biomass recalcitrance, enzymatic hydrolysis normally starts with an initial fast rate of hydrolysis followed by a rapid decrease of rate toward the end of hydrolysis. With majority of literature studies focusing on the effect of key substrate characteristics on the initial rate or finalmore » yield of enzymatic hydrolysis, information about physicochemical structural changes of cellulosic substrates during enzymatic hydrolysis is still quite limited. Consequently, what slows down the reaction rate toward the end of hydrolysis is not well understood. Lastly, this review highlights recent advances in understanding the structural changes of cellulosic substrates during the hydrolysis process, to better understand the fundamental mechanisms of enzymatic hydrolysis.« less

Physicochemical structural changes of cellulosic substrates during enzymatic saccharification

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

Meng, Xianzhi; Yoo, Chang Geun; Li, Mi

Enzymatic hydrolysis represents one of the major steps and barriers in the commercialization process of converting cellulosic substrates into biofuels and other value added products. It is usually achieved by a synergistic action of enzyme mixture typically consisting of multiple enzymes such as glucanase, cellobiohydrolase and β-glucosidase with different mode of actions. Due to the innate biomass recalcitrance, enzymatic hydrolysis normally starts with an initial fast rate of hydrolysis followed by a rapid decrease of rate toward the end of hydrolysis. With majority of literature studies focusing on the effect of key substrate characteristics on the initial rate or finalmore » yield of enzymatic hydrolysis, information about physicochemical structural changes of cellulosic substrates during enzymatic hydrolysis is still quite limited. Consequently, what slows down the reaction rate toward the end of hydrolysis is not well understood. Lastly, this review highlights recent advances in understanding the structural changes of cellulosic substrates during the hydrolysis process, to better understand the fundamental mechanisms of enzymatic hydrolysis.« less

Enzymatic induction of supramolecular order and bioactivity

NASA Astrophysics Data System (ADS)

Yang, Chengbiao; Ren, Xinrui; Ding, Dan; Wang, Ling; Yang, Zhimou

2016-05-01

We showed in this study that enzymatic triggering is a totally different pathway for the preparation of self-assembling nanomaterials to the heating-cooling process. Because the molecules were under lower energy levels and the molecular conformation was more ordered during the enzymatic triggeration under mild conditions, nanomaterials with higher supramolecular order could be obtained through biocatalytic control. In this study, nanoparticles were obtained by an enzymatic reaction and nanofibers were observed through the heating-cooling process. We observed a distinct trough at 318 nm from the CD spectrum of a particle sample but not a fiber sample, suggesting the long range arrangement of molecules and helicity in the nanoparticles. The nanoparticles with higher supramolecular order possessed much better potency as a protein vaccine adjuvant because it accelerated the DC maturation and elicited stronger T-cells cytokine production than the nanofibers. Our study demonstrated that biocatalytic triggering is a useful method for preparing supramolecular nanomaterials with higher supramolecular order and probably better bioactivity.We showed in this study that enzymatic triggering is a totally different pathway for the preparation of self-assembling nanomaterials to the heating-cooling process. Because the molecules were under lower energy levels and the molecular conformation was more ordered during the enzymatic triggeration under mild conditions, nanomaterials with higher supramolecular order could be obtained through biocatalytic control. In this study, nanoparticles were obtained by an enzymatic reaction and nanofibers were observed through the heating-cooling process. We observed a distinct trough at 318 nm from the CD spectrum of a particle sample but not a fiber sample, suggesting the long range arrangement of molecules and helicity in the nanoparticles. The nanoparticles with higher supramolecular order possessed much better potency as a protein vaccine

Enzymatic Synthesis of Psilocybin.

PubMed

Fricke, Janis; Blei, Felix; Hoffmeister, Dirk

2017-09-25

Psilocybin is the psychotropic tryptamine-derived natural product of Psilocybe carpophores, the so-called "magic mushrooms". Although its structure has been known for 60 years, the enzymatic basis of its biosynthesis has remained obscure. We characterized four psilocybin biosynthesis enzymes, namely i) PsiD, which represents a new class of fungal l-tryptophan decarboxylases, ii) PsiK, which catalyzes the phosphotransfer step, iii) the methyltransferase PsiM, catalyzing iterative N-methyl transfer as the terminal biosynthetic step, and iv) PsiH, a monooxygenase. In a combined PsiD/PsiK/PsiM reaction, psilocybin was synthesized enzymatically in a step-economic route from 4-hydroxy-l-tryptophan. Given the renewed pharmaceutical interest in psilocybin, our results may lay the foundation for its biotechnological production. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

In Situ Enzymatically Generated Photoswitchable Oxidase Mimetics and Their Application for Colorimetric Detection of Glucose Oxidase.

PubMed

Cao, Gen-Xia; Wu, Xiu-Ming; Dong, Yu-Ming; Li, Zai-Jun; Wang, Guang-Li

2016-07-09

In this study, a simple and amplified colorimetric assay is developed for the detection of the enzymatic activity of glucose oxidase (GOx) based on in situ formation of a photoswitchable oxidase mimetic of PO₄(3-)-capped CdS quantum dots (QDs). GOx catalyzes the oxidation of 1-thio-β-d-glucose to give 1-thio-β-d-gluconic acid which spontaneously hydrolyzes to β-d-gluconic acid and H₂S; the generated H₂S instantly reacts with Cd(2+) in the presence of Na₃PO₄ to give PO₄(3-)-stabilized CdS QDs in situ. Under visible-light (λ ≥ 400 nm) stimulation, the PO₄(3-)-capped CdS QDs are a new style of oxidase mimic derived by producing some active species, such as h⁺, (•)OH, O₂(•-) and a little H₂O₂, which can oxidize the typical substrate (3,3,5,5-tetramethylbenzydine (TMB)) with a color change. Based on the GOx-triggered growth of the oxidase mimetics of PO₄(3-)-capped CdS QDs in situ, we developed a simple and amplified colorimetric assay to probe the enzymatic activity of GOx. The proposed method allowed the detection of the enzymatic activity of GOx over the range from 25 μg/L to 50 mg/L with a low detection limit of 6.6 μg/L. We believe the PO₄(3-)-capped CdS QDs generated in situ with photo-stimulated enzyme-mimicking activity may find wide potential applications in biosensors.

Enzymatic Enantioselective Decarboxylative Protonation of Heteroaryl Malonates

PubMed Central

Lewin, Ross; Goodall, Mark; Thompson, Mark L; Leigh, James; Breuer, Michael; Baldenius, Kai; Micklefield, Jason

2015-01-01

The enzyme aryl/alkenyl malonate decarboxylase (AMDase) catalyses the enantioselective decarboxylative protonation (EDP) of a range of disubstituted malonic acids to give homochiral carboxylic acids that are valuable synthetic intermediates. AMDase exhibits a number of advantages over the non-enzymatic EDP methods developed to date including higher enantioselectivity and more environmentally benign reaction conditions. In this report, AMDase and engineered variants have been used to produce a range of enantioenriched heteroaromatic α-hydroxycarboxylic acids, including pharmaceutical precursors, from readily accessible α-hydroxymalonates. The enzymatic method described here represents an improvement upon existing synthetic chemistry methods that have been used to produce similar compounds. The relationship between the structural features of these new substrates and the kinetics associated with their enzymatic decarboxylation is explored, which offers further insight into the mechanism of AMDase. PMID:25766433

Relationship between the enzymatic browning and phenylalanine ammonia-lyase activity of cut lettuce, and the prevention of browning by inhibitors of polyphenol biosynthesis.

PubMed

Hisaminato, H; Murata, M; Homma, S

2001-05-01

Cut lettuce stored at 4 degrees C gradually turned brown on the cut section after several days of storage. Three factors for enzymatic browning, the polyphenol content, polyphenol oxidase activity, and phenylalanine ammonia-lyase (PAL) activity, were examined during the cold storage of cut lettuce. A relationship between the browning and PAL activity was apparent. We tried to prevent this browning by using the two enzyme inhibitors, 2-aminoindane-2-phosphonic acid (AIP), an inhibitor of the phenylpropanoid pathway, and glyphosate, an inhibitor of the shikimate pathway. AIP and glyphosate significantly inhibited the browning of cut lettuce. The polyphenol content and PAL activity were both reduced by the treatment with AIP. These results show that regulating the biosynthesis of polyphenols is essential to prevent the browning of cut lettuce.

How Mg2+ ions lower the SN2@P barrier in enzymatic triphosphate hydrolysis.

PubMed

van Bochove, Marc A; Roos, Goedele; Fonseca Guerra, Célia; Hamlin, Trevor A; Bickelhaupt, F Matthias

2018-04-03

Our quantum chemical activation strain analyses demonstrate how Mg2+ lowers the barrier of the enzymatic triphosphate hydrolysis through two distinct mechanisms: (a) weakening of the leaving-group bond, thereby decreasing activation strain; and (b) transition state (TS) stabilization through enhanced electrophilicity of the triphosphate PPP substrate, thereby strengthening the interaction with the nucleophile.

Performance of glucose/O2 enzymatic fuel cell based on supporting electrodes over-coated by polymer-nanogold particle composite with entrapped enzymes

NASA Astrophysics Data System (ADS)

Huo, W. S.; Zeng, H.; Yang, Y.; Zhang, Y. H.

2017-03-01

Enzymatic electrodes over-coated by thin film of nano-composite made up of polymer and functionalized nano-gold particle was prepared. Glucose/O2 membrane-free enzymatic fuel cell based on nano-composite based electrodes with incorporated glucose oxidase and laccase was assembled. This enzymatic fuel cell exhibited high energy out-put density even when applied in human serum. Catalytic cycle involved in enzymatic fuel cell was limited by oxidation of glucose occurred on bioanode resulting from impact of sophisticated interaction between active site in glucose oxidase and nano-gold particle on configuration of redox center of enzyme molecule which crippled catalytic efficiency of redox protein.

Enzymatic modification of schizophyllan

USDA-ARS?s Scientific Manuscript database

An enzymatic method was developed for the progressive modification of the polysaccharide schizophyllan. Fungal strains Hypocrea nigricans NRRL 62555, Penicillium crustosum NRRL 62558, and Penicillium simplicissimum NRRL 62550 were previously identified as novel sources of ß-endoglucanase with specif...

Polymersome nanoreactors for enzymatic ring-opening polymerization.

PubMed

Nallani, Madhavan; de Hoog, Hans-Peter M; Cornelissen, Jeroen J L M; Palmans, Anja R A; van Hest, Jan C M; Nolte, Roeland J M

2007-12-01

Polystyrene-polyisocyanopeptide (PS-PIAT) polymersomes containing CALB in two different locations, one in the aqueous inner compartment and one in the bilayer, were investigated for enzymatic ring-opening polymerization of lactones in water. It is shown that the monomers 8-octanolactone and dodecalactone yield oligomers with this polymersome system. It is also observed that the polymerization activity is dependent on the position of the enzyme in the polymersome. SEM investigations show that the polymersome structures were destabilized during the polymerization. Further investigations show that the vesicular morphology of the polymersomes was destabilized only in the case of polymer product formation.

Enzymatic biomarkers can portray nanoCuO-induced oxidative and neuronal stress in freshwater shredders.

PubMed

Pradhan, Arunava; Silva, Carla O; Silva, Carlos; Pascoal, Cláudia; Cássio, Fernanda

2016-11-01

Commercial applications of nanometal oxides have increased concern about their release into natural waters and consequent risks to aquatic biota and the processes they drive. In forest streams, the invertebrate shredder Allogamus ligonifer plays a key role in detritus food webs by transferring carbon and energy from plant litter to higher trophic levels. We assessed the response profiles of oxidative and neuronal stress enzymatic biomarkers in A. ligonifer after 96h exposure to nanoCuO at concentration ranges enzymatic responses to Cu 2+ exposure at similar effective concentrations were compared. The highest activities of superoxide dismutase (SOD), glutathione peroxidase (GPx) and glutathione reductase (GR) were observed at concentrations enzymatic activities decreased at effective concentrations between LC 10 and LC 30 . GR activity remained higher than in control at all concentrations. The activity of glutathione S-transferase (GST) increased whereas that of catalase (CAT) decreased at concentrations between LC 10 and LC 30 . The response patterns suggested that antioxidant enzymes could prevent oxidative stress at low concentrations (activities were concentration-dependent, and led to oxidative stress and even to animal death. The activity of acetylcholinesterase (AChE) was strongly inhibited even at concentrations

Unraveling the effects of laccase treatment on enzymatic hydrolysis of steam-exploded wheat straw.

PubMed

Oliva-Taravilla, Alfredo; Moreno, Antonio D; Demuez, Marie; Ibarra, David; Tomás-Pejó, Elia; González-Fernández, Cristina; Ballesteros, Mercedes

2015-01-01

Laccase enzymes are promising detoxifying agents during lignocellulosic bioethanol production from wheat straw. However, they affect the enzymatic hydrolysis of this material by lowering the glucose recovery yields. This work aimed at explaining the negative effects of laccase on enzymatic hydrolysis. Relative glucose recovery in presence of laccase (10IU/g substrate) with model cellulosic substrate (Sigmacell) at 10% (w/v) was almost 10% points lower (P<0.01) than in the absence of laccase. This fact could be due to an increase in the competition of cellulose binding sites between the enzymes and a slight inhibition of β-glucosidase activity. However, enzymatic hydrolysis and infrared spectra of laccase-treated and untreated wheat straw filtered pretreated residue (WS-FPR), revealed that a grafting process of phenoxy radicals onto the lignin fiber could be the cause of diminished accessibility of cellulases to cellulose in pretreated wheat straw. Copyright © 2014 Elsevier Ltd. All rights reserved.

Radical-Mediated Enzymatic Carbon Chain Fragmentation-Recombination

PubMed Central

Zhang, Qi; Li, Yuxue; Chen, Dandan; Yu, Yi; Duan, Lian; Shen, Ben; Liu, Wen

2010-01-01

The radical S-adenosylmethionine (S-AdoMet) superfamily contains thousands of proteins that catalyze highly diverse conversions, most of which are poorly understood due to a lack of information regarding chemical products and radical-dependent transformations. We here report that NosL, involved in forming the indole side ring of the thiopeptide nosiheptide (NOS), is a radical S-AdoMet 3-methyl-2-indolic acid (MIA) synthase. NosL catalyzed an unprecedented carbon chain reconstitution of L-Trp to give MIA, showing removal of the Cα-N unit and shift of the carboxylate to the indole ring. Dissection of the enzymatic process upon the identification of products and a putative glycyl intermediate uncovered a radical-mediated, unusual fragmentation-recombination reaction. This finding unveiled a key step in radical S-AdoMet enzyme-catalyzed structural rearrangements during complex biotransformations. Additionally, NosL tolerated fluorinated L-Trps as the substrates, allowing for production of a regiospecifically halogenated thiopeptide that has not been found in over 80 entity-containing, naturally occurring thiopeptide family. PMID:21240261

Fractionation of enzymatic hydrolysis lignin by sequential extraction for enhancing antioxidant performance.

PubMed

An, Liangliang; Wang, Guanhua; Jia, Hongyu; Liu, Cuiyun; Sui, Wenjie; Si, Chuanling

2017-06-01

The heterogeneity of lignin chemical structure and molecular weight results in the lignin inhomogeneous properties which also covers the antioxidant performance. In order to evaluate the effects of lignin heterogeneity on its antioxidant activity, four lignin fractions from enzymatic hydrolysis lignin were classified by sequential organic solvent extraction and further evaluated by DPPH (1,1-Diphenyl-2-Picrylhydrazyl) free radical scavenging capacity and reducing power analysis. The characterization including FTIR, 1 H NMR and GPC showed that the fractionation process could effectively separate lignin fractions with distinctly different molecular weight and weaken the heterogeneity of unfractionated lignin. The antioxidant performance comparison of lignin fractions indicated that the dichloromethane fraction (F1) with lowest molecular weight (4585g/mol) and highest total phenolics content (246.13mg GAE/g) exhibited the highest antioxidant activity whose value was close to commercial antioxidant BHT (butylated hydroxytoluene). Moreover, the relationship between the antioxidant activity and the structure of lignin was further discussed to elucidate the mechanism of antioxidant activity improvement of lignin fractionation. Consequently, this study suggested that the sequential extraction was an effective way to obtain relatively homogeneous enzymatic hydrolysis lignin fractions which showed the potential for the value-added antioxidant application. Copyright © 2017 Elsevier B.V. All rights reserved.

Ceramic microsystem incorporating a microreactor with immobilized biocatalyst for enzymatic spectrophotometric assays.

PubMed

Baeza, Mireia; López, Carmen; Alonso, Julián; López-Santín, Josep; Alvaro, Gregorio

2010-02-01

Low-temperature cofired ceramics (LTCC) technology is a versatile fabrication technique used to construct microflow systems. It permits the integration of several unitary operations (pretreatment, separation, (bio)chemical reaction, and detection stage) of an analytical process in a modular or monolithic way. Moreover, because of its compatibility with biological material, LTCC is adequate for analytical applications based on enzymatic reactions. Here we present the design, construction, and evaluation of a LTCC microfluidic system that integrates a microreactor (internal volume, 24.28 microL) with an immobilized beta-galactosidase from Escherichia coli (0.479 activity units) and an optical flow cell to measure the product of the enzymatic reaction. The enzyme was immobilized on a glyoxal-agarose support, maintaining its activity along the time of the study. As a proof of concept, the LTCC-beta-galactosidase system was tested by measuring the conversion of ortho-nitrophenyl beta-D-galactopyranoside, the substrate usually employed for activity determinations. Once packed in a monolithically integrated microcolumn, the miniaturized flow system was characterized, the operational conditions optimized (flow rate and injection volume), and its performance successfully evaluated by determining the beta-galactosidase substrate concentration at the millimolar level.

Kinetic study of the activation of banana juice enzymatic browning by the addition of maltosyl-beta-cyclodextrin.

PubMed

López-Nicolás, José M; Pérez-López, Antonio J; Carbonell-Barrachina, Angel; García-Carmona, Francisco

2007-11-14

In recent years, the use of cyclodextrins (CDs) as antibrowning agents in fruit juices has received growning attention. However, there has been no detailed study of the behavior of these molecules as substances, which can lead to the darkening of foods. In this paper, when the color of fresh banana juice was evaluated in the presence of different CDs, the evolution of several color parameters was the opposite of that observed in other fruit juices. Moreover, a kinetic model based on the complexation by CDs of the natural browning inhibitors present in banana is developed for the first time to clarify the enzymatic browning activation of banana juice. Finally, the apparent complexation constant between the natural polyphenoloxidase inhibitors present in banana juice and maltosyl-beta-CD was calculated (Kci = 27.026 +/- 0.212 mM (-1)).

Simultaneous sonochemical-enzymatic coating of medical textiles with antibacterial ZnO nanoparticles.

PubMed

Petkova, Petya; Francesko, Antonio; Perelshtein, Ilana; Gedanken, Aharon; Tzanov, Tzanko

2016-03-01

The antimicrobial finishing is a must for production of medical textiles, aiming at reducing the bioburden in clinical wards and consequently decreasing the risk of hospital-acquired infections. This work reports for the first time on a simultaneous sonochemical/enzymatic process for durable antibacterial coating of cotton with zinc oxide nanoparticles (ZnO NPs). The novel technology goes beyond the "stepwise" concept we proposed recently for enzymatic pre-activation of the fabrics and subsequent sonochemical nano-coating, and is designed to produce "ready-to-use" antibacterial medical textiles in a single step. A multilayer coating of uniformly dispersed NPs was obtained in the process. The enzymatic treatment provides better adhesion of the ZnO NPs and, as a consequence, enhanced coating stability during exploitation. The NPs-coated cotton fabrics inhibited the growth of the medically relevant Staphylococcus aureus and Escherichia coli respectively by 67% and 100%. The antibacterial efficiency of these textile materials resisted the intensive laundry regimes used in hospitals, though only 33% of the initially deposited NPs remained firmly fixed onto the fabrics after multiple washings. Copyright © 2015 Elsevier B.V. All rights reserved.

Electrochemical enzymatic biosensors using carbon nanofiber nanoelectrode arrays

NASA Astrophysics Data System (ADS)

Li, Jun; Li, Yi-fen; Swisher, Luxi Z.; Syed, Lateef U.; Prior, Allan M.; Nguyen, Thu A.; Hua, Duy H.

2012-10-01

The reduction of electrode size down to nanometers could dramatically enhance detection sensitivity and temporal resolution. Nanoelectrode arrays (NEAs) are of particular interest for ultrasensitive biosensors. Here we report the study of two types of biosensors for measuring enzyme activities using NEAs fabricated with vertically aligned carbon nanofibers (VACNFs). VACNFs of ~100 nm in average diameter and 3-5 μm in length were grown on conductive substrates as uniform vertical arrays which were then encapsulated in SiO2 matrix leaving only the tips exposed. We demonstrate that such VACNF NEAs can be used in profiling enzyme activities through monitoring the change in electrochemical signals induced by enzymatic reactions to the peptides attached to the VACNF tip. The cleavage of the tetrapeptide with a ferrocene tag by a cancerrelated protease (legumain) was monitored with AC voltammetry. Real-time electrochemical impedance spectroscopy (REIS) was used for fast label-free detection of two reversible processes, i.e. phosphorylation by c-Src tyrosine kinase and dephosphorylation by protein tyrosine phosphatase 1B (PTP1B). The REIS data of phosphorylation were slow and unreliable, but those of dephosphorylation showed large and fast exponential decay due to much higher activity of phosphatase PTP1B. The kinetic data were analyzed with a heterogeneous Michaelis-Menten model to derive the "specificity constant" kcat/Km, which is 8.2x103 M-1s-1 for legumain and (2.1 ± 0.1) x 107 M-1s-1 for phosphatase (PTP1B), well consistent with literature. It is promising to develop VACNF NEA based electrochemical enzymatic biosensors as portable multiplex electronic techniques for rapid cancer diagnosis and treatment monitoring.

Evaluation of the enzymatic activity and stability of commercial bromelain incorporated in topical formulations.

PubMed

Lourenço, C B; Ataide, J A; Cefali, L C; Novaes, L C D L; Moriel, P; Silveira, E; Tambourgi, E B; Mazzola, P G

2016-10-01

Bromelain is a mixture of proteolytic enzymes found in various tissues of the pineapple plant (Ananas comosus) and other species of Bromeliaceae. Owing to its proteolytic activity, bromelain has been used in the food, medical, pharmaceutical and cosmetic industries, for its cell renewal, anti-ageing, whitening and anti-cellulite properties. This study evaluated the stability of bromelain (commercial powder) incorporated in topical formulations. Bromelain was incorporated at three concentrations, 0.5%, 1.0% and 2.0%, in oil-in-water emulsion and gel, and stored for six months at varying stress conditions. Stability was accessed by measuring the changes in the protein content, enzymatic activity, viscosity, rheology, pH and colour of the selected formulations. The colour of all the samples changed after 180 days of incubation, indicating the concentration-dependence and temperature-sensitive nature of these formulations. No relationship was observed between the changes in the pH, temperature and luminosity exposure in all the samples. Gels proved to be the least preferred base for incorporation of bromelain for use as a topical formulation, owing to its inability to maintain the integrity of bromelain, thereby affecting the formulation characteristics. The emulsion-based formulations at all the concentrations of bromelain were more stable than the gel-based formulation over 180 days of evaluation, at a temperature of 5°C, protected from light. © 2016 Society of Cosmetic Scientists and the Société Française de Cosmétologie.

Sulfate radicals enable a non-enzymatic Krebs cycle precursor

PubMed Central

Keller, Markus A.; Kampjut, Domen; Harrison, Stuart A.; Ralser, Markus

2017-01-01

The evolutionary origins of the tricarboxylic acid cycle (TCA), or Krebs cycle, are so far unclear. Despite a few years ago, the existence of a simple non-enzymatic Krebs-cycle catalyst has been dismissed ‘as an appeal to magic’, citrate and other intermediates have meanwhile been discovered on a carbonaceous meteorite and do interconvert non-enzymatically. To identify the non-enzymatic Krebs cycle catalyst, we used combinatorial, quantitative high-throughput metabolomics to systematically screen iron and sulfate reaction milieus that orient on Archean sediment constituents. TCA cycle intermediates are found stable in water and in the presence of most iron and sulfate species, including simple iron-sulfate minerals. However, we report that TCA intermediates undergo 24 interconversion reactions in the presence of sulfate radicals that form from peroxydisulfate. The non-enzymatic reactions critically cover a topology as present in the Krebs cycle, the glyoxylate shunt and the succinic semialdehyde pathways. Assembled in a chemical network, the reactions achieve more than ninety percent carbon recovery. Our results show that a non-enzymatic precursor for the Krebs cycle is biologically sensible, efficient, and forms spontaneously in the presence of sulfate radicals. PMID:28584880

In situ enzymatic activity of transglutaminase isoforms on brain tissue sections of rodents: A new approach to monitor differences in post-translational protein modifications during neurodegeneration.

PubMed

Schulze-Krebs, Anja; Canneva, Fabio; Schnepf, Rebecca; Dobner, Julia; Dieterich, Walburga; von Hörsten, Stephan

2016-01-15

Mammalian transglutaminases (TGs) catalyze the irreversible post-translational modifications of proteins, the most prominent of which is the calcium-dependent formation of covalent acyl transfers between the γ-carboxamide group of glutamine and the ε-amino-group of lysine (GGEL-linkage). In the central nervous system, at least four TG isoforms are present and some of them are differentially expressed under pathological conditions in human patients. However, the precise TG-isoform-dependent enzymatic activities in the brain as well as their anatomical distribution are unknown. Specificity of the used biotinylated peptides was analyzed using an in vitro assay. Isoform-specific TG activity was evaluated in in vitro and in situ studies, using brain extracts and native brain tissue obtained from rodents. Our method allowed us to reveal in vitro and in situ TG-isoform-dependent enzymatic activity in brain extracts and tissue of rats and mice, with a specific focus on TG6. In situ activity of this isoform varied between BACHD mice in comparison to their wt controls. TG isozyme-specific activity can be detected by isoform-specific biotinylated peptides in brain tissue sections of rodents to reveal differences in the anatomical and/or subcellular distribution of TG activity. Our findings yield the basis for a broader application of this method for the screening of pathological expression and activity of TGs in a variety of animal models of human diseases, as in the case of neurodegenerative conditions such as Huntington׳s, Parkinson׳s and Alzheimer׳s, where protein modification is involved as a key mechanism of disease progression. Copyright © 2015 Elsevier B.V. All rights reserved.

Analysis of non-enzymatically glycated peptides: neutral-loss-triggered MS3 versus multi-stage activation tandem mass spectrometry

PubMed Central

Zhang, Qibin; Petyuk, Vladislav A.; Schepmoes, Athena A.; Orton, Daniel J.; Monroe, Matthew E.; Yang, Feng; Smith, Richard D.; Metz, Thomas O.

2009-01-01

Non-enzymatic glycation of tissue proteins has important implications in the development of complications of diabetes mellitus. While electron transfer dissociation (ETD) has been shown to outperform collision-induced dissociation (CID) in sequencing glycated peptides by tandem mass spectrometry, ETD instrumentation is not yet widely available and often suffers from significantly lower sensitivity than CID. In this study, we evaluated different advanced CID techniques (i.e., neutral-loss-triggered MS3 and multi-stage activation) during liquid chromatography/multi-stage mass spectrometric (LC/MSn) analyses of Amadori-modified peptides enriched from human serum glycated in vitro. During neutral-loss-triggered MS3 experiments, MS3 scans triggered by neutral losses of 3 H2O or 3 H2O + HCHO produced similar results in terms of glycated peptide identifications. However, neutral losses of 3 H2O resulted in significantly more glycated peptide identifications during multi-stage activation experiments. Overall, the multi-stage activation approach produced more glycated peptide identifications, while the neutral-loss-triggered MS3 approach resulted in much higher specificity. Both techniques are viable alternatives to ETD for identifying glycated peptides. PMID:18763275

Perturbation theory in the catalytic rate constant of the Henri-Michaelis-Menten enzymatic reaction.

PubMed

Bakalis, Evangelos; Kosmas, Marios; Papamichael, Emmanouel M

2012-11-01

The Henry-Michaelis-Menten (HMM) mechanism of enzymatic reaction is studied by means of perturbation theory in the reaction rate constant k (2) of product formation. We present analytical solutions that provide the concentrations of the enzyme (E), the substrate (S), as well as those of the enzyme-substrate complex (C), and the product (P) as functions of time. For k (2) small compared to k (-1), we properly describe the entire enzymatic activity from the beginning of the reaction up to longer times without imposing extra conditions on the initial concentrations E ( o ) and S ( o ), which can be comparable or much different.

Homogeneous, Heterogeneous, and Enzymatic Catalysis.

ERIC Educational Resources Information Center

Oyama, S. Ted; Somorjai, Gabor A.

1988-01-01

Discusses three areas of catalysis: homegeneous, heterogeneous, and enzymatic. Explains fundamentals and economic impact of catalysis. Lists and discusses common industrial catalysts. Provides a list of 107 references. (MVL)

The Use of Adenovirus Dodecahedron in the Delivery of an Enzymatic Activity in the Cell

PubMed Central

Sumarheni; Gallet, Benoit; Fender, Pascal

2016-01-01

Penton-dodecahedron (Pt-Dd) derived from adenovirus type 3 is a symmetric complex of pentameric penton base plus fiber which can be produced in the baculovirus system at a high concentration. The size of Pt-Dd is smaller than the virus, but this virus-like particle (VLP) has the major proteins recognized by specific receptors on the surface of almost all types of cell. In this study, by direct observation with fluorescence microscopy on a fixed and living cell, the intracellular trafficking and localization of Pt-Dd labeled with fluorescence dyes in the cytoplasm of HeLa Tub-GFP showed a rapid internalization characteristic. Subsequently, the linkage of horseradish peroxidase (HRP) with Pt-Dd as the vector demonstrated an efficient system to deliver this enzyme into the cell without interfering its enzymatic activity as shown by biochemical and cellular experiments. These results were supported by additional studies using Bs-Dd or free form of the HRP used as the control. Overall, this study strengthens the potential role of Pt-Dd as an alternative vector for delivering therapeutic agents. PMID:27242929

Development of enzymatically-active bacterial cellulose membranes through stable immobilization of an engineered β-galactosidase.

PubMed

Estevinho, Berta N; Samaniego, Nuria; Talens-Perales, David; Fabra, Maria José; López-Rubio, Amparo; Polaina, Julio; Marín-Navarro, Julia

2018-08-01

Enzymatically-active bacterial cellulose (BC) was prepared by non-covalent immobilization of a hybrid enzyme composed by a β-galactosidase from Thermotoga maritima (TmLac) and a carbohydrate binding module (CBM2) from Pyrococcus furiosus. TmLac-CBM2 protein was bound to BC, with higher affinity at pH 6.5 than at pH 8.5 and with high specificity compared to the non-engineered enzyme. Both hydrated (HBC) and freeze-dried (DBC) bacterial cellulose showed equivalent enzyme binding efficiencies. Initial reaction rate of HBC-bound enzyme was higher than DBC-bound and both of them were lower than the free enzyme. However, enzyme performance was similar in all three cases for the hydrolysis of 5% lactose to a high extent. Reuse of the immobilized enzyme was limited by the stability of the β-galactosidase module, whereas the CBM2 module provided stable attachment of the hybrid enzyme to the BC support, after long incubation periods (3 h) at 75 °C. Copyright © 2018 Elsevier B.V. All rights reserved.

Immobilization of proteins onto microbeads using a DNA binding tag for enzymatic assays.

PubMed

Kojima, Takaaki; Mizoguchi, Takuro; Ota, Eri; Hata, Jumpei; Homma, Keisuke; Zhu, Bo; Hitomi, Kiyotaka; Nakano, Hideo

2016-02-01

A novel DNA-binding protein tag, scCro-tag, which is a single-chain derivative of the bacteriophage lambda Cro repressor, has been developed to immobilize proteins of interest (POI) on a solid support through binding OR consensus DNA (ORC) that is tightly bound by the scCro protein. The scCro-tag successfully bound a transglutaminase 2 (TGase 2) substrate and manganese peroxidase (MnP) to microbeads via scaffolding DNA. The resulting protein-coated microbeads can be utilized for functional analysis of the enzymatic activity using flow cytometry. The quantity of bead-bound proteins can be enhanced by increasing the number of ORCs. In addition, proteins with the scCro-tag that were synthesized using a cell-free protein synthesis system were also immobilized onto the beads, thus indicating that this bead-based system would be applicable to high-throughput analysis of various enzymatic activities. Copyright © 2015 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Full inhibition of enzymatic browning in the presence of thiol-functionalised silica nanomaterial.

PubMed

Muñoz-Pina, Sara; Ros-Lis, José V; Argüelles, Ángel; Coll, Carmen; Martínez-Máñez, Ramón; Andrés, Ana

2018-02-15

Darkening processed fruits and vegetables is caused mainly by enzymatic browning through polyphenol oxidase (PPO) action. Accordingly, we explored the potential of four silica-based materials (MCM-41 nanometric size, MCM-41 micrometric size, UVM-7 and aerosil), non-functionalised and functionalised with thiol groups, to inhibit PPO activity in the model system and apple juice. All materials showed relevant performance when immobilising and inhibiting PPO in model systems, and support topology is a main factor for enzyme immobilisation and inhibition. Thiol-containing silica UVM7-SH showed the greatest inactivation, and similar browning values to those obtained by acidification. The enzyme's kinetic parameters in the presence of UVM-7-SH suggested non-competitive inhibition, which indicated that the material interacted with the enzyme, but beyond the active centre. In real systems, UVM-7-SH completely inhibited enzymatic browning in apple juice (cv. Granny Smith and cv. Golden Delicious) up to 9days after 5min of contact. Copyright © 2017 Elsevier Ltd. All rights reserved.

[The effect of enzymatic treatment using proteases on properties of persistent sodium current in CA1 pyramidal neurons of rat hippocampus].

PubMed

Lun'ko, O O; Isaiev, D S; Maxymiuk, O P; Kryshtal', O O; Isaieva, O V

2014-01-01

We investigated the effect of proteases, widely used for neuron isolation in electrophysiological studies, on the amplitude and kinetic characteristics of persistent sodium current (I(NaP)) in hippocampal CA1 pyramidal neurons. Properties of I(NaP) were studied on neurons isolated by mechanical treatment (control group) and by mechanical and enzymatic treatment using pronase E (from Streptomyces griseus) or protease type XXIII (from Aspergillus oryzae). We show that in neurons isolated with pronase E kinetic of activation and density of I(NaP) was unaltered. Enzymatic treatment with protease type XXIII did not alter I(NaP) activation but result in significant decrease in I(NaP) density. Our data indicates that enzymatic treatment using pronase E for neuron isolation is preferable for investigation of I(NaP).

Green-synthesized CdS nano-pesticides: Toxicity on young instars of malaria vectors and impact on enzymatic activities of the non-target mud crab Scylla serrata.

PubMed

Sujitha, Vasu; Murugan, Kadarkarai; Dinesh, Devakumar; Pandiyan, Amuthvalli; Aruliah, Rajasekar; Hwang, Jiang-Shiou; Kalimuthu, Kandasamy; Panneerselvam, Chellasamy; Higuchi, Akon; Aziz, Al Thabiani; Kumar, Suresh; Alarfaj, Abdullah A; Vaseeharan, Baskaralingam; Canale, Angelo; Benelli, Giovanni

2017-07-01

Currently, nano-formulated mosquito larvicides have been widely proposed to control young instars of malaria vector populations. However, the fate of nanoparticles in the aquatic environment is scarcely known, with special reference to the impact of nanoparticles on enzymatic activity of non-target aquatic invertebrates. In this study, we synthesized CdS nanoparticles using a green protocol relying on the cheap extract of Valoniopsis pachynema algae. CdS nanoparticles showed high toxicity on young instars of the malaria vectors Anopheles stephensi and A. sundaicus. The antimalarial activity of the nano-synthesized product against chloroquine-resistant (CQ-r) Plasmodium falciparum parasites was investigated. From a non-target perspective, we focused on the impact of this novel nano-pesticide on antioxidant enzymes acetylcholinesterase (AChE) and glutathione S-transferase (GST) activities of the mud crab Scylla serrata. The characterization of nanomaterials was carried out by UV-vis and FTIR spectroscopy, as well as SEM and XRD analyses. In mosquitocidal assays, LC 50 of V. pachynema-synthesized CdS nanoparticles on A. stephensi ranged from 16.856 (larva I), to 30.301μg/ml (pupa), while for An. sundaicus they ranged from 13.584 to 22.496μg/ml. The antiplasmodial activity of V. pachynema extract and CdS nanoparticles was evaluated against CQ-r and CQ-sensitive (CQ-s) strains of Plasmodium falciparum. IC 50 of V. pachynema extract was 58.1μg/ml (CQ-s) and 71.46μg/ml (CQ-r), while nano-CdS IC 50 was 76.14μg/ml (CQ-s) and 89.21μg/ml (CQ-r). In enzymatic assays, S. serrata crabs were exposed to sub-lethal concentrations, i.e. 4, 6 and 8μg/ml of CdS nanoparticles, assessing changes in GST and AChE activity after 16days. We observed significantly higher activity of GST, if compared to the control, during the whole experiment period. In addition, a single treatment with CdS nanoparticles led to a significant decrease in AChE activity over time. The toxicity of Cd

Efficacy of enzymatic debridement of deeply burned hands.

PubMed

Krieger, Yuval; Bogdanov-Berezovsky, Alexander; Gurfinkel, Reuven; Silberstein, Eldad; Sagi, Amiram; Rosenberg, Lior

2012-02-01

The burned hand is a common and difficult to care-for entity in the field of burns. Due to the anatomy of the hand (important and delicate structures crowded in a small limited space without sub-dermal soft tissue), surgical debridement of the burned tissue is technically difficult and may cause considerable complications and, therefore, should be performed judiciously. Selective enzymatic debridement of the burn wound can preserve the spontaneous epithelialisation potential and reduce the added injury to the traumatised tissue added by a surgical debridement. The aim of the study was to assess the implication of a selective enzymatic compound (Debrase(®) - Ds) in the special field of deep hand burns, by comparing the actual burn area that required surgical coverage after enzymatic debridement to the burn area clinically judged to require skin grafting prior to debridement. This was a retrospective data collection and analysis from 154 complete files of prospective, open-label study in 275 hospitalised, Ds-treated burn patients. A total of 69 hand burns diagnosed as 'deep' was analysed; 36% of the wounds required surgical intervention after enzymatic debridement; 28.6% of the total burned area estimated initially as deep was covered by skin graft (statistically significant p<0.001). Debridement of deep-hand burns with a selective enzymatic agent decreased the perceived full-thickness wound area and skin-graft use. Copyright © 2011 Elsevier Ltd and ISBI. All rights reserved.

Enzymatic and Pro-Inflammatory Activities of Bothrops lanceolatus Venom: Relevance for Envenomation

PubMed Central

Delafontaine, Marie; Villas-Boas, Isadora Maria; Mathieu, Laurence; Josset, Patrice; Blomet, Joël

2017-01-01

Bothrops lanceolatus, commonly named ‘Fer-de-Lance’, is an endemic snake of the French Caribbean Island of Martinique. Envenomations by B. lanceolatus present clinical aspects characterized by systemic thrombotic syndrome and important local inflammation, involving edema and pain but limited hemorrhage. To investigate mechanisms of venom-induced inflammation, B. lanceolatus venom was characterized, its cross-reactivity with bothropic antivenom explored, its cytotoxicity on human keratinocytes and vascular cells, and the production of cytokines and chemokines were analyzed. We used electrophoretic separation, zymography, colorimetric or fluorimetric enzymatic assays, and immunochemical assays. Therapeutic South American bothropic antivenom cross-reacted with B. lanceolatus venom and completely or partially abolished its PLA2, hyaluronidase, and proteolytic activities, as well as its cytotoxicity for keratinocytes. The substrate specificity of B. lanceolatus venom proteases was emphasized. B. lanceolatus venom cytotoxicity was compared to the B. jararaca venom. Both venoms were highly cytotoxic for keratinocytes (HaCaT), whereas B. lanceolatus venom showed particularly low toxicity for endothelial cells (EAhy926). Patterns of cytokine and chemokine production by cells exposed to the venoms were highly pro-inflammatory. Thus, the results presented here show that B. lanceolatus venom toxins share important antigenic similarities with South American Bothrops species toxins, although their proteases have acquired particular substrate specificity. Moreover, the venom displays important cytotoxic and pro-inflammatory action on human cell types such as keratinocytes and endothelial cells, which are important players in the local and systemic compartments affected by the envenomation. PMID:28783135

Immobilization of α-amylase onto a calix[4]arene derivative: Evaluation of its enzymatic activity.

PubMed

Veesar, Irshad Ali; Solangi, Imam Bakhsh; Memon, Shahabuddin

2015-06-01

In order to enhance the cost-effectiveness practicability of enzymes in many industries such as pharmaceutical, food, medical and some other technological processes, there is great need to immobilize them onto a solid supports. In this study, a new and efficient immobilization of α-amylase from Saccharomyces cerevisiae has been developed by using the surface functionalization of calix[4]arene as support. A glutaraldehyde-containing amino group functionalized calix[4]arene was used to immobilize α-amylase covalently. In this procedure, imide bonds are formed between amino groups on the protein and aldehyde groups on the calix[4]arene surface. The surface modified support was characterized using Fourier transform infrared spectroscopy (FT-IR), scanning electron microscopy (SEM). The effect of various preparation conditions on the immobilized α-amylase process such as immobilization time, enzyme concentration, temperature and pH were investigated. The influence of pH and temperature on the activity of free and immobilized α-amylase was also studied using starch as substrate. The optimum reaction temperature and pH value for the enzymatic conversion catalyzed by the immobilized α-amylase were 25°C and 7, respectively. Compared to the free enzyme, the immobilized α-amylase retained 85% of its original activity and exhibited significant thermal stability than the free one and excellent durability. Copyright © 2015 Elsevier Inc. All rights reserved.

The Pleckstrin Homology Domain of Phospholipase Cβ Transmits Enzymatic Activation through Modulation of Membrane - Domain Orientation§

PubMed Central

Drin, Guillaume; Douguet, Dominique; Scarlata, Suzanne

2008-01-01

Phospholipase C-beta (PLCβ) enzymes are activated by Gαq and Gβγ subunits and catalyze the hydrolysis of the minor membrane lipid phosphatidylinositol 4,5 bisphosphate (PI(4,5)P2). Activation of PLCβ2 by Gβγ subunits has been shown to be conferred through its N-terminal pleckstrin homology (PH) domain although the underlying mechanism is unclear. Also unclear are observations that the extent of Gβγ activation differs on different membrane surfaces. In this study, we have identified a unique region of the PH domain of PLCβ2 domain (residues 71-88) which, when added to the enzyme as a peptide, causes enzyme activation similar to Gβγ subunits. This PH domain segment interacts strongly with membranes composed of lipid mixtures but not those containing lipids with electrically neutral zwitterionic head groups. Moreso, addition of this segment perturbs interaction of the catalytic domain, but not the PH domain, with membrane surfaces. We monitored the orientation of the PH and catalytic domains of PLC by intermolecular fluorescence resonance energy transfer (FRET) using the Gβγ activatable mutant, PLCβ2/δ1(C193S). We find an increase in FRET with binding to membranes with mixed lipids but not to those containing only lipids with electrically neutral head groups. These results suggest that enzymatic activation can be conferred through optimal association of the PHβ71-88 region to specific membrane surfaces. These studies allow us to understand the basis of variations of Gβγ activation on different membrane surfaces. PMID:16669615

Aldehyde PEGylation of laccase from Trametes versicolor in route to increase its stability: effect on enzymatic activity.

PubMed

Mayolo-Deloisa, Karla; González-González, Mirna; Simental-Martínez, Jesús; Rito-Palomares, Marco

2015-03-01

Laccase is a multicopper oxidase that catalyzes the oxidation of phenolic compounds. Laccase can be used in bioremediation, beverage (wine, fruit juice, and beer) processing, ascorbic acid determination, sugar beet pectin gelation baking, and as a biosensor. Recently, the antiproliferative activity of laccase toward tumor cells has been reported. Because of the potential applications of this enzyme, the efforts for enhancing and stabilizing its activity have increased. Thus, the PEGylation of laccase can be an alternative. PEGylation is the covalent attachment of one or more molecules of methoxy poly(ethylene glycol) (mPEG) to a protein. Normally, during the PEGylation reaction, the activity is reduced but the stability increases; thus, it is important to minimize the loss of activity. In this work, the effects of molar ratio (1:4, 1:8, and 1:12), concentration of laccase (6 and 12 mg/ml), reaction time (4 and 17 h), molecular weight, and type of mPEG (20, 30, 40 kDa and 40 kDa-branched) were analyzed. The activity was measured using three substrates: ABTS, 2,6-dimethoxyphenol, and syringaldazine. The best conditions for laccase PEGylation were 12 mg/ml of laccase, molar ratio 1:4, and 4 h reaction time. Under these conditions, the enzyme was able to maintain nearly 100% of its enzymatic activity with ABTS. The PEGylation of laccase has not been extensively explored, so it is important to analyze the effects of this bioconjugation in route to produce a robust modified enzyme. Copyright © 2015 John Wiley & Sons, Ltd.

Dissipation of S-metolachlor in plant and soil and effect on enzymatic activities.

PubMed

Wołejko, Elżbieta; Kaczyński, Piotr; Łozowicka, Bożena; Wydro, Urszula; Borusiewicz, Andrzej; Hrynko, Izabela; Konecki, Rafał; Snarska, Krystyna; Dec, Dorota; Malinowski, Paweł

2017-07-01

The present study aimed at evaluating the dissipation of S-metolachlor (S-MET) at three doses in maize growing on diverse physico-chemical properties of soil. The effect of herbicide on dehydrogenase (DHA) and acid phosphatase (ACP) activity was estimated. A modified QuEChERS method using LC-MS/MS has been developed. The limit of quantification (0.001 mg kg -1 ) and detection (0.0005 mg kg -1 ) were very low for soil and maize samples. The mean recoveries and RSDs for the six spiked levels (0.001-0.5 mg kg -1 ) were 91.3 and 5.8%. The biggest differences in concentration of S-MET in maize were observed between the 28th and 63rd days. The dissipation of S-MET in the alkaline soil was the slowest between the 2nd and 7th days, and in the acidic soil between the 5th and 11th days. DT 50 of S-MET calculated according to the first-order kinetics model was 11.1-14.7 days (soil) and 9.6-13.9 days (maize). The enzymatic activity of soil was higher in the acidic environment. One observed the significant positive correlation of ACP with pH of soil and contents of potassium and magnesium and negative with contents of phosphorus and organic carbon. The results indicated that at harvest time, the residues of S-MET in maize were well below the safety limit for maize. The findings of this study will foster the research on main parameters influencing the dissipation in maize ecosystems.

Quantification of ricin, RCA and comparison of enzymatic activity in 18 Ricinus communis cultivars by isotope dilution mass spectrometry

PubMed Central

Schieltz, David M.; McWilliams, Lisa G.; Kuklenyik, Zsuzsanna; Prezioso, Samantha M.; Carter, Andrew J.; Williamson, Yulanda M.; McGrath, Sara C.; Morse, Stephen A.; Barr, John R.

2016-01-01

The seeds of the Ricinus communis (Castor bean) plant are the source of the economically important commodity castor oil. Castor seeds also contain the proteins ricin and R. communis agglutinin (RCA), two toxic lectins that are hazardous to human health. Radial immunodiffusion (RID) and the enzyme linked immunosorbent assay (ELISA) are two antibody-based methods commonly used to quantify ricin and RCA; however, antibodies currently used in these methods cannot distinguish between ricin and RCA due to the high sequence homology of the respective proteins. In this study, a technique combining antibody-based affinity capture with liquid chromatography and multiple reaction monitoring (MRM) mass spectrometry (MS) was used to quantify the amounts of ricin and RCA independently in extracts prepared from the seeds of eighteen representative cultivars of R. communis which were propagated under identical conditions. Additionally, liquid chromatography and MRM-MS was used to determine rRNA N-glycosidase activity for each cultivar and the overall activity in these cultivars was compared to a purified ricin standard. Of the cultivars studied, the average ricin content was 9.3 mg/g seed, the average RCA content was 9.9 mg/g seed, and the enzymatic activity agreed with the activity of a purified ricin reference within 35% relative activity. PMID:25576235

Alkali pretreated of wheat straw and its enzymatic hydrolysis.

PubMed

Han, Lirong; Feng, Juntao; Zhang, Shuangxi; Ma, Zhiqing; Wang, Yonghong; Zhang, Xing

2012-01-01

The efficiency of enzymatic hydrolysis of cellulose can be improved by various pretreatments of the substrate. In order to increase the efficiency of enzymatic saccharification of the wheat straw, we determined the effect of different pretreatments on the physical structure, chemical components and enzymatic saccharification of wheat straw. Our results showed that combination of grinding and sodium hydroxide (NaOH) treatment had high effect on the enzymatic hydrolysis of wheat straws. The optimal pretreatment condition was to grind the wheat straws into the sizes of 120 meshes followed by treatment with 1.0% NaOH for 1.5 h (121°C/15psi). Under this condition, the cellulose content of wheat straw was increased by 44.52%, while the content of hemicellulose and lignin was decreased by 44.15% and 42.52%, respectively. Scanning Electronic Microscopy and infrared spectrum analyses showed that significant changes occurred in the structure of wheat straws after pretreatment, which is favorable for the hydrolysis and saccharification. Cellulase produced by Penicillium waksmanii F10-2 was used to hydrolyze the pretreated wheat straw and the optimal condition was determined to be 30 h of enzymatic reaction under the temperature of 55°C, pH 5.5 and substrate concentration of 3%.

Effect of exogenous metal ions and mechanical stress on rice processed in thermal-solid enzymatic reaction system related to further alcoholic fermentation efficiency.

PubMed

Xu, Enbo; Wu, Zhengzong; Jiao, Aiquan; Jin, Zhengyu

2018-02-01

Metal-rich thermal-solid enzymatic processing of rice combined with yeast fermentation was investigated. 8 Metal ions were exogenously supplied at 0.05, 0.5 and 5mmol/100g (MG) rice prior to static high pressure enzymatic cooking (HPEC) and dynamic enzymatic extrusion cooking (EEC). Treated rice and its fermentation efficiency (FE) were characterized by rapid viscosity analyzer (RVA), UV-Vis, FT-IR and atomic absorption spectrophotometer (AAS). The optimum pH range of enzyme in solid system (>4.9) was broader than in a liquid system (>5.5). Cations decreased enzymatic activity in HPEC probably due to metal-induced aggregation of rice matrix with reduced reacting area as well as strengthened structure of starch/polysaccharides modified by metals. While using the EEC with mechanical mixing/shearing, relative activity was activated to 110 and 120% by Mg 2+ (0.05-0.5MG) and Ca 2+ (0.05-5MG), respectively. Furthermore, the effectiveness of residual ions to promote further FE was found to follow the order: Ca 2+ >K + >Zn 2+ >Mg 2+ >Mn 2+ >Na + ≈Control>Fe 2+ >Cu 2+ , individually. Copyright © 2017 Elsevier Ltd. All rights reserved.

Lack of beta-arrestin signaling in the absence of active G proteins.

PubMed

Grundmann, Manuel; Merten, Nicole; Malfacini, Davide; Inoue, Asuka; Preis, Philip; Simon, Katharina; Rüttiger, Nelly; Ziegler, Nicole; Benkel, Tobias; Schmitt, Nina Katharina; Ishida, Satoru; Müller, Ines; Reher, Raphael; Kawakami, Kouki; Inoue, Ayumi; Rick, Ulrike; Kühl, Toni; Imhof, Diana; Aoki, Junken; König, Gabriele M; Hoffmann, Carsten; Gomeza, Jesus; Wess, Jürgen; Kostenis, Evi

2018-01-23

G protein-independent, arrestin-dependent signaling is a paradigm that broadens the signaling scope of G protein-coupled receptors (GPCRs) beyond G proteins for numerous biological processes. However, arrestin signaling in the collective absence of functional G proteins has never been demonstrated. Here we achieve a state of "zero functional G" at the cellular level using HEK293 cells depleted by CRISPR/Cas9 technology of the Gs/q/12 families of Gα proteins, along with pertussis toxin-mediated inactivation of Gi/o. Together with HEK293 cells lacking β-arrestins ("zero arrestin"), we systematically dissect G protein- from arrestin-driven signaling outcomes for a broad set of GPCRs. We use biochemical, biophysical, label-free whole-cell biosensing and ERK phosphorylation to identify four salient features for all receptors at "zero functional G": arrestin recruitment and internalization, but-unexpectedly-complete failure to activate ERK and whole-cell responses. These findings change our understanding of how GPCRs function and in particular of how they activate ERK1/2.

Enzymatic lipid oxidation by eosinophils propagates coagulation, hemostasis, and thrombotic disease

PubMed Central

Uderhardt, Stefan; Ackermann, Jochen A.; Fillep, Tobias; Hammond, Victoria J.; Willeit, Johann; Stark, Konstantin; Rossaint, Jan; Schubert, Irene; Mielenz, Dirk; Dietel, Barbara; Raaz-Schrauder, Dorette; Ay, Cihan; Thaler, Johannes; Heim, Christian; Collins, Peter W.; Schabbauer, Gernot; Mackman, Nigel; Voehringer, David; Nadler, Jerry L.; Lee, James J.; Massberg, Steffen; Rauh, Manfred; O’Donnell, Valerie B.

2017-01-01

Blood coagulation is essential for physiological hemostasis but simultaneously contributes to thrombotic disease. However, molecular and cellular events controlling initiation and propagation of coagulation are still incompletely understood. In this study, we demonstrate an unexpected role of eosinophils during plasmatic coagulation, hemostasis, and thrombosis. Using a large-scale epidemiological approach, we identified eosinophil cationic protein as an independent and predictive risk factor for thrombotic events in humans. Concurrent experiments showed that eosinophils contributed to intravascular thrombosis by exhibiting a strong endogenous thrombin-generation capacity that relied on the enzymatic generation and active provision of a procoagulant phospholipid surface enriched in 12/15-lipoxygenase–derived hydroxyeicosatetraenoic acid–phosphatidylethanolamines. Our findings reveal a previously unrecognized role of eosinophils and enzymatic lipid oxidation as regulatory elements that facilitate both hemostasis and thrombosis in response to vascular injury, thus identifying promising new targets for the treatment of thrombotic disease. PMID:28566277

Enzymatic Synthesis of Self-assembled Dicer Substrate RNA Nanostructures for Programmable Gene Silencing.

PubMed

Jang, Bora; Kim, Boyoung; Kim, Hyunsook; Kwon, Hyokyoung; Kim, Minjeong; Seo, Yunmi; Colas, Marion; Jeong, Hansaem; Jeong, Eun Hye; Lee, Kyuri; Lee, Hyukjin

2018-06-08

Enzymatic synthesis of RNA nanostructures is achieved by isothermal rolling circle transcription (RCT). Each arm of RNA nanostructures provides a functional role of Dicer substrate RNA inducing sequence specific RNA interference (RNAi). Three different RNAi sequences (GFP, RFP, and BFP) are incorporated within the three-arm junction RNA nanostructures (Y-RNA). The template and helper DNA strands are designed for the large-scale in vitro synthesis of RNA strands to prepare self-assembled Y-RNA. Interestingly, Dicer processing of Y-RNA is highly influenced by its physical structure and different gene silencing activity is achieved depending on its arm length and overhang. In addition, enzymatic synthesis allows the preparation of various Y-RNA structures using a single DNA template offering on demand regulation of multiple target genes.

Enzymatic membrane reactors for biodegradation of recalcitrant compounds. Application to dye decolourisation.

PubMed

López, C; Mielgo, I; Moreira, M T; Feijoo, G; Lema, J M

2002-11-13

Membrane bioreactors are being increasingly used in enzymatic catalysed transformations. However, the application of enzymatic-based treatment systems in the environmental field is rather unusual. The aim of this paper is to overview the application of enzymatic membrane reactors to wastewater treatment, more specifically to dye decolourisation. Firstly, the basic aspects such as different configurations of enzymatic reactors, advantages and disadvantages associated to their utilisation are revised as well as the application of this technology to wastewater treatment. Secondly, dye decolourisation by white-rot fungi and their oxidative enzymes are discussed, presenting an overall view from for in vivo and in vitro systems. Finally, dye decolourisation by manganese peroxidase in an enzymatic membrane reactor in continuous operation is presented.

Effect of micro-stirring on enzymatic reaction kinetics inside a biomimetic container

NASA Astrophysics Data System (ADS)

Gozen, Irep; Horowitz, Viva; Chambers, Zachary; Manoharan, Vinothan

The intracellular environment is dynamic, influenced by the motion of active machinery such as cytoskeleton filaments and molecular motors. To understand whether and how such activity affects the rates of diffusion-limited reactions, we construct a model system consisting of a phospholipid vesicle encapsulating a small number of micro- or nanoparticles, the active motion of which can be induced by chemical or magnetic cues. We aim to determine a relation between active motion of particles and rates of enzymatic reactions in the confined volume. Our findings might illuminate how active motion influences cytoplasmic reaction dynamics, or may have played a role in protocell genetics.

Overcoming bottlenecks of enzymatic biofuel cell cathodes: crude fungal culture supernatant can help to extend lifetime and reduce cost.

PubMed

Sané, Sabine; Jolivalt, Claude; Mittler, Gerhard; Nielsen, Peter J; Rubenwolf, Stefanie; Zengerle, Roland; Kerzenmacher, Sven

2013-07-01

Enzymatic biofuel cells (BFCs) show great potential for the direct conversion of biochemically stored energy from renewable biomass resources into electricity. However, enzyme purification is time-consuming and expensive. Furthermore, the long-term use of enzymatic BFCs is hindered by enzyme degradation, which limits their lifetime to only a few weeks. We show, for the first time, that crude culture supernatant from enzyme-secreting microorganisms (Trametes versicolor) can be used without further treatment to supply the enzyme laccase to the cathode of a mediatorless BFC. Polarization curves show that there is no significant difference in the cathode performance when using crude supernatant that contains laccase compared to purified laccase in culture medium or buffer solution. Furthermore, we demonstrate that the oxygen reduction activity of this enzymatic cathode can be sustained over a period of at least 120 days by periodic resupply of crude culture supernatant. This is more than five times longer than control cathodes without the resupply of culture supernatant. During the operation period of 120 days, no progressive loss of potential is observed, which suggests that significantly longer lifetimes than shown in this work may be possible. Our results demonstrate the possibility to establish simple, cost efficient, and mediatorless enzymatic BFC cathodes that do not require expensive enzyme purification procedures. Furthermore, they show the feasibility of an enzymatic BFC with an extended lifetime, in which self-replicating microorganisms provide the electrode with catalytically active enzymes in a continuous or periodic manner. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

A non-enzymatic function of 17β-hydroxysteroid dehydrogenase type 10 is required for mitochondrial integrity and cell survival

PubMed Central

Rauschenberger, Katharina; Schöler, Katja; Sass, Jörn Oliver; Sauer, Sven; Djuric, Zdenka; Rumig, Cordula; Wolf, Nicole I; Okun, Jürgen G; Kölker, Stefan; Schwarz, Heinz; Fischer, Christine; Grziwa, Beate; Runz, Heiko; Nümann, Astrid; Shafqat, Naeem; Kavanagh, Kathryn L; Hämmerling, Günter; Wanders, Ronald J A; Shield, Julian P H; Wendel, Udo; Stern, David; Nawroth, Peter; Hoffmann, Georg F; Bartram, Claus R; Arnold, Bernd; Bierhaus, Angelika; Oppermann, Udo; Steinbeisser, Herbert; Zschocke, Johannes

2010-01-01

Deficiency of the mitochondrial enzyme 2-methyl-3-hydroxybutyryl-CoA dehydrogenase involved in isoleucine metabolism causes an organic aciduria with atypical neurodegenerative course. The disease-causing gene is HSD17B10 and encodes 17β-hydroxysteroid dehydrogenase type 10 (HSD10), a protein also implicated in the pathogenesis of Alzheimer's disease. Here we show that clinical symptoms in patients are not correlated with residual enzymatic activity of mutated HSD10. Loss-of-function and rescue experiments in Xenopus embryos and cells derived from conditional Hsd17b10−/− mice demonstrate that a property of HSD10 independent of its enzymatic activity is essential for structural and functional integrity of mitochondria. Impairment of this function in neural cells causes apoptotic cell death whilst the enzymatic activity of HSD10 is not required for cell survival. This finding indicates that the symptoms in patients with mutations in the HSD17B10 gene are unrelated to accumulation of toxic metabolites in the isoleucine pathway and, rather, related to defects in general mitochondrial function. Therefore alternative therapeutic approaches to an isoleucine-restricted diet are required. PMID:20077426

Lack of induction of tissue transglutaminase but activation of the preexisting enzyme in c-Myc-induced apoptosis of CHO cells.

PubMed

Balajthy, Z; Kedei, N; Nagy, L; Davies, P J; Fésüs, L

1997-07-18

The intracellular activity and expression of tissue transglutaminase, which crosslinks proteins through epsilon(gamma-glutamyl)lysine isodipeptide bond, was investigated in CHO cells and those stably transfected with either inducible c-Myc (which leads to apoptosis) or with c-myc and the apoptosis inhibitor Bcl-2. Protein-bound cross-link content was significantly higher when apoptosis was induced by c-Myc while the concomitant presence of Bcl-2 markedly reduced both apoptosis and enzymatic protein cross-linking. The expression of tissue transglutaminase did not change following the initiation of apoptosis by c-Myc or when it was blocked by Bcl-2. Studying transiently co-transfected elements of the mouse tissue transglutaminase promoter linked to a reporter enzyme revealed their overall repression in cells expressing c-Myc. This repression was partially suspended in cells also carrying Bcl-2. Our data suggest that tissue transglutaminase is not induced when c-Myc initiates apoptosis but the pre-existing endogenous enzyme is activated.

Effects of vitamin E supplementation on renal non-enzymatic antioxidants in young rats submitted to exhaustive exercise stress.

PubMed

Bucioli, Sérvio A; de Abreu, Luiz Carlos; Valenti, Vitor E; Leone, Claudio; Vannucchi, Helio

2011-12-20

Exercise stress was shown to increase oxidative stress in rats. It lacks reports of increased protection afforded by dietary antioxidant supplements against ROS production during exercise stress. We evaluated the effects of vitamin E supplementation on renal non-enzymatic antioxidants in young rats submitted to exhaustive exercise stress. Wistar rats were divided into three groups: 1) control group; 2) exercise stress group and; 3) exercise stress + Vitamin E group. Rats from the group 3 were treated with gavage administration of 1 mL of Vitamin E (5 mg/kg) for seven consecutive days. Animals from groups 2 and 3 were submitted to a bout of swimming exhaustive exercise stress. Kidney samples were analyzed for Thiobarbituric Acid Reactive Substances to (TBARS) by malondialdehyde (MDA), reduced glutathione (GSH) and vitamin-E levels. The group treated with vitamin E and submitted to exercise stress presented the lowest levels of renal MDA (1: 0.16+0.02 mmmol/mgprot vs. 2: 0.34+0.07 mmmol/mgprot vs. 3: 0.1+0.01 mmmol/mgprot; p < 0.0001), the highest levels of renal GSH (1: 23+4 μmol/gprot vs. 2: 23+2 μmol/gprot vs. 3: 58+9 μmol/gprot; p < 0.0001) and the highest levels of renal vitamin E (1: 24+6 μM/gtissue vs. 2: 28+2 μM/gtissue vs. 3: 43+4 μM/gtissue; p < 0.001). Vitamin E supplementation improved non-enzymatic antioxidant activity in young rats submitted to exhaustive exercise stress.

Effects of vitamin E supplementation on renal non-enzymatic antioxidants in young rats submitted to exhaustive exercise stress

PubMed Central

2011-01-01

Background Exercise stress was shown to increase oxidative stress in rats. It lacks reports of increased protection afforded by dietary antioxidant supplements against ROS production during exercise stress. We evaluated the effects of vitamin E supplementation on renal non-enzymatic antioxidants in young rats submitted to exhaustive exercise stress. Methods Wistar rats were divided into three groups: 1) control group; 2) exercise stress group and; 3) exercise stress + Vitamin E group. Rats from the group 3 were treated with gavage administration of 1 mL of Vitamin E (5 mg/kg) for seven consecutive days. Animals from groups 2 and 3 were submitted to a bout of swimming exhaustive exercise stress. Kidney samples were analyzed for Thiobarbituric Acid Reactive Substances to (TBARS) by malondialdehyde (MDA), reduced glutathione (GSH) and vitamin-E levels. Results The group treated with vitamin E and submitted to exercise stress presented the lowest levels of renal MDA (1: 0.16+0.02 mmmol/mgprot vs. 2: 0.34+0.07 mmmol/mgprot vs. 3: 0.1+0.01 mmmol/mgprot; p < 0.0001), the highest levels of renal GSH (1: 23+4 μmol/gprot vs. 2: 23+2 μmol/gprot vs. 3: 58+9 μmol/gprot; p < 0.0001) and the highest levels of renal vitamin E (1: 24+6 μM/gtissue vs. 2: 28+2 μM/gtissue vs. 3: 43+4 μM/gtissue; p < 0.001). Conclusion Vitamin E supplementation improved non-enzymatic antioxidant activity in young rats submitted to exhaustive exercise stress. PMID:22185374

An air-breathing enzymatic cathode with extended lifetime by continuous laccase supply.

PubMed

Kipf, Elena; Sané, Sabine; Morse, Daniel; Messinger, Thorsten; Zengerle, Roland; Kerzenmacher, Sven

2018-04-22

We present a novel concept of an air-breathing enzymatic biofuel cell cathode combined with continuous supply of unpurified laccase-containing supernatant of the white-rot fungus Trametes versicolor for extended lifetime. The air-breathing cathode design obviates the need for energy-intensive active aeration. In a corresponding long-term experiment at a constant current density of 50 µA cm -2 , we demonstrated an increased lifetime of 33 days (cathode potential above 0.430 V vs. SCE), independent of enzyme degradation. The obtained data suggest that theoretically a longer lifetime is feasible. However, further engineering efforts are required to prevent clogging and fouling of the supply tubes. These results represent an important step towards the realization of enzymatic biofuel cell cathodes with extended lifetime and enhanced performance. Copyright © 2018 Elsevier Ltd. All rights reserved.

Molecular and enzymatic characterization of two enzymes BmPCD and BmDHPR involving in the regeneration pathway of tetrahydrobiopterin from the silkworm Bombyx mori.

PubMed

Li, Wentian; Gong, Meixia; Shu, Rui; Li, Xin; Gao, Junshan; Meng, Yan

2015-08-01

Tetrahydrobiopterin (BH4) is an essential cofactor of aromatic amino acid hydroxylases and nitric oxide synthase so that BH4 plays a key role in many biological processes. BH4 deficiency is associated with numerous metabolic syndromes and neuropsychological disorders. BH4 concentration in mammals is maintained through a de novo synthesis pathway and a regeneration pathway. Previous studies showed that the de novo pathway of BH4 is similar between insects and mammals. However, knowledge about the regeneration pathway of BH4 (RPB) is very limited in insects. Several mutants in the silkworm Bombyx mori have been approved to be associated with BH4 deficiency, which are good models to research on the RPB in insects. In this study, homologous genes encoding two enzymes, pterin-4a-carbinolamine dehydratase (PCD) and dihydropteridine reductase (DHPR) involving in RPB have been cloned and identified from B. mori. Enzymatic activity of DHPR was found in the fat body of wild type silkworm larvae. Together with the transcription profiles, it was indicated that BmPcd and BmDhpr might normally act in the RPB of B. mori and the expression of BmDhpr was activated in the brain and sexual glands while BmPcd was expressed in a wider special pattern when the de novo pathway of BH4 was lacked in lemon. Biochemical analyses showed that the recombinant BmDHPR exhibited high enzymatic activity and more suitable parameters to the coenzyme of NADH in vitro. The results in this report give new information about the RPB in B. mori and help in better understanding insect BH4 biosynthetic networks. Copyright © 2015 Elsevier Inc. All rights reserved.

Structural Elucidation of Enzymatically Synthesized Galacto-oligosaccharides Using Ion-Mobility Spectrometry-Tandem Mass Spectrometry.

PubMed

Carević, Milica; Bezbradica, Dejan; Banjanac, Katarina; Milivojević, Ana; Fanuel, Mathieu; Rogniaux, Hélène; Ropartz, David; Veličković, Dušan

2016-05-11

Galacto-oligosaccharides (GOS) represent a diverse group of well-characterized prebiotic ingredients derived from lactose in a reaction catalyzed with β-galactosidases. Enzymatic transgalactosylation results in a mixture of compounds of various degrees of polymerization and types of linkages. Because structure plays an important role in terms of prebiotic activity, it is of crucial importance to provide an insight into the mechanism of transgalactosylation reaction and occurrence of different types of β-linkages during GOS synthesis. Our study proved that a novel one-step method, based on ion-mobility spectrometry-tandem mass spectrometry (IMS-MS/MS), enables complete elucidation of GOS structure. It has been shown that β-galactosidase from Aspergillus oryzae has the highest affinity toward formation of β-(1→3) or β-(1→6) linkages. Additionally, it was observed that the occurrence of different linkages varies during the reaction course, indicating that tailoring favorable GOS structures with improved prebiotic activity can be achieved by adequate control of enzymatic synthesis.

The Differential Gibbs Free Energy of Activation and its Implications in the Transition-State of Enzymatic Reactions

NASA Astrophysics Data System (ADS)

Maggi, F.; Riley, W. J.

2016-12-01

We propose a mathematical framework to introduce the concept of differential free energy of activation in enzymatically catalyzed reactions, and apply it to N uptake by microalgae and bacteria. This framework extends the thermodynamic capabilities of the classical transition-state theory in and harmonizes the consolidated definitions of kinetic parameters with their thermodynamic and physical meaning. Here, the activation energy is assumed to be a necessary energetic level for equilibrium complexation between reactants and activated complex; however, an additional energy contribution is required for the equilibrium activated complex to release reaction products. We call this "differential free energy of activation"; it can be described by a Boltzmann distribution, and corresponds to a free energy level different from that of complexation. Whether this level is above or below the free energy of activation depends on the reaction, and defines energy domains that correspond to "superactivated", "activated", and "subactivated" complexes. The activated complex reaching one of those states will eventually release the products from an energy level different than that of activation. The concept of differential free energy of activation was tested on 57 independent experiments of NH­4+ and NO3- uptake by various microalgae and bacteria at temperatures ranging between 1 and 45oC. Results showed that the complexation equilibrium always favored the activated complex, but the differential energy of activation led to an apparent energy barrier consistent with observations. Temperature affected all energy levels within this framework but did not alter substantially these thermodynamic features. Overall the approach: (1) provides a thermodynamic and mathematical link between Michaelis-Menten and rate constants; (2) shows that both kinetic parameters can be described or approximated by Arrhenius' like equations; (3) describes the likelihood of formation of sub-, super-, and

Enzymatic Transition States, Transition-State Analogs, Dynamics, Thermodynamics, and Lifetimes

PubMed Central

Schramm, Vern L.

2017-01-01

Experimental analysis of enzymatic transition-state structures uses kinetic isotope effects (KIEs) to report on bonding and geometry differences between reactants and the transition state. Computational correlation of experimental values with chemical models permits three-dimensional geometric and electrostatic assignment of transition states formed at enzymatic catalytic sites. The combination of experimental and computational access to transition-state information permits (a) the design of transition-state analogs as powerful enzymatic inhibitors, (b) exploration of protein features linked to transition-state structure, (c) analysis of ensemble atomic motions involved in achieving the transition state, (d) transition-state lifetimes, and (e) separation of ground-state (Michaelis complexes) from transition-state effects. Transition-state analogs with picomolar dissociation constants have been achieved for several enzymatic targets. Transition states of closely related isozymes indicate that the protein’s dynamic architecture is linked to transition-state structure. Fast dynamic motions in catalytic sites are linked to transition-state generation. Enzymatic transition states have lifetimes of femtoseconds, the lifetime of bond vibrations. Binding isotope effects (BIEs) reveal relative reactant and transition-state analog binding distortion for comparison with actual transition states. PMID:21675920

The Enzymatic Oxidation of Graphene Oxide

PubMed Central

Kotchey, Gregg P.; Allen, Brett L.; Vedala, Harindra; Yanamala, Naveena; Kapralov, Alexander A.; Tyurina, Yulia Y.; Klein-Seetharaman, Judith; Kagan, Valerian E.; Star, Alexander

2011-01-01

Two-dimensional graphitic carbon is a new material with many emerging applications, and studying its chemical properties is an important goal. Here, we reported a new phenomenon – the enzymatic oxidation of a single layer of graphitic carbon by horseradish peroxidase (HRP). In the presence of low concentrations of hydrogen peroxide (~40 µM), HRP catalyzed the oxidation of graphene oxide, which resulted in the formation of holes on its basal plane. During the same period of analysis, HRP failed to oxidize chemically reduced graphene oxide (RGO). The enzymatic oxidation was characterized by Raman, UV-Vis, EPR and FT-IR spectroscopy, TEM, AFM, SDS-PAGE, and GC-MS. Computational docking studies indicated that HRP was preferentially bound to the basal plane rather than the edge for both graphene oxide and RGO. Due to the more dynamic nature of HRP on graphene oxide, the heme active site of HRP was in closer proximity to graphene oxide compared to RGO, thereby facilitating the oxidation of the basal plane of graphene oxide. We also studied the electronic properties of the reduced intermediate product, holey reduced graphene oxide (hRGO), using field-effect transistor (FET) measurements. While RGO exhibited a V-shaped transfer characteristic similar to a single layer of graphene that was attributed to its zero band gap, hRGO demonstrated a p-type semiconducting behavior with a positive shift in the Dirac points. This p-type behavior rendered hRGO, which can be conceptualized as interconnected graphene nanoribbons, as a potentially attractive material for FET sensors. PMID:21344859

Enzymatic sulfation of tocopherols and tocopherol metabolites by human cytosolic sulfotransferases.

PubMed

Hashiguchi, Takuyu; Kurogi, Katsuhisa; Sakakibara, Yoichi; Yamasaki, Masao; Nishiyama, Kazuo; Yasuda, Shin; Liu, Ming-Cheh; Suiko, Masahito

2011-01-01

Tocopherols are essential micronutrients for mammals widely known as potent lipid-soluble antioxidants that are present in cell membranes. Recent studies have demonstrated that most of the carboxychromanol (CEHC), a tocopherol metabolite, in the plasma exists primarily in sulfate- and glucuronide-conjugated forms. To gain insight into the enzymatic sulfation of tocopherols and their metabolites, a systematic investigation was performed using all 14 known human cytosolic sulfotransferases (SULTs). The results showed that the members of the SULT1 family displayed stronger sulfating activities toward tocopherols and their metabolites. These enzymes showed a substrate preference for γ-tocopherol over α-tocopherol and for γ-CEHC over other CEHCs. Using A549 human lung epithelial cells in a metabolic labeling study, a similar trend in the sulfation of tocopherols and CEHCs was observed. Collectively, the results obtained indicate that SULT-mediated enzymatic sulfation of tocopherols and their metabolites is a significant pathway for regulation of the homeostasis and physiological functions of these important compounds.

Three-dimensional Structure and Enzymatic Function of Proapoptotic Human p53-inducible Quinone Oxidoreductase PIG3*

PubMed Central

Porté, Sergio; Valencia, Eva; Yakovtseva, Evgenia A.; Borràs, Emma; Shafqat, Naeem; Debreczeny, Judit É.; Pike, Ashley C. W.; Oppermann, Udo; Farrés, Jaume; Fita, Ignacio; Parés, Xavier

2009-01-01

Tumor suppressor p53 regulates the expression of p53-induced genes (PIG) that trigger apoptosis. PIG3 or TP53I3 is the only known member of the medium chain dehydrogenase/reductase superfamily induced by p53 and is used as a proapoptotic marker. Although the participation of PIG3 in the apoptotic pathway is proven, the protein and its mechanism of action were never characterized. We analyzed human PIG3 enzymatic function and found NADPH-dependent reductase activity with ortho-quinones, which is consistent with the classification of PIG3 in the quinone oxidoreductase family. However, the activity is much lower than that of ζ-crystallin, a better known quinone oxidoreductase. In addition, we report the crystallographic structure of PIG3, which allowed the identification of substrate- and cofactor-binding sites, with residues fully conserved from bacteria to human. Tyr-59 in ζ-crystallin (Tyr-51 in PIG3) was suggested to participate in the catalysis of quinone reduction. However, kinetics of Tyr/Phe and Tyr/Ala mutants of both enzymes demonstrated that the active site Tyr is not catalytic but may participate in substrate binding, consistent with a mechanism based on propinquity effects. It has been proposed that PIG3 contribution to apoptosis would be through oxidative stress generation. We found that in vitro activity and in vivo overexpression of PIG3 accumulate reactive oxygen species. Accordingly, an inactive PIG3 mutant (S151V) did not produce reactive oxygen species in cells, indicating that enzymatically active protein is necessary for this function. This supports that PIG3 action is through oxidative stress produced by its enzymatic activity and provides essential knowledge for eventual control of apoptosis. PMID:19349281

Quantification of ricin, RCA and comparison of enzymatic activity in 18 Ricinus communis cultivars by isotope dilution mass spectrometry.

PubMed

Schieltz, David M; McWilliams, Lisa G; Kuklenyik, Zsuzsanna; Prezioso, Samantha M; Carter, Andrew J; Williamson, Yulanda M; McGrath, Sara C; Morse, Stephen A; Barr, John R

2015-03-01

The seeds of the Ricinus communis (Castor bean) plant are the source of the economically important commodity castor oil. Castor seeds also contain the proteins ricin and R. communis agglutinin (RCA), two toxic lectins that are hazardous to human health. Radial immunodiffusion (RID) and the enzyme linked immunosorbent assay (ELISA) are two antibody-based methods commonly used to quantify ricin and RCA; however, antibodies currently used in these methods cannot distinguish between ricin and RCA due to the high sequence homology of the respective proteins. In this study, a technique combining antibody-based affinity capture with liquid chromatography and multiple reaction monitoring (MRM) mass spectrometry (MS) was used to quantify the amounts of ricin and RCA independently in extracts prepared from the seeds of eighteen representative cultivars of R. communis which were propagated under identical conditions. Additionally, liquid chromatography and MRM-MS was used to determine rRNA N-glycosidase activity for each cultivar and the overall activity in these cultivars was compared to a purified ricin standard. Of the cultivars studied, the average ricin content was 9.3 mg/g seed, the average RCA content was 9.9 mg/g seed, and the enzymatic activity agreed with the activity of a purified ricin reference within 35% relative activity. Published by Elsevier Ltd.

Recalcitrant carbohydrates after enzymatic hydrolysis of pretreated lignocellulosic biomass

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

Alcantara, Maria Angeles Bermudez; Dobruchowska, Justyna; Azadi, Parastoo

To reduce the cost of the enzymes for the hydrolysis of lignocellulosic biomass, two main strategies have been followed: one, the reduction of enzyme dosing by the use of more efficient and stable enzymatic cocktails; another, to include accessory enzymes in the cocktails to increase yields by reducing the recalcitrant carbohydrate fraction remaining at the end of the process. To guide this second strategy, we have explored the chemical bond composition of different fractions of recalcitrant carbohydrates after enzymatic hydrolysis. As a result, two lignocellulosic feedstocks of relevance for the biofuels industry have been analyzed, corn stover and sugarcane straw.more » On comparing the composition of chemical bonds of the starting pretreated material with samples after standard and forced hydrolysis (with enzyme overdosing), we obtained similar sugar and chemical bond composition. In conclusion, this suggests that the current enzymatic cocktails bear the set of enzymes needed to hydrolyze these feedstocks. From our point of view, the results show the need for a parallel fine-tuning of the enzymatic cocktails with the pretreatment process to maximize sugar release yield.« less

Recalcitrant carbohydrates after enzymatic hydrolysis of pretreated lignocellulosic biomass

DOE PAGES

Alcantara, Maria Angeles Bermudez; Dobruchowska, Justyna; Azadi, Parastoo; ...

2016-10-06

To reduce the cost of the enzymes for the hydrolysis of lignocellulosic biomass, two main strategies have been followed: one, the reduction of enzyme dosing by the use of more efficient and stable enzymatic cocktails; another, to include accessory enzymes in the cocktails to increase yields by reducing the recalcitrant carbohydrate fraction remaining at the end of the process. To guide this second strategy, we have explored the chemical bond composition of different fractions of recalcitrant carbohydrates after enzymatic hydrolysis. As a result, two lignocellulosic feedstocks of relevance for the biofuels industry have been analyzed, corn stover and sugarcane straw.more » On comparing the composition of chemical bonds of the starting pretreated material with samples after standard and forced hydrolysis (with enzyme overdosing), we obtained similar sugar and chemical bond composition. In conclusion, this suggests that the current enzymatic cocktails bear the set of enzymes needed to hydrolyze these feedstocks. From our point of view, the results show the need for a parallel fine-tuning of the enzymatic cocktails with the pretreatment process to maximize sugar release yield.« less

Recalcitrant carbohydrates after enzymatic hydrolysis of pretreated lignocellulosic biomass.

PubMed

Alcántara, María Ángeles Bermúdez; Dobruchowska, Justyna; Azadi, Parastoo; García, Bruno Díez; Molina-Heredia, Fernando P; Reyes-Sosa, Francisco Manuel

2016-01-01

To reduce the cost of the enzymes for the hydrolysis of lignocellulosic biomass, two main strategies have been followed: one, the reduction of enzyme dosing by the use of more efficient and stable enzymatic cocktails; another, to include accessory enzymes in the cocktails to increase yields by reducing the recalcitrant carbohydrate fraction remaining at the end of the process. To guide this second strategy, we have explored the chemical bond composition of different fractions of recalcitrant carbohydrates after enzymatic hydrolysis. Two lignocellulosic feedstocks of relevance for the biofuels industry have been analyzed, corn stover and sugarcane straw. On comparing the composition of chemical bonds of the starting pretreated material with samples after standard and forced hydrolysis (with enzyme overdosing), we obtained similar sugar and chemical bond composition. This suggests that the current enzymatic cocktails bear the set of enzymes needed to hydrolyze these feedstocks. From our point of view, the results show the need for a parallel fine-tuning of the enzymatic cocktails with the pretreatment process to maximize sugar release yield.

Modelling Tethered Enzymatic Reactions

NASA Astrophysics Data System (ADS)

Solis Salas, Citlali; Goyette, Jesse; Coker-Gordon, Nicola; Bridge, Marcus; Isaacson, Samuel; Allard, Jun; Maini, Philip; Dushek, Omer

Enzymatic reactions are key to cell functioning, and whilst much work has been done in protein interaction in cases where diffusion is possible, interactions of tethered proteins are poorly understood. Yet, because of the large role cell membranes play in enzymatic reactions, several reactions may take place where one of the proteins is bound to a fixed point in space. We develop a model to characterize tethered signalling between the phosphatase SHP-1 interacting with a tethered, phosphorylated protein. We compare our model to experimental data obtained using surface plasmon resonance (SPR). We show that a single SPR experiment recovers 5 independent biophysical/biochemical constants. We also compare the results between a three dimensional model and a two dimensional model. The work gives the opportunity to use known techniques to learn more about signalling processes, and new insights into how enzyme tethering alters cellular signalling. With support from the Mexican Council for Science and Technology (CONACyT), the Public Education Secretariat (SEP), and the Mexican National Autonomous University's Foundation (Fundacion UNAM).

Production of MAG via enzymatic glycerolysis

NASA Astrophysics Data System (ADS)

Jamlus, Norul Naziraa Ahmad; Derawi, Darfizzi; Salimon, Jumat

2015-09-01

Enzymatic glycerolysis of a medium chain methyl ester, methyl laurate was performed using lipase Candida antarctica (Novozyme 435) for 6 hours at 55°C. The percentage of components mixture of product were determined by using gas chromatography technique. The enzymatic reaction was successfully produced monolaurin (45.9 %), dilaurin (47.1 %) and trilaurin (7.0 %) respectively. Thin layer chromatography (TLC) plate also showed a good separation of component spots. Fourier transformation infra-red (FTIR) spectrum showed the presence of ester carbonyl at wavenumber 1739.99 cm-1 and hydrogen bonded O-H at 3512.03 cm-1. The product is potentially to be used as emulsifier and additive in food industry, pharmaceutical, as well as antibacterial.

Longitudinal Changes in PON1 Enzymatic Activities in Mexican-American Mothers and Children with Different Genotypes and Haplotypes

PubMed Central

Huen, Karen; Harley, Kim; Bradman, Asa; Eskenazi, Brenda; Holland, Nina

2010-01-01

The paraoxonase 1 (PON1) enzyme prevents low density lipoprotein oxidation and also detoxifies the oxon derivatives of certain neurotoxic organophosphate (OP) pesticides. PON1 activity in infants is low compared to adults, rendering them with lower metabolic and antioxidant capacities. We made a longitudinal comparison of the role of genetic variability on control of PON1 phenotypes in Mexican-American mothers and their children at the time of delivery (n=388 and 338, respectively) and again seven years later (n=280 and 281, respectively) using generalized estimating equations models. At age seven, children’s mean PON1 activities were still lower than those of mothers. This difference was larger in children with genotypes associated with low PON1 activities (PON1−108TT, PON1192QQ, and PON1−909CC). In mothers, PON1 activities were elevated at delivery and during pregnancy compared to seven years later when they were not pregnant (p<0.001). In non-pregnant mothers, PON1 polymorphisms and haplotypes accounted for almost 2-fold more variation of arylesterase (AREase) and chlorpyrifos-oxonase (CPOase) activity than in mothers at delivery. In both mothers and children, the five PON1 polymorphisms (192, 55, −108, −909, −162) explained a noticeably larger proportion of variance of paraoxonase activity (62–78%) than AREase activity (12.3–26.6%). Genetic control of PON1 enzymatic activity varies in children compared to adults and is also affected by pregnancy status. In addition to known PON1 polymorphisms, unidentified environmental, genetic, or epigenetic factors may also influence variability of PON1 expression and therefore susceptibility to OPs and oxidative stress. PMID:20045427

Lack of mitochondrial thioredoxin o1 is compensated by antioxidant components under salinity in Arabidopsis thaliana plants.

PubMed

Calderón, Aingeru; Sánchez-Guerrero, Antonio; Ortiz-Espín, Ana; Martínez-Alcalá, Isabel; Camejo, Daymi; Jiménez, Ana; Sevilla, Francisca

2018-02-15

In a changing environment, plants are able to acclimate to the new conditions by regulating their metabolism through the antioxidant and redox systems involved in the stress response. Here we studied a mitochondrial thioredoxin in wild type (WT) Arabidopis thaliana and two Attrxo1 mutant lines grown in the absence or presence of 100 mM NaCl. Compared to WT plants, no evident phenotype was observed in the mutant plants in control condition, although they had higher number of stomata, loss of water, nitric oxide and carbonyl protein contents as well as higher activity of superoxide dismutase (SOD) and catalase enzymes than WT plants. Under salinity, the mutants presented lower water loss and higher stomatal closure, H 2 O 2 and lipid peroxidation levels accompanied by higher enzymatic activity of catalase and the different SOD isoenzymes compared to WT plants. These inductions may collaborate in the maintenance of plant integrity and growth observed under saline conditions, possibly as a way to compensate the lack of TRXo1. We discuss the potential of TRXo1 to influence the development of the whole plant under saline conditions, which have great value for the agronomy of plants growing under unfavourable environment. This article is protected by copyright. All rights reserved.

Lignosulfonate and elevated pH can enhance enzymatic saccharification of lignocelluloses

PubMed Central

2013-01-01

Background Nonspecific (nonproductive) binding (adsorption) of cellulase by lignin has been identified as a key barrier to reduce cellulase loading for economical sugar and biofuel production from lignocellulosic biomass. Sulfite Pretreatment to Overcome Recalcitrance of Lignocelluloses (SPORL) is a relatively new process, but demonstrated robust performance for sugar and biofuel production from woody biomass especially softwoods in terms of yields and energy efficiencies. This study demonstrated the role of lignin sulfonation in enhancing enzymatic saccharification of lignocelluloses – lignosulfonate from SPORL can improve enzymatic hydrolysis of lignocelluloses, contrary to the conventional belief that lignin inhibits enzymatic hydrolysis due to nonspecific binding of cellulase. Results The study found that lignosulfonate from SPORL pretreatment and from a commercial source inhibits enzymatic hydrolysis of pure cellulosic substrates at low concentrations due to nonspecific binding of cellulase. Surprisingly, the reduction in enzymatic saccharification efficiency of a lignocellulosic substrate was fully recovered as the concentrations of these two lignosulfonates increased. We hypothesize that lignosulfonate serves as a surfactant to enhance enzymatic hydrolysis at higher concentrations and that this enhancement offsets its inhibitive effect from nonspecific binding of cellulase, when lignosulfonate is applied to lignocellulosic solid substrates. Lignosulfonate can block nonspecific binding of cellulase by bound lignin on the solid substrates, in the same manner as a nonionic surfactant, to significantly enhance enzymatic saccharification. This enhancement is linearly proportional to the amount of lignosulfonate applied which is very important to practical applications. For a SPORL-pretreated lodgepole pine solid, 90% cellulose saccharification was achieved at cellulase loading of 13 FPU/g glucan with the application of its corresponding pretreatment hydrolysate

Bridging Enzymatic Structure Function via Mechanics: A Coarse-Grain Approach.

PubMed

Sacquin-Mora, S

2016-01-01

Flexibility is a central aspect of protein function, and ligand binding in enzymes involves a wide range of structural changes, ranging from large-scale domain movements to small loop or side-chain rearrangements. In order to understand how the mechanical properties of enzymes, and the mechanical variations that are induced by ligand binding, relate to enzymatic activity, we carried out coarse-grain Brownian dynamics simulations on a set of enzymes whose structures in the unbound and ligand-bound forms are available in the Protein Data Bank. Our results show that enzymes are remarkably heterogeneous objects from a mechanical point of view and that the local rigidity of individual residues is tightly connected to their part in the protein's overall structure and function. The systematic comparison of the rigidity of enzymes in their unbound and bound forms highlights the fact that small conformational changes can induce large mechanical effects, leading to either more or less flexibility depending on the enzyme's architecture and the location of its ligand-biding site. These mechanical variations target a limited number of specific residues that occupy key locations for enzymatic activity, and our approach thus offers a mean to detect perturbation-sensitive sites in enzymes, where the addition or removal of a few interactions will lead to important changes in the proteins internal dynamics. © 2016 Elsevier Inc. All rights reserved.

Surface plasmon aided high sensitive non-enzymatic glucose sensor using Au/NiAu multilayered nanowire arrays.

PubMed

Wang, Lanfang; Zhu, Weiqi; Lu, Wenbo; Qin, Xiufang; Xu, Xiaohong

2018-07-15

A novel plasmon aided non-enzymatic glucose sensor was first constructed based on the unique half-rough Au/NiAu multilayered nanowire arrays. These multilayered and half-rough nanowires provide high chemical activity and large surface area for glucose oxidation in an alkaline solution. Under visible light irradiation, the surface plasmons originated from Au part enhance the electron transfer in the vertically aligned nanowires, leading to high sensitivity and wide detection range. The resulting sensor exhibits a wide glucose detection concentration range, low detection limit, and high sensitivity for plasmon aided non-enzymatic glucose sensor. Moreover, the detection sensitivity is enhanced by almost 2 folds compared to that in the dark, which significantly enhanced the performance of Au/NiAu multilayered nanowire arrays sensor. An excellent selectivity and acceptable stability were also achieved. These results indicate that surface plasmon aided nanostructures are promising new platforms for the construction of non-enzymatic glucose sensors. Copyright © 2018 Elsevier B.V. All rights reserved.

Fentanyl-related designer drugs W-18 and W-15 lack appreciable opioid activity in vitro and in vivo.

PubMed

Huang, Xi-Ping; Che, Tao; Mangano, Thomas J; Le Rouzic, Valerie; Pan, Ying-Xian; Majumdar, Susruta; Cameron, Michael D; Baumann, Michael H; Pasternak, Gavril W; Roth, Bryan L

2017-11-16

W-18 (4-chloro-N-[1-[2-(4-nitrophenyl)ethyl]-2-piperidinylidene]-benzenesulfonamide) and W-15 (4-chloro-N-[1-(2-phenylethyl)-2-piperidinylidene]-benzenesulfonamide) represent two emerging drugs of abuse chemically related to the potent opioid agonist fentanyl (N-(1-(2-phenylethyl)-4-piperidinyl)-N-phenylpropanamide). Here, we describe the comprehensive pharmacological profiles of W-18 and W-15, as examination of their structural features predicted that they might lack opioid activity. We found W-18 and W-15 to be without detectible activity at μ, δ, κ, and nociception opioid receptors in a variety of assays. We also tested W-18 and W-15 for activity as allosteric modulators at opioid receptors and found them devoid of significant positive or negative allosteric modulatory activity. Comprehensive profiling at essentially all the druggable GPCRs in the human genome using the PRESTO-Tango platform revealed no significant activity. Weak activity at the sigma receptors and the peripheral benzodiazepine receptor was found for W-18 (Ki = 271 nM). W-18 showed no activity in either the radiant heat tail-flick or the writhing assays and also did not induce classical opioid behaviors. W-18 is extensively metabolized, but its metabolites also lack opioid activity. Thus, although W-18 and W-15 have been suggested to be potent opioid agonists, our results reveal no significant activity at these or other known targets for psychoactive drugs.

Enzymatic transformation of nonfood biomass to starch

PubMed Central

You, Chun; Chen, Hongge; Myung, Suwan; Sathitsuksanoh, Noppadon; Ma, Hui; Zhang, Xiao-Zhou; Li, Jianyong; Zhang, Y.-H. Percival

2013-01-01

The global demand for food could double in another 40 y owing to growth in the population and food consumption per capita. To meet the world’s future food and sustainability needs for biofuels and renewable materials, the production of starch-rich cereals and cellulose-rich bioenergy plants must grow substantially while minimizing agriculture’s environmental footprint and conserving biodiversity. Here we demonstrate one-pot enzymatic conversion of pretreated biomass to starch through a nonnatural synthetic enzymatic pathway composed of endoglucanase, cellobiohydrolyase, cellobiose phosphorylase, and alpha-glucan phosphorylase originating from bacterial, fungal, and plant sources. A special polypeptide cap in potato alpha-glucan phosphorylase was essential to push a partially hydrolyzed intermediate of cellulose forward to the synthesis of amylose. Up to 30% of the anhydroglucose units in cellulose were converted to starch; the remaining cellulose was hydrolyzed to glucose suitable for ethanol production by yeast in the same bioreactor. Next-generation biorefineries based on simultaneous enzymatic biotransformation and microbial fermentation could address the food, biofuels, and environment trilemma. PMID:23589840

Isomerization of 1-O-indol-3-ylacetyl-beta-D-glucose. Enzymatic hydrolysis of 1-O, 4-O, and 6-O-indol-3-ylacetyl-beta-D-glucose and the enzymatic synthesis of indole-3-acetyl glycerol by a hormone metabolizing complex

NASA Technical Reports Server (NTRS)

Kowalczyk, S.; Bandurski, R. S.

1990-01-01

The first compound in the series of reactions leading to the ester conjugates of indole-3-acetic acid (IAA) in kernels of Zea mays sweet corn is the acyl alkyl acetal, 1-O-indol-3-ylacetyl-beta-D-glucose (1-O-IAGlu). The enzyme catalyzing the synthesis of this compound is UDP-glucose:indol-3-ylacetate glucosyl-transferase (IAGlu synthase). The IAA moiety of the high energy compound 1-O-IAGlu may be enzymatically transferred to myo-inositol or to glycerol or the 1-O-IAGlu may be enzymatically hydrolyzed. Alternatively, nonenzymatic acyl migration may occur to yield the 2-O, 4-O, and 6-O esters of IAA and glucose. The 4-O and 6-O esters may then be enzymatically hydrolyzed to yield free IAA and glucose. This work reports new enzymatic activities, the transfer of IAA from 1-O-IAGlu to glycerol, and the enzyme-catalyzed hydrolysis of 4-O and 6-O-IAGlu. Data is also presented on the rate of non-enzymatic acyl migration of IAA from the 1-O to the 4-O and 6-O positions of glucose. We also report that enzymes catalyzing the synthesis of 1-O-IAGlu and the hydrolysis of 1-O, 4-O, and 6-O-IAGlu fractionate as a hormone metabolizing complex. The association of synthetic and hydrolytic capabilities in enzymes which cofractionate may have physiological significance.

Construction of graphene oxide magnetic nanocomposites-based on-chip enzymatic microreactor for ultrasensitive pesticide detection.

PubMed

Liang, Ru-Ping; Wang, Xiao-Ni; Liu, Chun-Ming; Meng, Xiang-Ying; Qiu, Jian-Ding

2013-11-08

A new strategy for facile construction of graphene oxide magnetic nanocomposites (GO/Fe3O4 MNCs)-based on-chip enzymatic microreactor and ultrasensitive pesticide detection has been proposed. GO/Fe3O4 MNCs were first prepared through an in situ chemical deposition strategy. Then, acetylcholinesterase (AChE) was adsorbed onto the GO/Fe3O4 surface to form GO/Fe3O4/AChE MNCs which was locally packed into PDMS microchannel simply with the help of external magnetic field to form an on-chip enzymatic microreactor. The constructed GO/Fe3O4/AChE MNCs-based enzymatic microreactor not only have the magnetism of Fe3O4 NPs that make them conveniently manipulated by an external magnetic field, but also have the larger surface and excellent biocompatibility of graphene which can incorporate much more AChE molecules and well maintain their biological activity. On the basis of the AChE inhibition principle, a novel on-chip enzymatic microreactor was constructed for analyzing dimethoate which is usually used as a model of organophosphorus pesticides. Under optimal conditions, a linear relationship between the inhibition rates of AChE and the concentration of dimethoate from 1 to 20 μgL(-1) with a detection limit of 0.18 μgL(-1) (S/N=3) was obtained. The developed electrophoretic and magnetic-based chip exhibited excellent reproducibility and stability with no decrease in the activity of enzyme for more than 20 repeated measurements over one week period, which provided a new and promising tool for the analysis of enzyme inhibitors with low cost and excellent performance. Copyright © 2013 Elsevier B.V. All rights reserved.

Hormonal enzymatic systems in normal and cancerous human breast: control, prognostic factors, and clinical applications.

PubMed

Pasqualini, Jorge R; Chetrite, Gérard S

2012-04-01

The bioformation and transformation of estrogens and other hormones in the breast tissue as a result of the activity of the various enzymes involved attract particular attention for the role they play in the development and pathogenesis of hormone-dependent breast cancer. The enzymatic process concerns the aromatase, which transforms androgens into estrogens; the sulfatase, which hydrolyzes the biologically inactive sulfates to the active hormone; the 17β-hydroxysteroid dehydrogenases, which are involved in the interconversion estradiol/estrone or testosterone/androstenedione; hydroxylases, which transform estrogens into mitotic and antimitotic derivatives; and sulfotransferases and glucuronidases, which, respectively convert into the biologically inactive sulfates and glucuronides. These enzymatic activities are more intense in the carcinoma than in the normal tissue. Concerning aromatase, the application of antiaromatase agents has been largely developed in the treatment of breast cancer patients, with very positive results. Various studies have shown that the activity levels of these enzymes and their mRNA can be involved as interesting prognostic factors for breast cancer. In conclusion, the application of new antienzymatic molecules can open attractive perspectives in the treatment of hormone-dependent breast cancer.

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.

Bioluminescence methods for enzymatic determinations

DOEpatents

Bostick, William D.; Denton, Mark S.; Dinsmore, Stanley R.

1982-01-01

An enzymatic method for continuous, on-line and rapid detection of diagnostically useful biomarkers, which are symptomatic of disease or trauma-related tissue damage, is disclosed. The method is characterized by operability on authentic samples of complex biological fluids which contain the biomarkers.

Non-enzymatic interactions of glyoxylate with lysine, arginine, and glucosamine: a study of advanced non-enzymatic glycation like compounds.

PubMed

Dutta, Udayan; Cohenford, Menashi A; Guha, Madhumita; Dain, Joel A

2007-02-01

Glyoxylate is a 2 carbon aldo acid that is formed in hepatic tissue from glycolate. Once formed, the molecule can be converted to glycine by alanine-glyoxylate aminotransferase (AGAT). In defects of AGAT, glyoxylate is transformed to oxalate, resulting in high levels of oxalate in the body. The objective of this study was 2-fold. First, it was to determine, if akin to D-glucose, D-fructose or DL-glyceraldehyde, glyoxylate was susceptible to non-enzymatic attack by amino containing molecules such as lysine, arginine or glucosamine. Second, if by virtue of its molecular structure and size, glyoxylate was as reactive a reagent in non-enzymatic reactions as DL-glyceraldehyde; i.e., a glycose that we previously demonstrated to be a more effective glycating agent than D-glucose or D-fructose. Using capillary electrophoresis (CE), high performance liquid chromatography and UV and fluorescence spectroscopy, glyoxylate was found to be a highly reactive precursor of advanced glycation like end products (AGLEs) and a more effective promoter of non-enzymatic end products than D-glucose, D-fructose or DL-glyceraldehyde.

Effects of treated industrial wastewaters and temperatures on growth and enzymatic activities of duckweed (Lemna minor L.).

PubMed

Basiglini, E; Pintore, M; Forni, C

2018-05-30

The efficacy of the removal of contaminants from wastewater depends on physico-chemical properties of pollutants and the efficiency of treatment plant. Sometimes, low amounts of toxic compounds can be still present in the treated sewage. In this work we considered the effects of contaminant residues in treated wastewaters and of temperatures on Lemna minor L. Treated effluent waters were collected, analyzed and used as duckweed growth medium. In order to better understand the effects of micropollutants and seasonal variation, the plants were grown under ambient conditions for seven days in summer and winter. Relative growth rate, pigments and phenolic compounds concentrations were determined, as well as the activities of catalase (CAT), ascorbate peroxidase (APX), guaiacol peroxidase (G-POD) and polyphenol oxidase (PPO). The pollutant concentrations varied in the two seasons, depending on the industrial and municipal activities and efficiency of treatments. Treated waters contained heavy metals, nitrogenous and phosphorus compounds, surfactants and hydrocarbons. Compared to the control, duckweed growth of treated plants decreased by 25% in summer, while in the winter due to the lower temperatures and the presence of pollutants was completely impeded. The amounts of photosynthetic pigments of treated plants were not significantly affected in the summer, while they were higher than the control in the winter when the effluent had a high nitrogen amount. High CAT activity was registered in both seasons. Treated plants had significantly lower APX activity in the summer (53%) and winter (59%) respect to the controls. The observed inhibition of the peroxidase activities in the exposed plants, confirms the controversy existing in the literature about the variability of enzymatic response in stress condition. Copyright © 2018 Elsevier Inc. All rights reserved.

Lacking "Lack": A Reply to Joldersma

ERIC Educational Resources Information Center

Marshall, James D.

2007-01-01

First I would like to thank Clarence Joldersma for his review of our "Poststructuralism, Philosophy, Pedagogy" (Marshall, 2004-PPP). In particular, I would thank him for his opening sentence: "[t]his book is a response to a lack." It is the notion of a lack, noted again later in his review, which I wish to take up mainly in this response. Rather…

Enzymatic desulfurization of coal: Third quarterly report

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

Marquis, Judith K.; Kitchell, Judith P.

Our current efforts to develop clean coal technology emphasize the advantages of enzymatic desulfurization techniques and have specifically addressed the potential of using partially-purified extracellular microbial enzymes or commercially available enzymes. Our work is focused on the treatment of ''model'' organic sulfur compounds such as dibenzothiophene (DBT) and ethylphenylsulfide (EPS). Furthermore, we are designing experiments to facilitate the enzymatic process by means of a hydrated organic solvent matrix. In this quarter we obtained important results both with the development of our understanding of the enzyme reaction systems and also with the microbial work at Woods Hole. 12 figs., 11 tabs.

Discovery of HDAC Inhibitors That Lack an Active Site Zn(2+)-Binding Functional Group.

PubMed

Vickers, Chris J; Olsen, Christian A; Leman, Luke J; Ghadiri, M Reza

2012-06-14

Natural and synthetic histone deacetylase (HDAC) inhibitors generally derive their strong binding affinity and high potency from a key functional group that binds to the Zn(2+) ion within the enzyme active site. However, this feature is also thought to carry the potential liability of undesirable off-target interactions with other metalloenzymes. As a step toward mitigating this issue, here, we describe the design, synthesis, and structure-activity characterizations of cyclic α3β-tetrapeptide HDAC inhibitors that lack the presumed indispensable Zn(2+)-binding group. The lead compounds (e.g., 15 and 26) display good potency against class 1 HDACs and are active in tissue culture against various human cancer cell lines. Importantly, enzymological analysis of 26 indicates that the cyclic α3β-tetrapeptide is a fast-on/off competitive inhibitor of HDACs 1-3 with K i values of 49, 33, and 37 nM, respectively. Our proof of principle study supports the idea that novel classes of HDAC inhibitors, which interact at the active-site opening, but not with the active site Zn(2+), can have potential in drug design.

Isolation of phenolic compounds from iceberg lettuce and impact on enzymatic browning.

PubMed

Mai, Franziska; Glomb, Marcus A

2013-03-20

Enzymatic browning is generally reported as the reaction between phenolic substances and enzymes. The quality of iceberg lettuce is directly linked to this discoloration. In particular, the color change of lettuce stems considerably reduces consumer acceptance and thus decreases sales revenue of iceberg lettuce. Ten phenolic compounds (caffeic acid, chlorogenic acid, phaseolic acid, chicoric acid, isochlorogenic acid, luteolin-7-O-glucuronide, quercetin-3-O-glucuronide, quercetin-3-O-galactoside, quercetin-3-O-glucoside, and quercetin-3-O-(6″-malonyl)-glucoside) were isolated from Lactuca sativa var. capitata by multilayer countercurrent chromatography (MLCCC) and preparative high-performance liquid chromatography (HPLC). In addition, syringin was identified for the first time in iceberg lettuce. This polyphenolic ingredient was previously not mentioned for the family of Cichorieae in general. The purity and identity of isolated compounds were confirmed by different NMR experiments, HPLC-DAD-MS, and HR-MS techniques. Furthermore, the relationship between discoloration of iceberg lettuce and enzymatic browning was thoroughly investigated. Unexpectedly, the total concentration of phenolic compounds and the activity of polyphenol oxidase were not directly related to the browning processes. Results of model incubation experiments of plant extract solutions led to the conclusion that in addition to the typical enzymatic browning induced by polyphenol oxidases, further mechanisms must be involved to explain total browning of lettuce.

Xylan extraction from pretreated sugarcane bagasse using alkaline and enzymatic approaches.

PubMed

Sporck, Daniele; Reinoso, Felipe A M; Rencoret, Jorge; Gutiérrez, Ana; Del Rio, José C; Ferraz, André; Milagres, Adriane M F

2017-01-01

New biorefinery concepts are necessary to drive industrial use of lignocellulose biomass components. Xylan recovery before enzymatic hydrolysis of the glucan component is a way to add value to the hemicellulose fraction, which can be used in papermaking, pharmaceutical, and food industries. Hemicellulose removal can also facilitate subsequent cellulolytic glucan hydrolysis. Sugarcane bagasse was pretreated with an alkaline-sulfite chemithermomechanical process to facilitate subsequent extraction of xylan by enzymatic or alkaline procedures. Alkaline extraction methods yielded 53% (w/w) xylan recovery. The enzymatic approach provided a limited yield of 22% (w/w) but produced the xylan with the lowest contamination with lignin and glucan components. All extracted xylans presented arabinosyl side groups and absence of acetylation. 2D-NMR data suggested the presence of O -methyl-glucuronic acid and p -coumarates only in enzymatically extracted xylan. Xylans isolated using the enzymatic approach resulted in products with molecular weights (Mw) lower than 6 kDa. Higher Mw values were detected in the alkali-isolated xylans. Alkaline extraction of xylan provided a glucan-enriched solid readily hydrolysable with low cellulase loads, generating hydrolysates with a high glucose/xylose ratio. Hemicellulose removal before enzymatic hydrolysis of the cellulosic fraction proved to be an efficient manner to add value to sugarcane bagasse biorefining. Xylans with varied yield, purity, and structure can be obtained according to the extraction method. Enzymatic extraction procedures produce high-purity xylans at low yield, whereas alkaline extraction methods provided higher xylan yields with more lignin and glucan contamination. When xylan extraction is performed with alkaline methods, the residual glucan-enriched solid seems suitable for glucose production employing low cellulase loadings.

Fundamentals and Bioengineering of Enzymatic Fuel Cells. Part 1. Bioengineering of Enzymes as Electrocatalysts

DTIC Science & Technology

2012-01-31

assembles to form a thermostable. 3-dimensionaI supramolecular hydrogel that has aldo-keto reductase ( AKR ) activity. This is again accomplished... AKR activity, AdhD from Pyrococcus furiosus2*. The monomers are able to self-assemble into a bioactive enzymatic hydrogel that is stable to...temperatures in excess of 60 °C. AdhD is a member of the AKR superfamily that catalyzes the oxidation of secondary alcohols under basic conditions (optimum pH

Cross-cultural comparison of lack of regular physical activity among college students: Universal versus transversal.

PubMed

Seo, Dong-Chul; Torabi, Mohammad R; Jiang, Nan; Fernandez-Rojas, Xinia; Park, Bock-Hee

2009-01-01

This study examined cultural influence on personal and behavioral correlates of lack of regular physical activity (PA) among college students in four countries, i.e., the United States, Costa Rica, India, and South Korea. Public universities were randomly chosen among the four countries. A total of 4,685 students participated in the study during the 2006-2007 academic year with a response rate of 90.1%. The vast majority of the questions on the instrument were adopted from the Youth Risk Behavior Survey and the Behavioral Risk Factor Surveillance System questionnaires. The instrument was translated into Spanish and Korean and then back-translated into English to check accuracy of the translation. Low fruit consumption was a culture-universal predictor of lack of regular PA. Gender, perceived body weight, vegetable consumption, and cigarette smoking were culture-specific predictors, indicating PA might be a transversal value. Body mass index, binge drinking, and TV/video watching were not associated with lack of regular PA in any of the four countries. While PA is valued across different segments of many cultures, given the several culture-specific predictors, PA appears to be more transversal than universal. Therefore, culturally sensitive interventions are necessary to promote PA among young adults.

Inhibition of enzymatic browning in actual food systems by the Maillard reaction products.

PubMed

Mogol, Burçe Ataç; Yildirim, Asli; Gökmen, Vural

2010-12-01

The Maillard reaction occurring between amino acids and sugars produces neo-formed compounds having certain levels of antioxidant activity depending on the reaction conditions and the type of reactants. The objective of this study was to investigate enzymatic browning inhibition capacity of Maillard reaction products (MRPs) formed from different amino acids including arginine (Arg), histidine (His), lysine (Lys) and proline (Pro). The inhibitory effects of the MRPs on polyphenol oxidase (PPO) were determined. The total antioxidant capacity (TAC) of MRPs derived from different amino acids were in the order Arg > His > Lys > Pro. The TAC and PPO inhibition of MRPs were evaluated as a function of temperature (80-120 °C), time (1-6 h) and pH (2-12). Arg-Glc and His-Glc MRPs exhibited strong TAC and PPO inhibition. Increasing temperature (up to 100 °C) and time also increased TAC and PPO inhibition. Kinetics analysis indicated a mixed type inhibition of PPO by MRPs. The results indicate that the MRPs derived from Arg and His under certain reaction conditions significantly prevent enzymatic browning in actual food systems. The intermediate compounds capable of preventing enzymatic browning are reductones and dehydroreductones, as confirmed by liquid chromatographic-mass spectrometric analyses. Copyright © 2010 Society of Chemical Industry.

Unraveling the Enzymatic Basis of Wine “Flavorome”: A Phylo-Functional Study of Wine Related Yeast Species

PubMed Central

Belda, Ignacio; Ruiz, Javier; Alastruey-Izquierdo, Ana; Navascués, Eva; Marquina, Domingo; Santos, Antonio

2016-01-01

Non-Saccharomyces yeasts are a heterogeneous microbial group involved in the early stages of wine fermentation. The high enzymatic potential of these yeasts makes them a useful tool for increasing the final organoleptic characteristics of wines in spite of their low fermentative power. Their physiology and contribution to wine quality are still poorly understood, with most current knowledge being acquired empirically and in most cases based in single species and strains. This work analyzed the metabolic potential of 770 yeast isolates from different enological origins and representing 15 different species, by studying their production of enzymes of enological interest and linking phylogenetic and enzymatic data. The isolates were screened for glycosidase enzymes related to terpene aroma release, the β-lyase activity responsible for the release of volatile thiols, and sulfite reductase. Apart from these aroma-related activities, protease, polygalacturonase and cellulase activities were also studied in the entire yeast collection, being related to the improvement of different technological and sensorial features of wines. In this context, and in terms of abundance, two different groups were established, with α-L-arabinofuranosidase, polygalacturonase and cellulase being the less abundant activities. By contrast, β-glucosidase and protease activities were widespread in the yeast collection studied. A classical phylogenetic study involving the partial sequencing of 26S rDNA was conducted in conjunction with the enzymatic profiles of the 770 yeast isolates for further typing, complementing the phylogenetic relationships established by using 26S rDNA. This has rendered it possible to foresee the contribution different yeast species make to wine quality and their potential applicability as pure inocula, establishing species-specific behavior. These consistent results allowed us to design future targeted studies on the impact different non-Saccharomyces yeast species

Oral administration of Saccharomyces boulardii alters duodenal morphology, enzymatic activity and cytokine production response in broiler chickens.

PubMed

Sun, Yajing; Rajput, Imran Rashid; Arain, Muhammad Asif; Li, Yanfei; Baloch, Dost Muhammad

2017-08-01

The present study evaluated the effects of Saccharomyces boulardii on duodenal digestive enzymes, morphology and cytokine induction response in broiler chicken. A total of 200 birds were allotted into two groups (n = 100) and each group divided into five replications (n = 20). The control group was fed basal diet in addition to antibiotic (virginiamycin 20 mg/kg), and treatment group received (1 × 10 8  colony-forming units/kg feed) S. boulardii in addition to basal diet lasting for 72 days. The results compared to control group revealed that adenosine triphosphatase, gamma glutamyl transpeptidase, lipase and trypsin activities were higher, while, no significant improvement was observed in amylase activities in the duodenum of the treatment group. Moreover, morphological findings showed that villus height, width and number of goblet cells markedly increased. Additionally, transmission electron microscopy visualized that villus height, width and structural condensation significantly increased in the treatment group. The immunohistological observations showed increased numbers of immunoglobulin A (IgA)-positive cells in the duodenum of the treatment group. Meanwhile, cytokine production levels of tumor necrosis factor-α, interleukin (IL)-10, transforming growth factor-β and secretory IgA markedly increased, and IL-6 statistically remained unchanged as compared to the control group. These findings illustrated that initial contact of S. boulardii to the duodenum has significant impact in improving enzymatic activity, intestinal morphology and cytokine response in broiler chicken. © 2016 Japanese Society of Animal Science.

The effects of fermentation and enzymatic treatment of pea on nutrient digestibility and growth performance of broilers.

PubMed

Goodarzi Boroojeni, F; Senz, M; Kozłowski, K; Boros, D; Wisniewska, M; Rose, D; Männer, K; Zentek, J

2017-10-01

The present study examined the impacts of native, fermented or enzymatically treated peas (Pisum sativum L.) inclusion in broiler diets, on growth performance and nutrient digestibility. For the fermentation process, Madonna pea was mixed with water (1/1) containing 2.57×108 Bacillus subtilis (GalliPro®) spores/kg pea and then, incubated for 48 h at 30 °C. For the enzymatic treatment process, the used water for dough production contained three enzymes, AlphaGalTM (α-galactosidase), RONOZYME® ProAct and VP (protease and pectinases respectively - DSM, Switzerland) and the pea dough incubated for 24 h at 30°C. Nine corn-wheat-soybean diets were formulated by supplying 10%, 20% and 30% of the required CP with either native, fermented or enzymatically treated peas. Performance was recorded weekly and at the end of the experiment (day 35), apparent ileal digestibility (AID) of CP, amino acids (AA), crude fat, starch, Ca, P and K were determined. Data were subjected to ANOVA using GLM procedure with a 3×3 factorial arrangement of treatments. Both processes reduced α-galactosides, phytate, trypsin inhibitor activity and resistant starch in peas. Increasing levels of pea products up to 300 g/kg diet, reduced BW gain and feed intake (P⩽0.05). Broilers fed diets containing enzymatically treated pea had the best feed conversion ratio at day 35. Different types of pea product and their inclusion levels had no effect on AID of all nutrients. The interaction between type of the pea products and inclusion levels was significant for AID of starch. For native pea diets, 10% group showed similar AID of starch to 20% native pea but it had higher AID than 30% native pea. For fermented and enzymatically treated groups, all three levels displayed similar AID of starch. In conclusion, enzymatic treatment and fermentation could improve the nutritional quality of pea. Inclusion of enzymatically treated pea in broiler diets could improve broiler performance compared with other pea

Lack of activity of cadmium in in vitro estrogenicity assays

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

Silva, Elisabete; Lopez-Espinosa, Maria Jose; Molina-Molina, Jose-Manuel

2006-10-01

Prompted by reports about strong estrogenic effects of cadmium, attempts were made to reproduce these observations using the yeast estrogen screen (YES) and the E-Screen assays. For the first time, possible activation of the Src/MAPK pathway was also investigated. In the YES, only a slight activation (10% of a maximal effect) of the estrogen receptor alpha (ER{alpha}) was observed at cadmium concentrations between 5 x 10{sup -7} M and 5 x 10{sup -6} M. In the E-Screen assay, carried out by two laboratories, the heavy metal was without observable cell proliferative effects when tested in the range between 6 xmore » 10{sup -11} M and 1 x 10{sup -5} M. However, in both assays, cadmium led to a reduction of the effects of 17{beta}-estradiol (E2). Treatment of MCF-7 human breast cancer cells with 1 x 10{sup -7} M cadmium failed to induce phosphorylation of Src and the MAP kinases Erk1 and Erk2-effects shown to occur with E2 and epidermal growth factor (EGF). In summary, we were unable to confirm the strong estrogenicity of cadmium reported recently by a number of laboratories. This apparent absence of effects in our hands is not due to a lack of uptake of the metal or to effective protection against cadmium by high levels of glutathione or metallothionein, since toxicity and an antagonism of E2 responses were observed both in the YES and the E-Screen.« less

Evaluation of physical structural features on influencing enzymatic hydrolysis efficiency of micronized wood

Treesearch

Jinxue Jiang; Jinwu Wang; Xiao Zhang; Michael Wolcott

2016-01-01

Enzymatic hydrolysis of lignocellulosic biomass is highly dependent on the changes in structural features after pretreatment. Mechanical milling pretreatment is an effective approach to alter the physical structure of biomass and thus improve enzymatic hydrolysis. This study examined the influence of structural characteristics on the enzymatic hydrolysis of micronized...

Kinetic approach for the enzymatic determination of levodopa and carbidopa assisted by multivariate curve resolution-alternating least squares.

PubMed

Grünhut, Marcos; Garrido, Mariano; Centurión, Maria E; Fernández Band, Beatriz S

2010-07-12

A combination of kinetic spectroscopic monitoring and multivariate curve resolution-alternating least squares (MCR-ALS) was proposed for the enzymatic determination of levodopa (LVD) and carbidopa (CBD) in pharmaceuticals. The enzymatic reaction process was carried out in a reverse stopped-flow injection system and monitored by UV-vis spectroscopy. The spectra (292-600 nm) were recorded throughout the reaction and were analyzed by multivariate curve resolution-alternating least squares. A small calibration matrix containing nine mixtures was used in the model construction. Additionally, to evaluate the prediction ability of the model, a set with six validation mixtures was used. The lack of fit obtained was 4.3%, the explained variance 99.8% and the overall prediction error 5.5%. Tablets of commercial samples were analyzed and the results were validated by pharmacopeia method (high performance liquid chromatography). No significant differences were found (alpha=0.05) between the reference values and the ones obtained with the proposed method. It is important to note that a unique chemometric model made it possible to determine both analytes simultaneously. Copyright 2010 Elsevier B.V. All rights reserved.

Complete localization of disulfide bonds in GM2 activator protein.

PubMed Central

Schütte, C. G.; Lemm, T.; Glombitza, G. J.; Sandhoff, K.

1998-01-01

Lysosomal degradation of ganglioside GM2 by hexosaminidase A requires the presence of a small, non-enzymatic cofactor, the GM2-activator protein (GM2AP). Lack of functional protein leads to the AB variant of GM2-gangliosidosis, a fatal lysosomal storage disease. Although its possible mode of action and functional domains have been discussed frequently in the past, no structural information about GM2AP is available so far. Here, we determine the complete disulfide bond pattern of the protein. Two of the four disulfide bonds present in the protein were open to classical determination by enzymatic cleavage and mass spectrometry. The direct localization of the remaining two bonds was impeded by the close vicinity of cysteines 136 and 138. We determined the arrangement of these disulfide bonds by MALDI-PSD analysis of disulfide linked peptides and by partial reduction, cyanylation and fragmentation in basic solution, as described recently (Wu F, Watson JT, 1997, Protein Sci 6:391-398). PMID:9568910

Autoantibodies against aromatic amino acid hydroxylases in patients with autoimmune polyendocrine syndrome type 1 target multiple antigenic determinants and reveal regulatory regions crucial for enzymatic activity.

PubMed

Bratland, Eirik; Magitta, Ng'weina Francis; Bøe Wolff, Anette Susanne; Ekern, Trude; Knappskog, Per Morten; Kämpe, Olle; Haavik, Jan; Husebye, Eystein Sverre

2013-06-01

Patients with autoimmune polyendocrine syndrome type 1 (APS-1) frequently have autoantibodies directed against the aromatic amino acid hydroxylases tryptophan hydroxylase (TPH) and tyrosine hydroxylase (TH). We aimed to characterize these autoantibodies with regard to their antigenic determinants, their influence on enzymatic activity and their clinical associations. In particular, we wanted to compare autoantibodies against the two different isoforms of TPH, which display different tissue distribution. Using sera from 48 Scandinavian APS-1 patients we identified 36 patients (75%) with antibodies against one or more of these three enzymes. Antibodies against TPH1, but not TPH2, were associated with malabsorption in the whole Scandinavian cohort, while TH antibodies were associated with dental enamel hypoplasia in Norwegian patients. Subsequent experiments with selected patient sera indicated that while the C-terminal domain was the immunodominant part of TPH1, the epitopes of TPH2 and TH were mainly located in the N-terminal regulatory domains. We also identified a TPH1 specific epitope involved in antibody mediated inhibition of enzyme activity, a finding that provides new insight into the enzymatic mechanisms of the aromatic amino acid hydroxylases and knowledge about structural determinants of enzyme autoantigens. In conclusion, TPH1, TPH2 and TH all have unique antigenic properties in spite of their structural similarity. Copyright © 2012 Elsevier GmbH. All rights reserved.

The enzymatic oxidation of graphene oxide.

PubMed

Kotchey, Gregg P; Allen, Brett L; Vedala, Harindra; Yanamala, Naveena; Kapralov, Alexander A; Tyurina, Yulia Y; Klein-Seetharaman, Judith; Kagan, Valerian E; Star, Alexander

2011-03-22

Two-dimensional graphitic carbon is a new material with many emerging applications, and studying its chemical properties is an important goal. Here, we reported a new phenomenon--the enzymatic oxidation of a single layer of graphitic carbon by horseradish peroxidase (HRP). In the presence of low concentrations of hydrogen peroxide (∼40 μM), HRP catalyzed the oxidation of graphene oxide, which resulted in the formation of holes on its basal plane. During the same period of analysis, HRP failed to oxidize chemically reduced graphene oxide (RGO). The enzymatic oxidation was characterized by Raman, ultraviolet-visible, electron paramagnetic resonance, Fourier transform infrared spectroscopy, transmission electron microscopy, atomic force microscopy, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and gas chromatography-mass spectrometry. Computational docking studies indicated that HRP was preferentially bound to the basal plane rather than the edge for both graphene oxide and RGO. Owing to the more dynamic nature of HRP on graphene oxide, the heme active site of HRP was in closer proximity to graphene oxide compared to RGO, thereby facilitating the oxidation of the basal plane of graphene oxide. We also studied the electronic properties of the reduced intermediate product, holey reduced graphene oxide (hRGO), using field-effect transistor (FET) measurements. While RGO exhibited a V-shaped transfer characteristic similar to a single layer of graphene that was attributed to its zero band gap, hRGO demonstrated a p-type semiconducting behavior with a positive shift in the Dirac points. This p-type behavior rendered hRGO, which can be conceptualized as interconnected graphene nanoribbons, as a potentially attractive material for FET sensors.

[Extraction and analysis of the essential oil in Pogostemon cablin by enzymatic hydrolysis and inhibitory activity against Hela cell proliferation].

PubMed

Yu, Jing; Qi, Yue; Luo, Gang; Duan, Hong-quan; Zhou, Jing

2012-05-01

To optimize the extraction method of essential oil in Pogostemon cablin and analyze its inhibitory activity against Hela cell proliferation. The Pogostemon cablin was treated by hemicellulase before steam distillation. The enzyme dosage, treatment time, treatment temperature, pH were optimized through orthogonal experimental design. The components of essential oil were identified by gas chromatography-mass spectrometry (GC-MS). Inhibitory activity of patchouli oil against Hela cell proliferation was determined by MTP method. The optimum extraction process was as follows: pH 4.5, temperature 45 degrees C, the ratio of hemicellulase to Pogostemon cablin was 1% and enzymatic hydrolysis for 1.0 hour. Extraction ratio of the patchouli oil in steam distillation and hemicellulase extraction method was 2.2220 mg/g, 3.1360 mg/g respectively. Patchouli oil could inhibit Hela cell proliferation. IC50 of the patchouli oil in steam distillation and hemicellulase extraction method was 12.2 +/- 0.46 microg/mL and 0.36 +/- 0.03 microg/mL respectively. In comparison with steam distillation method, extraction ratios of essential oil and the inhibitory activity against Hela cell proliferation can be increased by the hemicellulase extraction method.

Dynamics of microbiological parameters, enzymatic activities and worm biomass production during vermicomposting of effluent treatment plant sludge of bakery industry.

PubMed

Yadav, Anoop; Suthar, S; Garg, V K

2015-10-01

This paper reports the changes in microbial parameters and enzymatic activities during vermicomposting of effluent treatment plant sludge (ETPS) of bakery industry spiked with cow dung (CD) by Eisenia fetida. Six vermibins containing different ratios of ETPS and CD were maintained under controlled laboratory conditions for 15 weeks. Total bacterial and total fungal count increased upto 7th week and declined afterward in all the bins. Maximum bacterial and fungal count was 31.6 CFU × 10(6) g(-1) and 31 CFU × 10(4) g(-1) in 7th week. Maximum dehydrogenase activity was 1921 μg TPF g(-1) h(-1) in 9th week in 100 % CD containing vermibin, whereas maximum urease activity was 1208 μg NH4 (-)N g(-1) h(-1) in 3rd week in 100 % CD containing vermibin. The enzyme activity and microbial counts were lesser in ETPS containing vermibins than control (100 % CD). The growth and fecundity of the worms in different vermibins were also investigated. The results showed that initially biomass and fecundity of the worms increased but decreased at the later stages due to non-availability of the palatable feed. This showed that quality and palatability of food directly affect biological parameters of the system.

An update on enzymatic cocktails for lignocellulose breakdown.

PubMed

de Mello Lopes, Andreza; Ferreira Filho, Edivaldo Ximenes; de Souza Moreira, Leonora Rios

2018-05-22

Alternative energy sources have received increasing attention in recent years. The possibility of adding value to agricultural wastes, by producing biofuels and other products with economic value from lignocellulosic biomass by enzymatic hydrolysis, has been widely explored. Lignocellulosic biomass, as well as being an abundant residue, is a complex recalcitrant structure that requires a consortium of enzymes for its complete degradation. Pools of enzymes with different specificities acting together usually produce an increase in hydrolysis yield. Enzymatic cocktails have been widely studied due to their potential industrial application for the bioconversion of lignocellulosic biomass. This review presents an overview of enzymes required to degrade the plant cell wall, paying particular attention to the latest advances in enzymatic cocktail production and the main results obtained with cocktails used to degrade a variety of types of biomass, as well as some future perspectives within this field. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Inhibition of non-enzymatic glycation by silk extracts from a Mexican land race and modern inbred lines of maize (Zea mays).

PubMed

Farsi, Darius Arthur; Harris, Cory S; Reid, Lana; Bennett, Steffany A L; Haddad, Pierre S; Martineau, Louis C; Arnason, John Thor

2008-01-01

Non-enzymatic glycation and the accumulation of advanced glycation end products (AGEs) are associated with various disease states, including complications of diabetes and aging. Secondary metabolites from several plant species are known to inhibit non-enzymatic glycation and the formation of AGEs, including flavonoids found in the style (silk) of Zea mays (maize). Thirteen modern maize inbreds and one land race were tested for in vitro inhibition of non-enzymatic glycation of bovine serum albumin. Many of the tested extracts exhibited inhibitory activity, in particular the newest inbreds, which were bred for resistance to gibberella ear rot (Fusarium graminearum) and common smut (Ustilago maydis). The most active maize genotype (CO441), displaying an IC50 of 9.5 microg/mL, was more effective than aminoguanidine, a known inhibitor of glycation. Zapalote chico, a land race with high maysin content, showed only moderate inhibitory activity compared with the modern maize genotypes. Antiglycation activity was highly correlated with the total phenolic content of silk extracts and mildly correlated with resistance to certain fungal infections. The results identify modern resistant and high phenolic maize inbreds as promising candidates for the development of natural AGE inhibitors for the prevention and treatment of diabetic complications and the degenerative effects of aging. Copyright (c) 2007 John Wiley & Sons, Ltd.

The influence of cyclomaltooligosaccharides (cyclodextrins) on the enzymatic decomposition of l-phenylalanine catalyzed by phenylalanine ammonia-lyase.

PubMed

Gubica, Tomasz; Pełka, Agnieszka; Pałka, Katarzyna; Temeriusz, Andrzej; Kańska, Marianna

2011-09-27

Cyclomaltohexaose (α-cyclodextrin) and cyclomaltoheptaose (β-cyclodextrin) as well as their four methyl ether derivatives, that is, hexakis(2,3-di-O-methyl)cyclomaltohexaose, hexakis(2,3,6-tri-O-methyl)cyclomaltohexaose, heptakis(2,3-di-O-methyl)cyclomaltoheptaose, and heptakis(2,3,6-tri-O-methyl)cyclomaltoheptaose were investigated as the additives in the course of enzymatic decomposition of l-phenylalanine catalyzed by phenylalanine ammonia-lyase. Only a few of those additives behaved like classical inhibitors of the enzymatic reaction under investigation because the values of the Michaelis constants that were obtained, as well as the maximum velocity values depended mostly atypically on the concentrations of those additives. In most cases cyclodextrins caused mixed inhibition, both competitive and noncompetitive, but they also acted as activators for selected concentrations. This atypical behaviour of cyclodextrins is caused by three different and independent effects. The inhibitory effect of cyclodextrins is connected with the decrease of substrate concentration and unfavourable influence on the flexibility of the enzyme molecules. On the other hand, the activating effect is connected with the decrease of product concentration (the product is an inhibitor of the enzymatic reaction under investigation). All these effects are caused by the ability of the cyclodextrins to form inclusion complexes. Copyright © 2011 Elsevier Ltd. All rights reserved.

Enhancing bioactive peptide release and identification using targeted enzymatic hydrolysis of milk proteins.

PubMed

Nongonierma, Alice B; FitzGerald, Richard J

2018-06-01

Milk proteins have been extensively studied for their ability to yield a range of bioactive peptides following enzymatic hydrolysis/digestion. However, many hurdles still exist regarding the widespread utilization of milk protein-derived bioactive peptides as health enhancing agents for humans. These mostly arise from the fact that most milk protein-derived bioactive peptides are not highly potent. In addition, they may be degraded during gastrointestinal digestion and/or have a low intestinal permeability. The targeted release of bioactive peptides during the enzymatic hydrolysis of milk proteins may allow the generation of particularly potent bioactive hydrolysates and peptides. Therefore, the development of milk protein hydrolysates capable of improving human health requires, in the first instance, optimized targeted release of specific bioactive peptides. The targeted hydrolysis of milk proteins has been aided by a range of in silico tools. These include peptide cutters and predictive modeling linking bioactivity to peptide structure [i.e., molecular docking, quantitative structure activity relationship (QSAR)], or hydrolysis parameters [design of experiments (DOE)]. Different targeted enzymatic release strategies employed during the generation of milk protein hydrolysates are reviewed herein and their limitations are outlined. In addition, specific examples are provided to demonstrate how in silico tools may help in the identification and discovery of potent milk protein-derived peptides. It is anticipated that the development of novel strategies employing a range of in silico tools may help in the generation of milk protein hydrolysates containing potent and bioavailable peptides, which in turn may be used to validate their health promoting effects in humans. Graphical abstract The targeted enzymatic hydrolysis of milk proteins may allow the generation of highly potent and bioavailable bioactive peptides.

Effects of lignin-metal complexation on enzymatic hydrolysis of cellulose

Treesearch

H. Liu; Junyong Zhu; S.Y. Fu

2010-01-01

This study investigated the inhibition of enzymatic hydrolysis by unbound lignin (soluble and insoluble) with or without the addition of metal compounds. Sulfonated, Organosolv, and Kraft lignin were added in aqueous enzyme-cellulose systems at different concentrations before hydrolysis. The measured substrate enzymatic digestibility (SED) of cellulose was decreased by...

Enhancement of enzymatic saccharification of Eucalyptus globulus: steam explosion versus steam treatment.

PubMed

Martin-Sampedro, Raquel; Revilla, Esteban; Villar, Juan C; Eugenio, Maria E

2014-09-01

Steam explosion and steam pre-treatment have proved capable of enhancing enzymatic saccharification of lignocellulosic materials. However, until now, these methods had not been compared under the same operational conditions and using the same raw material. Both pre-treatments lead to increased yields in the saccharification of Eucalyptus globulus; but results have been better with steam pre-treatments, despite the more accessible surface of exploded samples. The reason for this finding could be enzymatic inhibition: steam explosion causes a more extensive extraction of hemicelluloses and releases a greater amount of degradation products which can inhibit enzymatic action. Enzymatic inhibition is also dependent on the amount and chemical structure of lignin, which was also a contributing factor to the lower enzymatic yields obtained with the most severe pre-treatment. Thus, the highest yields (46.7% glucose and 73.4% xylose yields) were obtained after two cycle of steam treatment, of 5 and 3 min, at 183°C. Copyright © 2014 Elsevier Ltd. All rights reserved.

Phytase production by Rhizopus microsporus var. microsporus biofilm: characterization of enzymatic activity after spray drying in presence of carbohydrates and nonconventional adjuvants.

PubMed

Sato, Vanessa Sayuri; Jorge, João Atílio; Oliveira, Wanderley Pereira; Souza, Claudia Regina Fernandez; Guimarães, Luis Henrique Souza

2014-02-28

Microbial phytases are enzymes with biotechnological interest for the feed industry. In this article, the effect of spray-drying conditions on the stability and activity of extracellular phytase produced by R. microsporus var. microsporus biofilm is described. The phytase was spray-dried in the presence of starch, corn meal (>150 μm), soy bean meal (SB), corn meal (<150 μm) (CM), and maltodextrin as drying adjuvants. The residual enzyme activity after drying ranged from 10.7% to 60.4%, with SB and CM standing out as stabilizing agents. Water concentration and residual enzyme activity were determined in obtained powders as a function of the drying condition. When exposed to different pH values, the SB and CM products were stable, with residual activity above 50% in the pH range from 4.5 to 8.5 for 60 min. The use of CM as drying adjuvant promoted the best retention of enzymatic activity compared with SB. Spray drying of the R. microsporus var. microsporus phytase using different drying adjuvants showed interesting results, being quite feasible with regards their biotechnological applications, especially for poultry diets.

Mannanase hydrolysis of spruce galactoglucomannan focusing on the influence of acetylation on enzymatic mannan degradation.

PubMed

Arnling Bååth, Jenny; Martínez-Abad, Antonio; Berglund, Jennie; Larsbrink, Johan; Vilaplana, Francisco; Olsson, Lisbeth

2018-01-01

Galactoglucomannan (GGM) is the most abundant hemicellulose in softwood, and consists of a backbone of mannose and glucose units, decorated with galactose and acetyl moieties. GGM can be hydrolyzed into fermentable sugars, or used as a polymer in films, gels, and food additives. Endo -β-mannanases, which can be found in the glycoside hydrolase families 5 and 26, specifically cleave the mannan backbone of GGM into shorter oligosaccharides. Information on the activity and specificity of different mannanases on complex and acetylated substrates is still lacking. The aim of this work was to evaluate and compare the modes of action of two mannanases from Cellvibrio japonicus ( Cj Man5A and Cj Man26A) on a variety of mannan substrates, naturally and chemically acetylated to varying degrees, including naturally acetylated spruce GGM. Both enzymes were evaluated in terms of cleavage patterns and their ability to accommodate acetyl substitutions. Cj Man5A and Cj Man26A demonstrated different substrate preferences on mannan substrates with distinct backbone and decoration structures. Cj Man5A action resulted in higher amounts of mannotriose and mannotetraose than that of Cj Man26A, which mainly generated mannose and mannobiose as end products. Mass spectrometric analysis of products from the enzymatic hydrolysis of spruce GGM revealed that an acetylated hexotriose was the shortest acetylated oligosaccharide produced by Cj Man5A, whereas Cj Man26A generated acetylated hexobiose as well as diacetylated oligosaccharides. A low degree of native acetylation did not significantly inhibit the enzymatic action. However, a high degree of chemical acetylation resulted in decreased hydrolyzability of mannan substrates, where reduced substrate solubility seemed to reduce enzyme activity. Our findings demonstrate that the two mannanases from C. japonicus have different cleavage patterns on linear and decorated mannan polysaccharides, including the abundant and industrially important

Enzymatic degradation of somatostatin by rat plasma and hypothalamus.

PubMed

Dupont, A; Alvarado-Urbina, G; Côté, J; Labrie, F

1978-10-01

A highly sensitive and specific radioimmunoassay for somatostatin has been used to study inactivation of the neurohormone by plasma and hypothalamic peptidase(s). Specificity of the inactivation process was indicated by the absence of interference by addition of luteinizing hormone releasing hormone, thyrotropin-releasing hormone, oxytocin, or substance P. The inactivating ability of hypothalamic tissue and plasma was destroyed by heating and the protease inhibitor benzamidine prevented plasma activity, thus suggesting the enzymatic nature of the processes involved. The present data suggest that the inactivation of somatostatin by hypothalamus and plasma could be an important factor in the regulation of circulating somatostatin levels.

Plasmodium falciparum SERA5 plays a non-enzymatic role in the malarial asexual blood-stage lifecycle

PubMed Central

Stallmach, Robert; Kavishwar, Manoli; Withers-Martinez, Chrislaine; Hackett, Fiona; Collins, Christine R; Howell, Steven A; Yeoh, Sharon; Knuepfer, Ellen; Atid, Avshalom J; Holder, Anthony A; Blackman, Michael J

2015-01-01

The malaria parasite Plasmodium falciparum replicates in an intraerythrocytic parasitophorous vacuole (PV). The most abundant P. falciparum PV protein, called SERA5, is essential in blood stages and possesses a papain-like domain, prompting speculation that it functions as a proteolytic enzyme. Unusually however, SERA5 possesses a Ser residue (Ser596) at the position of the canonical catalytic Cys of papain-like proteases, and the function of SERA5 or whether it performs an enzymatic role is unknown. In this study, we failed to detect proteolytic activity associated with the Ser596-containing parasite-derived or recombinant protein. However, substitution of Ser596 with a Cys residue produced an active recombinant enzyme with characteristics of a cysteine protease, demonstrating that SERA5 can bind peptides. Using targeted homologous recombination in P. falciparum, we substituted Ser596 with Ala with no phenotypic consequences, proving that SERA5 does not perform an essential enzymatic role in the parasite. We could also replace an internal segment of SERA5 with an affinity-purification tag. In contrast, using almost identical targeting constructs, we could not truncate or C-terminally tag the SERA5 gene, or replace Ser596 with a bulky Arg residue. Our findings show that SERA5 plays an indispensable but non-enzymatic role in the P. falciparum blood-stage life cycle. PMID:25599609

Pharmacologic inhibition of the enzymatic effects of tissue transglutaminase reduces cardiac fibrosis and attenuates cardiomyocyte hypertrophy following pressure overload.

PubMed

Shinde, Arti V; Su, Ya; Palanski, Brad A; Fujikura, Kana; Garcia, Mario J; Frangogiannis, Nikolaos G

2018-04-01

Tissue transglutaminase (tTG) is a multifunctional protein with a wide range of enzymatic and non-enzymatic functions. We have recently demonstrated that tTG expression is upregulated in the pressure-overloaded myocardium and exerts fibrogenic actions promoting diastolic dysfunction, while preventing chamber dilation. Our current investigation dissects the in vivo and in vitro roles of the enzymatic effects of tTG on fibrotic remodeling in pressure-overloaded myocardium. Using a mouse model of transverse aortic constriction, we demonstrated perivascular and interstitial tTG activation in the remodeling pressure-overloaded heart. tTG inhibition through administration of the selective small molecule tTG inhibitor ERW1041E attenuated left ventricular diastolic dysfunction and reduced cardiomyocyte hypertrophy and interstitial fibrosis in the pressure-overloaded heart, without affecting chamber dimensions and ejection fraction. In vivo, tTG inhibition markedly reduced myocardial collagen mRNA and protein levels and attenuated transcription of fibrosis-associated genes. In contrast, addition of exogenous recombinant tTG to fibroblast-populated collagen pads had no significant effects on collagen transcription, and instead increased synthesis of matrix metalloproteinase (MMP)3 and tissue inhibitor of metalloproteinases (TIMP)1 through transamidase-independent actions. However, enzymatic effects of matrix-bound tTG increased the thickness of pericellular collagen in fibroblast-populated pads. tTG exerts distinct enzymatic and non-enzymatic functions in the remodeling pressure-overloaded heart. The enzymatic effects of tTG are fibrogenic and promote diastolic dysfunction, but do not directly modulate the pro-fibrotic transcriptional program of fibroblasts. Targeting transamidase-dependent actions of tTG may be a promising therapeutic strategy in patients with heart failure and fibrosis-associated diastolic dysfunction. Copyright © 2018 Elsevier Ltd. All rights reserved.

Isohexide and Sorbitol-Derived, Enzymatically Synthesized Renewable Polyesters with Enhanced Tg.

PubMed

Gustini, Liliana; Lavilla, Cristina; de Ilarduya, Antxon Martínez; Muñoz-Guerra, Sebastián; Koning, Cor E

2016-10-10

Sugar-based polyesters derived from sorbitol and isohexides were obtained via solvent-free enzymatic catalysis. Pendant hydroxyl groups, coming from the sorbitol units, were present along the polyester backbone, whereas the two isohexides, namely, isomannide and isoidide dimethyl ester monomers, were selected to introduce rigidity into the polyester chains. The feasibility of incorporating isomannide as a diol compared to the isoidide dimethyl ester as acyl-donor via lipase-catalyzed polycondensation was investigated. The presence of bicyclic units resulted in enhanced T g with respect to the parent sorbitol-containing polyester lacking isohexides. The different capability of the two isohexides to boost the thermal properties confirmed the more flexible character provided by the isoidide diester derivative. Solvent-borne coatings were prepared by cross-linking the sugar-based polyester polyols with polyisocyanates. The increased rigidity of the obtained sugar-based polyester polyols led to an enhancement in hardness of the resulting coatings.

Evaluation of in vitro enzymatic and non-enzymatic antioxidant properites of leaf extract from Alpinia Purpurata (Vieill.) K. Schum.

PubMed

Raj, Chinthamony Arul; Ragavendran, Paramasivam; Sophia, Dominic; Starlin, Thangarajan; Rathi, Muthian Ahalliya; Gopalakrishnan, Velliyur Kanniappan

2016-09-01

To evaluate the enzymatic and non-enzymatic antioxidants of leaf extract from Alpinia purpurata. One gram of fresh leaf of Alpinia purpurata was grinded in 2 mL of 50% ethanol and centrifuged at 10,000×g at 4°C for 10 min. The supernatant obtained was used within 4 h for various enzymatic antioxidants assays like superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione S-transferase (GST), ascorbate oxidase, peroxidase, polyphenol oxidase (PPO) and non-enzymatic antioxidants such as vitamin C, total reduced glutathione (TRG) and lipid peroxidation (LPO). The leaf extract of Alpinia purpurata possess antioxidants like vitamin C 472.92±6.80 μg/mg protein, GST 372.11±5.70 μmol of 1-chloro 2,4 dinitrobenzene (CDNB)-reduced glutathione (GSH) conjugate formed/min/mg protein, GPx 281.69±6.43 μg of glutathione oxidized/min/mg protein, peroxidases 173.12±9.40 μmol/g tissue, TRG 75.27±3.55 μg/mg protein, SOD 58.03±2.11 U/mg protein, CAT 46.70±2.35 μmol of H2O2 consumed/min/mg protein in high amount whereas ascorbate oxidase 17.41±2.46 U/g tissue, LPO 2.71±0.14 nmol/L of malondialdehyde formed/min/mg protein and PPO 1.14±0.11 μmol/g tissue in moderate amount. Alpinia purpurata has the potential to scavenge the free radicals and protect against oxidative stress causing diseases. In future, Alpinia purpurata may serve as a good pharmacotherapeutic agent.

Biocompatible enzymatic roller pens for direct writing of biocatalytic materials: "do-it-yourself" electrochemical biosensors.

PubMed

Bandodkar, Amay J; Jia, Wenzhao; Ramírez, Julian; Wang, Joseph

2015-06-03

The development of enzymatic-ink-based roller pens for direct drawing of biocatalytic sensors, in general, and for realizing renewable glucose sensor strips, in particular, is described. The resulting enzymatic-ink pen allows facile fabrication of high-quality inexpensive electrochemical biosensors of any design by the user on a wide variety of surfaces having complex textures with minimal user training. Unlike prefabricated sensors, this approach empowers the end user with the ability of "on-demand" and "on-site" designing and fabricating of biocatalytic sensors to suit their specific requirement. The resulting devices are thus referred to as "do-it-yourself" sensors. The bio-active pens produce highly reproducible biocatalytic traces with minimal edge roughness. The composition of the new enzymatic inks has been optimized for ensuring good biocatalytic activity, electrical conductivity, biocompati-bility, reproducible writing, and surface adherence. The resulting inks are characterized using spectroscopic, viscometric, electrochemical, thermal and microscopic techniques. Applicability to renewable blood glucose testing, epidermal glucose monitoring, and on-leaf phenol detection are demonstrated in connection to glucose oxidase and tyrosinase-based carbon inks. The "do-it-yourself" renewable glucose sensor strips offer a "fresh," reproducible, low-cost biocatalytic sensor surface for each blood test. The ability to directly draw biocatalytic conducting traces even on unconventional surfaces opens up new avenues in various sensing applications in low-resource settings and holds great promise for diverse healthcare, environmental, and defense domains. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

An enzymatic assay based on luciferase Ebola virus-like particles for evaluation of virolytic activity of antimicrobial peptides.

PubMed

Peskova, Marie; Heger, Zbynek; Janda, Petr; Adam, Vojtech; Pekarik, Vladimir

2017-02-01

Antimicrobial peptides are currently considered as promising antiviral compounds. Current assays to evaluate the effectivity of peptides against enveloped viruses based on liposomes or hemolysis are encumbered by the artificial nature of liposomes or distinctive membrane composition of used erythrocytes. We propose a novel assay system based on enzymatic Ebola virus-like particles containing sensitive luciferase reporter. The assay was validated with several cationic and anionic peptides and compared with lentivirus inactivation and hemolytic assays. The assay is sensitive and easy to perform in standard biosafety level laboratory with potential for high-throughput screens. The use of virus-like particles in the assay provides a system as closely related to the native viruses as possible eliminating some issues associated with other more artificial set ups. We have identified CAM-W (KWKLWKKIEKWGQGIGAVLKWLTTWL) as a peptide with the greatest antiviral activity against infectious lentiviral vectors and filoviral virus-like particles. Copyright © 2016 Elsevier Inc. All rights reserved.

Enzymatic oxidative biodegradation of nanoparticles: Mechanisms, significance and applications

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

Vlasova, Irina I.

Biopersistence of carbon nanotubes, graphene oxide (GO) and several other types of carbonaceous nanomaterials is an essential determinant of their health effects. Successful biodegradation is one of the major factors defining the life span and biological responses to nanoparticles. Here, we review the role and contribution of different oxidative enzymes of inflammatory cells – myeloperoxidase, eosinophil peroxidase, lactoperoxidase, hemoglobin, and xanthine oxidase – to the reactions of nanoparticle biodegradation. We further focus on interactions of nanomaterials with hemoproteins dependent on the specific features of their physico-chemical and structural characteristics. Mechanistically, we highlight the significance of immobilized peroxidase reactive intermediates vsmore » diffusible small molecule oxidants (hypochlorous and hypobromous acids) for the overall oxidative biodegradation process in neutrophils and eosinophils. We also accentuate the importance of peroxynitrite-driven pathways realized in macrophages via the engagement of NADPH oxidase- and NO synthase-triggered oxidative mechanisms. We consider possible involvement of oxidative machinery of other professional phagocytes such as microglial cells, myeloid-derived suppressor cells, in the context of biodegradation relevant to targeted drug delivery. We evaluate the importance of genetic factors and their manipulations for the enzymatic biodegradation in vivo. Finally, we emphasize a novel type of biodegradation realized via the activation of the “dormant” peroxidase activity of hemoproteins by the nano-surface. This is exemplified by the binding of GO to cyt c causing the unfolding and ‘unmasking’ of the peroxidase activity of the latter. We conclude with the strategies leading to safe by design carbonaceous nanoparticles with optimized characteristics for mechanism-based targeted delivery and regulatable life-span of drugs in circulation. - Highlights: • Nanoparticles can be degraded

Enzymatic reactivity of glucose oxidase confined in nanochannels.

PubMed

Yu, Jiachao; Zhang, Yuanjian; Liu, Songqin

2014-05-15

The construction of nanodevices coupled with an integrated real-time detection system for evaluation of the function of biomolecules in biological processes, and enzymatic reaction kinetics occurring at the confined space or interface is a significant challenge. In this work, a nanochannel-enzyme system in which the enzymatic reaction could be investigated with an electrochemical method was constructed. The model system was established by covalently linking glucose oxidase (GOD) onto the inner wall of the nanochannels of the porous anodic alumina (PAA) membrane. An Au disc was attached at the end of the nanochannels of the PAA membrane as the working electrode for detection of H2O2 product of enzymatic reaction. The effects of ionic strength, amount of immobilized enzyme and pore diameter of the nanochannels on the enzymatic reaction kinetics were illustrated. The GOD confined in nanochannels showed high stability and reactivity. Upon addition of glucose to the nanochannel-enzyme system, the current response had a calibration range span from 0.005 to 2 mM of glucose concentration. The apparent Michaelis-Menten constant (K(m)(app)) of GOD confined in nanochannel was 0.4 mM. The presented work provided a platform for real-time monitoring of the enzyme reaction kinetics confined in nanospaces. Such a nanochannel-enzyme system could also help design future biosensors and enzyme reactors with high sensitivity and efficiency. Copyright © 2013 Elsevier B.V. All rights reserved.

p48 Activates a UV-Damaged-DNA Binding Factor and Is Defective in Xeroderma Pigmentosum Group E Cells That Lack Binding Activity

PubMed Central

Hwang, Byung Joon; Toering, Stephanie; Francke, Uta; Chu, Gilbert

1998-01-01

A subset of xeroderma pigmentosum (XP) group E cells lack a factor that binds to DNA damaged by UV radiation. This factor can be purified to homogeneity as p125, a 125-kDa polypeptide. However, when cDNA encoding p125 is translated in vitro, only a small fraction binds to UV-damaged DNA, suggesting that a second factor is required for the activation of p125. We discovered that most hamster cell lines expressed inactive p125, which was activated in somatic cell hybrids containing human chromosome region 11p11.2-11cen. This region excluded p125 but included p48, which encodes a 48-kDa polypeptide known to copurify with p125 under some conditions. Expression of human p48 activated p125 binding in hamster cells and increased p125 binding in human cells. No such effects were observed from expression of p48 containing single amino acid substitutions from XP group E cells that lacked binding activity, demonstrating that the p48 gene is defective in those cells. Activation of p125 occurred by a “hit-and-run” mechanism, since the presence of p48 was not required for subsequent binding. Nevertheless, p48 was capable of forming a complex with p125 either bound to UV-damaged DNA or in free solution. It is notable that hamster cells fail to efficiently repair cyclobutane pyrimidine dimers in nontranscribed DNA and fail to express p48, which contains a WD motif with homology to proteins that reorganize chromatin. We propose that p48 plays a role in repairing lesions that would otherwise remain inaccessible in nontranscribed chromatin. PMID:9632823

Enzymatic characterization of a novel bovine liver dihydrodiol dehydrogenase--reaction mechanism and bile acid dehydrogenase activity.

PubMed

Nanjo, H; Adachi, H; Morihana, S; Mizoguchi, T; Nishihara, T; Terada, T

1995-05-11

Bovine liver cytosolic dihydrodiol dehydrogenase (DD3) has been characterized by its unique dihydrodiol dehydrogenase activity for trans-benzenedihydrodiol (trans-1,2-dihydrobenzene-1,2-diol) with the highest affinity and the greatest velocity among three multiple forms of dihydrodiol dehydrogenases (DD1-DD3). It is the first time that DD3 has shown a significant dehydrogenase activity for (S)-(+)-1-indanol with low Km value (0.33 +/- 0.022 mM) and high K(cat) value (25 +/- 0.79 min-1). The investigation of the product inhibition of (S)-(+)-1-indanol with NADP+ versus 1-indanone and NADPH clearly showed that the enzymatic reaction of DD3 may follow a typical ordered Bi Bi mechanism similar to many aldo/keto reductases. Additionally, DD3 was shown to catalyze the dehydrogenation of bile acids (lithocholic acid, taurolithocholic acid and taurochenodeoxycholic acid) having no 12-hydroxy groups with low Km values (17 +/- 0.65, 33 +/- 1.9 and 890 +/- 73 microM, respectively). In contrast, DD1, 3 alpha-hydroxysteroid dehydrogenase, shows a broad substrate specificity for many bile acids with higher affinity than those of DD3. Competitive inhibition of DD3 with androsterone against dehydrogenase activity for (S)-(+)-1-indanol, trans-benzenedihydrodiol or lithocholic acid suggests that these three substrates bind to the same substrate binding site of DD3, different from the case of human liver bile acid binder/dihydrodiol dehydrogenase (Takikawa, H., Stolz, A., Sugiyama, Y., Yoshida, H., Yamamoto, M. and Kaplowitz, N. (1990) J. Biol. Chem. 265, 2132-2136). Considering the reaction mechanism, DD3 may also play an important role in bile acids metabolism as well as the detoxication of aromatic hydrocarbons.

Does a lack of physical activity explain the rheumatoid arthritis lipid profile?

PubMed

AbouAssi, Hiba; Connelly, Margery A; Bateman, Lori A; Tune, K Noelle; Huebner, Janet L; Kraus, Virginia B; Winegar, Deborah A; Otvos, James D; Kraus, William E; Huffman, Kim M

2017-02-10

In rheumatoid arthritis (RA), cardiovascular risk is associated with paradoxical reductions in total cholesterol, low density lipoprotein-cholesterol (LDL-C), and high density lipoprotein-cholesterol (HDL-C). Concentrations of small LDL (LDL-P) and HDL (HDL-P) particles are also reduced with increased inflammation and disease activity in RA patients. Here we sought to identify which measure(s) of inflammation, disease activity and cardiometabolic risk contribute most to the RA-associated lipoprotein profile. NMR lipoprotein measurements were obtained for individuals with RA (n = 50) and age-, gender-, and body mass index (BMI)-matched controls (n = 39). Groups were compared using 39 matched pairs with 11 additional subjects used in RA only analyses. Among RA patients, relationships were determined for lipoprotein parameters with measures of disease activity, disability, pain, inflammation, body composition, insulin sensitivity and exercise. Percentage of time spent in basal activity (<1 metabolic equivalent) and exercise (≥3 metabolic equivalents) were objectively-determined. Subjects with RA had fewer total and small LDL-P as well as larger LDL and HDL size (P < 0.05). Among RA patients, pain and disability were associated with fewer small HDL-P (P < 0.05), while interleukin (IL)-6, IL-18, and TNF-α were associated with LDL size (P < 0.05). BMI, waist circumference, abdominal visceral adiposity and insulin resistance were associated with more total and small LDL-P, fewer large HDL-P, and a reduction in HDL size (P < 0.05). Most similar to the RA lipoprotein profile, more basal activity (minimal physical activity) and less exercise time were associated with fewer small LDL-P and total and small HDL-P (P < 0.05). The RA-associated lipoprotein profile is associated with a lack of physical activity. As this was a cross-sectional investigation and not an intervention and was performed from 2008-13, this study was not registered in

Effect of enzymatic treatment of extracted sunflower proteins on solubility, amino acid composition, and surface activity.

PubMed

Conde, José Miñones; Escobar, María del Mar Yust; Pedroche Jiménez, Justo J; Rodríguez, Francisco Millán; Rodríguez Patino, Juan M

2005-10-05

Industrial proteins from agriculture of either animal or vegetable origin, including their peptide derivatives, are of great importance, from the qualitative and quantitative point of view, in food formulations (emulsions and foams). A fundamental understanding of the physical, chemical, and functional properties of these proteins is essential if the performance of proteins in foods is to be improved and if underutilized proteins, such as plant proteins (and their hydrolysates and peptides derivatives), are to be increasingly used in traditional and new processed food products (safe, high-quality, health foods with good nutritional value). In this contribution we have determined the main physicochemical characteristics (solubility, composition, and analysis of amino acids) of a sunflower protein isolate (SPI) and its hydrolysates with low (5.62%), medium (23.5%), and high (46.3%) degrees of hydrolysis. The hydrolysates were obtained by enzymatic treatment with Alcalase 2.4 L for DH 5.62 and 23.5% and with Alcalase 2.4 L and Flavorzyme 1000 MG sequentially for DH 46.3%. The protein concentration dependence on surface pressure (surface pressure isotherm), a measure of the surface activity of the products (SPI and its hydrolysates), was obtained by tensiometry. We have observed that the degree of hydrolysis has an effect on solubility, composition, and content of the amino acids of the SPI and its hydrolysates. The superficial activity and the adsorption efficiency were also affected by the degree of hydrolysis.

Structural basis for lack of ADP-ribosyltransferase activity in poly(ADP-ribose) polymerase-13/zinc finger antiviral protein.

PubMed

Karlberg, Tobias; Klepsch, Mirjam; Thorsell, Ann-Gerd; Andersson, C David; Linusson, Anna; Schüler, Herwig

2015-03-20

The mammalian poly(ADP-ribose) polymerase (PARP) family includes ADP-ribosyltransferases with diphtheria toxin homology (ARTD). Most members have mono-ADP-ribosyltransferase activity. PARP13/ARTD13, also called zinc finger antiviral protein, has roles in viral immunity and microRNA-mediated stress responses. PARP13 features a divergent PARP homology domain missing a PARP consensus sequence motif; the domain has enigmatic functions and apparently lacks catalytic activity. We used x-ray crystallography, molecular dynamics simulations, and biochemical analyses to investigate the structural requirements for ADP-ribosyltransferase activity in human PARP13 and two of its functional partners in stress granules: PARP12/ARTD12, and PARP15/BAL3/ARTD7. The crystal structure of the PARP homology domain of PARP13 shows obstruction of the canonical active site, precluding NAD(+) binding. Molecular dynamics simulations indicate that this closed cleft conformation is maintained in solution. Introducing consensus side chains in PARP13 did not result in 3-aminobenzamide binding, but in further closure of the site. Three-dimensional alignment of the PARP homology domains of PARP13, PARP12, and PARP15 illustrates placement of PARP13 residues that deviate from the PARP family consensus. Introducing either one of two of these side chains into the corresponding positions in PARP15 abolished PARP15 ADP-ribosyltransferase activity. Taken together, our results show that PARP13 lacks the structural requirements for ADP-ribosyltransferase activity. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Oxidative Stress and Digestive Enzyme Activity of Flatfish Larvae in a Changing Ocean

PubMed Central

Pimentel, Marta S.; Faleiro, Filipa; Diniz, Mário; Machado, Jorge; Pousão-Ferreira, Pedro; Peck, Myron A.; Pörtner, Hans O.; Rosa, Rui

2015-01-01

Until now, it is not known how the antioxidant and digestive enzymatic machinery of fish early life stages will change with the combined effects of future ocean acidification and warming. Here we show that high pCO2 (~1600 μatm) significantly decreased metabolic rates (up to 27.4 %) of flatfish larvae, Solea senegalensis, at both present (18 °C) and warmer temperatures (+4 °C). Moreover, both warming and hypercapnia increased the heat shock response and the activity of antioxidant enzymes, namely catalase (CAT) and glutathione S-transferase (GST), mainly in post-metamorphic larvae (30 dph). The lack of changes in the activity of CAT and GST of pre-metamorphic larvae (10 dph) seems to indicate that earlier stages lack a fully-developed antioxidant defense system. Nevertheless, the heat shock and antioxidant responses of post-metamorphic larvae were not enough to avoid the peroxidative damage, which was greatly increased under future environmental conditions. Digestive enzymatic activity of S. senegalensis larvae was also affected by future predictions. Hypercapnic conditions led to a decrease in the activity of digestive enzymes, both pancreatic (up to 26.1 % for trypsin and 74.5 % for amylase) and intestinal enzymes (up to 36.1 % for alkaline phosphatase) in post-metamorphic larvae. Moreover, the impact of ocean acidification and warming on some of these physiological and biochemical variables (namely, lower OCR and higher HSP and MDA levels) were translated into larvae performance, being significantly correlated with decreased larval growth and survival or increased incidence of skeletal deformities. The increased vulnerability of flatfish early life stages under future ocean conditions is expected to potentially determine recruitment and population dynamics in marine ecosystems. PMID:26221723

Oxidative Stress and Digestive Enzyme Activity of Flatfish Larvae in a Changing Ocean.

PubMed

Pimentel, Marta S; Faleiro, Filipa; Diniz, Mário; Machado, Jorge; Pousão-Ferreira, Pedro; Peck, Myron A; Pörtner, Hans O; Rosa, Rui

2015-01-01

Until now, it is not known how the antioxidant and digestive enzymatic machinery of fish early life stages will change with the combined effects of future ocean acidification and warming. Here we show that high pCO2 (~1600 μatm) significantly decreased metabolic rates (up to 27.4 %) of flatfish larvae, Solea senegalensis, at both present (18 °C) and warmer temperatures (+4 °C). Moreover, both warming and hypercapnia increased the heat shock response and the activity of antioxidant enzymes, namely catalase (CAT) and glutathione S-transferase (GST), mainly in post-metamorphic larvae (30 dph). The lack of changes in the activity of CAT and GST of pre-metamorphic larvae (10 dph) seems to indicate that earlier stages lack a fully-developed antioxidant defense system. Nevertheless, the heat shock and antioxidant responses of post-metamorphic larvae were not enough to avoid the peroxidative damage, which was greatly increased under future environmental conditions. Digestive enzymatic activity of S. senegalensis larvae was also affected by future predictions. Hypercapnic conditions led to a decrease in the activity of digestive enzymes, both pancreatic (up to 26.1 % for trypsin and 74.5 % for amylase) and intestinal enzymes (up to 36.1 % for alkaline phosphatase) in post-metamorphic larvae. Moreover, the impact of ocean acidification and warming on some of these physiological and biochemical variables (namely, lower OCR and higher HSP and MDA levels) were translated into larvae performance, being significantly correlated with decreased larval growth and survival or increased incidence of skeletal deformities. The increased vulnerability of flatfish early life stages under future ocean conditions is expected to potentially determine recruitment and population dynamics in marine ecosystems.

Enzymatic cell disruption of microalgae biomass in biorefinery processes.

PubMed

Demuez, Marie; Mahdy, Ahmed; Tomás-Pejó, Elia; González-Fernández, Cristina; Ballesteros, Mercedes

2015-10-01

When employing biotechnological processes for the procurement of biofuels and bio-products from microalgae, one of the most critical steps affecting economy and yields is the "cell disruption" stage. Currently, enzymatic cell disruption has delivered effective and cost competitive results when compared to mechanical and chemical cell disruption methods. However, the introduction of enzymes implies additional associated cost within the overall process. In order to reduce this cost, autolysis of microalgae is proposed as alternative enzymatic cell disruption method. This review aims to provide the state of the art of enzymatic cell disruption treatments employed in biorefinery processes and highlights the use of endopeptidases. During the enzymatic processes of microalgae life cycle, some lytic enzymes involved in cell division and programmed cell death have been proven useful in performing cell lysis. In this context, the role of endopeptidases is emphasized. Mirroring these natural events, an alternative cell disruption approach is proposed and described with the potential to induce the autolysis process using intrinsic cell enzymes. Integrating induced autolysis within biofuel production processes offers a promising approach to reduce overall global costs and energetic input associated with those of current cell disruption methods. A number of options for further inquiry are also discussed. © 2015 Wiley Periodicals, Inc.

Screening of plants used in the European traditional medicine to treat memory disorders for acetylcholinesterase inhibitory activity and anti amyloidogenic activity.

PubMed

Lobbens, Eva S B; Vissing, Karina J; Jorgensen, Lene; van de Weert, Marco; Jäger, Anna K

2017-03-22

Plants used in the traditional medicine of Europe to treat memory dysfunction and/or to enhance memory were investigated for activity against the underlying mechanisms of Alzheimer's disease. To investigate 35 ethanolic extracts of plants, selected using an ethnopharmacological approach, for anti-amyloidogenic activity as well as an ability to inhibit the enzymatic activity of acetylcholinesterase. The anti-amyloidogenic activity of the extracts against amyloid beta was investigated by Thioflavin T fibrillation assays and the ability to inhibit the enzymatic activity of acetylcholinesterase was evaluated monitoring the hydrolysis of acetylthiocholine RESULTS: Under the experimental conditions investigated, extracts of two plants, Carum carvi and Olea sylvestris, inhibited amyloid beta fibrillation considerably, eight plant extracts inhibited amyloid beta fibrillation to some extent, 16 plant extracts had no effect on amyloid beta fibrillation and nine extracts accelerated fibrillation of amyloid beta. Furthermore, five plant extracts from Corydalis species inhibited the enzymatic activity of acetylcholinesterase considerably, one plant extract inhibited the enzymatic activity of acetylcholinesterase to some extent and 29 plant extract had no effect on the enzymatic activity of acetylcholinesterase. An optimal extract in this study would possess acetylcholinesterase inhibitory activity as well as anti-amyloidogenic activity in order to address multiple facets of Alzheimer's disease, until the molecular origin of the disease is unraveled. Unfortunately no such extract was found. Copyright © 2017 Elsevier Ireland Ltd. All rights reserved.

Adenovirus vectors lacking virus-associated RNA expression enhance shRNA activity to suppress hepatitis C virus replication

NASA Astrophysics Data System (ADS)

Pei, Zheng; Shi, Guoli; Kondo, Saki; Ito, Masahiko; Maekawa, Aya; Suzuki, Mariko; Saito, Izumu; Suzuki, Tetsuro; Kanegae, Yumi

2013-12-01

First-generation adenovirus vectors (FG AdVs) expressing short-hairpin RNA (shRNA) effectively downregulate the expressions of target genes. However, this vector, in fact, expresses not only the transgene product, but also virus-associated RNAs (VA RNAs) that disturb cellular RNAi machinery. We have established a production method for VA-deleted AdVs lacking expression of VA RNAs. Here, we showed that the highest shRNA activity was obtained when the shRNA was inserted not at the popularly used E1 site, but at the E4 site. We then compared the activities of shRNAs against hepatitis C virus (HCV) expressed from VA-deleted AdVs or conventional AdVs. The VA-deleted AdVs inhibited HCV production much more efficiently. Therefore, VA-deleted AdVs were more effective than the currently used AdVs for shRNA downregulation, probably because of the lack of competition between VA RNAs and the shRNAs. These VA-deleted AdVs might enable more effective gene therapies for chronic hepatitis C.

Investigation of trypsin-CdSe quantum dot interactions via spectroscopic methods and effects on enzymatic activity.

PubMed

Kaur, Gurvir; Tripathi, S K

2015-01-05

The paper presents the interactions between trypsin and water soluble cadmium selenide (CdSe) quantum dots investigated by spectrophotometric methods. CdSe quantum dots have strong ability to quench the intrinsic fluorescence of trypsin by a static quenching mechanism. The quenching has been studied at three different temperatures where the results revealed that electrostatic interactions exist between CdSe quantum dots and trypsin and are responsible to stabilize the complex. The Scatchard plot from quenching revealed 1 binding site for quantum dots by trypsin, the same has been confirmed by making isothermal titrations of quantum dots against trypsin. The distance between donor and acceptor for trypsin-CdSe quantum dot complexes is calculated to be 2.8 nm by energy transfer mechanisms. The intrinsic fluorescence of CdSe quantum dots has also been enhanced by the trypsin, and is linear for concentration of trypsin ranging 1-80 μl. All the observations evidence the formation of trypsin-CdSe quantum dot conjugates, where trypsin retains the enzymatic activity which in turn is temperature and pH dependent. Copyright © 2014 Elsevier B.V. All rights reserved.

Conferring specificity in redox pathways by enzymatic thiol/disulfide exchange reactions.

PubMed

Netto, Luis Eduardo S; de Oliveira, Marcos Antonio; Tairum, Carlos A; da Silva Neto, José Freire

2016-01-01

Thiol-disulfide exchange reactions are highly reversible, displaying nucleophilic substitutions mechanism (S(N)2 type). For aliphatic, low molecular thiols, these reactions are slow, but can attain million times faster rates in enzymatic processes. Thioredoxin (Trx) proteins were the first enzymes described to accelerate thiol-disulfide exchange reactions and their high reactivity is related to the high nucleophilicity of the attacking thiol. Substrate specificity in Trx is achieved by several factors, including polar, hydrophobic, and topological interactions through a groove in the active site. Glutaredoxin (Grx) enzymes also contain the Trx fold, but they do not share amino acid sequence similarity with Trx. A conserved glutathione binding site is a typical feature of Grx that can reduce substrates by two mechanisms (mono and dithiol). The high reactivity of Grx enzymes is related to the very acid pK(a) values of reactive Cys that plays roles as good leaving groups. Therefore, although distinct oxidoreductases catalyze similar thiol–disulfide exchange reactions, their enzymatic mechanisms vary. PDI and DsbA are two other oxidoreductases, but they are involved in disulfide bond formation, instead of disulfide reduction, which is related to the oxidative environment where they are found. PDI enzymes and DsbC are endowed with disulfide isomerase activity, which is related with their tetra-domain architecture. As illustrative description of specificity in thiol-disulfide exchange, redox aspects of transcription activation in bacteria, yeast, and mammals are presented in an evolutionary perspective. Therefore, thiol-disulfide exchange reactions play important roles in conferring specificity to pathways, a required feature for signaling.

Anti-oxidant, anti-inflammatory and immunomodulating properties of an enzymatic protein hydrolysate from yellow field pea seeds.

PubMed

Ndiaye, Fatou; Vuong, Tri; Duarte, Jairo; Aluko, Rotimi E; Matar, Chantal

2012-02-01

Enzymatic protein hydrolysates of yellow pea seed have been shown to possess high anti-oxidant and anti-bacterial activities. The aim of this work was to confirm the anti-oxidant, anti-inflammatory and immunomodulating activities of an enzymatic protein hydrolysate of yellow field pea seeds. The anti-oxidant and anti-inflammatory properties of peptides from yellow field pea proteins (Pisum sativum L.) were investigated in LPS/IFN-γ-activated RAW 264.7 NO⁻ macrophages. The immunomodulating potential of pea protein hydrolysate (PPH) was then studied in a murine model. Pea protein hydrolysate, after a 12 h pre-treatment, showed significant inhibition of NO production by activated macrophages up to 20%. Moreover, PPH significantly inhibited their secretion of pro-inflammatory cytokines, TNF-α- and IL-6, up to 35 and 80%, respectively. Oral administration of PPH in mice enhanced the phagocytic activity of their peritoneal macrophages and stimulated the gut mucosa immune response. The number of IgA+ cells was elevated in the small intestine lamina propria, accompanied by an increase in the number of IL-4+, IL-10+ and IFN-γ+ cells. This was correlated to up-regulation of IL-6 secretion by small intestine epithelial cells (IEC), probably responsible for B-cell terminal differentiation to IgA-secreting cells. Moreover, PPH might have increased IL-6 production in IECs via the stimulation of toll-like receptors (TLRs) family, especially TLR2 and TLR4 since either anti-TLR2 or anti-TLR4 was able to completely abolish PPH-induced IL-6 secretion. Enzymatic protein degradation confers anti-oxidant, anti-inflammatory and immunomodulating potentials to pea proteins, and the resulted peptides could be used as an alternative therapy for the prevention of inflammatory-related diseases.

Platelets lacking PIP5KIγ have normal integrin activation but impaired cytoskeletal-membrane integrity and adhesion

PubMed Central

Wang, Yanfeng; Zhao, Liang; Suzuki, Aae; Lian, Lurong; Min, Sang H.; Wang, Ziqian; Litvinov, Rustem I.; Stalker, Timothy J.; Yago, Tadayuki; Klopocki, Arkadiusz G.; Schmidtke, David W.; Yin, Helen; Choi, John K.; McEver, Rodger P.; Weisel, John W.; Hartwig, John H.; Abrams, Charles S.

2013-01-01

Three isoforms of phosphatidylinositol-4-phosphate 5-kinase (PIP5KIα, PIP5KIβ, and PIP5KIγ) can each catalyze the final step in the synthesis of phosphatidylinositol-4,5-bisphosphate (PIP2), which in turn can be either converted to second messengers or bind directly to and thereby regulate proteins such as talin. A widely quoted model speculates that only p90, a longer splice form of platelet-specific PIP5KIγ, but not the shorter p87 PIP5KIγ, regulates the ligand-binding activity of integrins via talin. However, when we used mice genetically engineered to lack only p90 PIP5KIγ, we found that p90 PIP5KIγ is not critical for integrin activation or platelet adhesion on collagen. However, p90 PIP5KIγ-null platelets do have impaired anchoring of their integrins to the underlying cytoskeleton. Platelets lacking both the p90 and p87 PIP5KIγ isoforms had normal integrin activation and actin dynamics, but impaired anchoring of their integrins to the cytoskeleton. Most importantly, they formed weak shear-resistant adhesions ex vivo and unstable vascular occlusions in vivo. Together, our studies demonstrate that, although PIP5KIγ is essential for normal platelet function, individual isoforms of PIP5KIγ fulfill unique roles for the integrin-dependent integrity of the membrane cytoskeleton and for the stabilization of platelet adhesion. PMID:23372168

Enzymatic properties and localization of motopsin (PRSS12), a protease whose absence causes mental retardation.

PubMed

Mitsui, Shinichi; Yamaguchi, Nozomi; Osako, Yoji; Yuri, Kazunari

2007-03-09

Motopsin (PRSS12) is a mosaic protease expressed in the central nervous system. Truncation of the human motopsin gene causes nonsyndromic mental retardation. Understanding the enzymatic properties and localization of motopsin protein in the central nervous system will help identify the molecular mechanism by which the loss of motopsin function causes mental retardation. Recombinant motopsin showed amidolytic activity against the synthetic substrate benzyloxycarbonyl-l-phenylalanyl-l-arginine 4-methyl-coumaryl-7-amide. Motopsin activated the single-chain tissue plasminogen activator precursor and exhibited gelatinolytic activity. This enzymatic activity was inhibited by typical serine protease inhibitors such as aprotinin, leupeptin, and (4-amidinophenyl) methanesulfonyl fluoride. Immunocytochemistry using anti-motopsin IgG revealed that both human and mouse motopsin proteins were distributed in discrete puncta along the dendrites and soma as well as axons in cultured hippocampal neurons. In the limbic system, including the cingulate and hippocampal pyramidal neurons and piriform cortex, high level of motopsin protein was expressed at postnatal day 10, but a very low level at 10-week-old mice. Motopsin and tissue plasminogen activator were co-expressed in the cingulate pyramidal neurons at postnatal day 10 and were distributed along dendrites of cultured pyramidal neurons. In cranial nuclei, a moderate level of motopsin protein was detected independently on the developmental stage. Our results suggest that motopsin has multiple functions, such as axon outgrowth, arranging perineuronal environment, and maintaining neuronal plasticity, partly in coordination with other proteases including tissue plasminogen activator.

Enzymatic cascade bioreactor

DOEpatents

Simmons, Blake A.; Volponi, Joanne V.; Ingersoll, David; Walker, Andrew

2007-09-04

Disclosed is an apparatus and method for continuously converting sucrose to .beta.-D-glucose. The method comprises a three stage enzymatic reactor in which an aqueous solution of sucrose is first converted into a solution of fructose and .alpha.-D-glucose by passing it through a porous, packed column containing an inert media on which invertase is immobilized. This solution is then sent through a second packed column containing glucose isomerase and finally a third packed column containing mutarotase. Solution temperature and pH are adjusted to maximize glucose output.

A comparison of the enzymatic properties of three recombinant isoforms of thrombolytic and antibacterial protein--Destabilase-Lysozyme from medicinal leech.

PubMed

Kurdyumov, Alexey S; Manuvera, Valentin A; Baskova, Isolda P; Lazarev, Vassili N

2015-11-21

Destabilase-Lysozyme (mlDL) is a multifunctional i-type enzyme that has been found in the secretions from the salivary glands of medicinal leeches. mlDL has been shown to exhibit isopeptidase, muramidase and antibacterial activity. This enzyme attracts interest because it expresses thrombolytic activity through isopeptidolysis of the ε-(γ-Glu)-Lys bonds that cross-link polypeptide chains in stabilised fibrin. To date, three isoforms of mlDL have been identified. The enzymatic properties of pure mlDL isoforms have not yet been described because only destabilase complexes containing other proteins could be isolated from the salivary gland secretion and because low product yield from the generation of recombinant proteins has made comprehensive testing difficult. In the present study, we optimised the procedures related to the expression, isolation and purification of active mlDL isoforms (mlDL-Ds1, mlDL-Ds2, mlDL-Ds3) using an Escherichia coli expression system, and we detected and compared their muramidase, lytic, isopeptidase and antimicrobial activities. After optimisation, the product yield was 30 mg per litre of culture. The data obtained in our study led to the suggestion that the recombinant mlDL isoforms isolated from inclusion bodies form stable oligomeric complexes. Analyses of the tested activities revealed that all isoforms exhibited almost identical patterns of pH and ionic strength effects on the activities. We determined that mlDL-Ds1, 2, 3 possessed non-enzymatic antibacterial activity independent of their muramidase activity. For the first time, we demonstrated the fibrinolytic activity of the recombinant mlDL and showed that only intact proteins possessed this activity, suggesting their enzymatic nature. The recombinant Destabilase-Lysozyme isoforms obtained in our study may be considered potential thrombolytic agents that act through a mechanism different from that of common thrombolytics.

Microbial Enzymatic Degradation of Biodegradable Plastics.

PubMed

Roohi; Bano, Kulsoom; Kuddus, Mohammed; Zaheer, Mohammed R; Zia, Qamar; Khan, Mohammed F; Ashraf, Ghulam Md; Gupta, Anamika; Aliev, Gjumrakch

2017-01-01

The renewable feedstock derived biodegradable plastics are important in various industries such as packaging, agricultural, paper coating, garbage bags and biomedical implants. The increasing water and waste pollution due to the available decomposition methods of plastic degradation have led to the emergence of biodegradable plastics and biological degradation with microbial (bacteria and fungi) extracellular enzymes. The microbes utilize biodegradable polymers as the substrate under starvation and in unavailability of microbial nutrients. Microbial enzymatic degradation is suitable from bioremediation point of view as no waste accumulation occurs. It is important to understand the microbial interaction and mechanism involved in the enzymatic degradation of biodegradable plastics under the influence of several environmental factors such as applied pH, thermo-stability, substrate molecular weight and/or complexity. To study the surface erosion of polymer film is another approach for hydrolytic degradation characteristion. The degradation of biopolymer is associated with the production of low molecular weight monomer and generation of carbon dioxide, methane and water molecule. This review reported the degradation study of various existing biodegradable plastics along with the potent degrading microbes (bacteria and fungi). Patents available on plastic biodegradation with biotechnological significance is also summarized in this paper. This paper assesses that new disposal technique should be adopted for the degradation of polymers and further research is required for the economical production of biodegradable plastics along with their enzymatic degradation. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

IMPORTANCE OF ENZYMATIC BIOTRANSFORMATION IN IMMUNOTOXICOLOGY

EPA Science Inventory

Many immunotoxic compounds, such as benzene and other organic solvents, pesticides, mycotoxins and polycyclic aromatic hydrocarbons, can alter immune function only after undergoing enzyme-mediated reactions within various tissues. In the review that follows, the role of enzymatic...

Enzymatic catalysis of formation of Z-aspartame in ionic liquid - An alternative to enzymatic catalysis in organic solvents.

PubMed

Erbeldinger, M; Mesiano, A J; Russell, A J

2000-01-01

We present the first report of enzymatic catalysis in an ionic liquid. The virtually nonexistent vapor pressure makes ionic liquids an exciting new alternative for enzyme-catalyzed syntheses in environmentally friendly environments. Z-aspartame was synthesized in a thermolysin-catalyzed reaction of carbobenzoxy-L-aspartate and L-phenylalanine methyl ester hydrochloride in 1-butyl-3-methylimidazolium hexafluorophosphate (BP6). Ionic liquids such as BP6 are thermally stable and have a remarkable range of temperatures over which they remain liquid (300 degrees C). With an initial rate of 1.2 +/- 0.1 nmol min(-)(1) mg(-)(1), we observed a competitive rate in comparison to that of enzymatic synthesis in organic solvent. Additionally, the enzyme exhibits outstanding stability, which would normally require immobilization.

Secretion profiles of fungi as potential tools for metal ecotoxicity assessment: a study of enzymatic system in Trametes versicolor.

PubMed

Lebrun, Jérémie D; Demont-Caulet, Nathalie; Cheviron, Nathalie; Laval, Karine; Trinsoutrot-Gattin, Isabelle; Mougin, Christian

2011-01-01

The relationship between the expression of extracellular enzymatic system and a metal stress is scarce in fungi, hence limiting the possible use of secretion profiles as tools for metal ecotoxicity assessment. In the present study, we investigated the effect of Zn, Cu, Pb and Cd, tested alone or in equimolar cocktail, on the secretion profiles at enzymatic and protein levels in Trametesversicolor. For that purpose, extracellular hydrolases (acid phosphatase, β-glucosidase, β-galactosidase and N-acetyl-β-glucosaminidase) and ligninolytic oxidases (laccase, Mn-peroxidase) were monitored in liquid cultures. Fungal secretome was analyzed by electrophoresis and laccase secretion was characterized by western-blot and mass spectrometry analyses. Our results showed that all hydrolase activities were inhibited by the metals tested alone or in cocktail, whereas oxidase activities were specifically stimulated by Cu, Cd and metal cocktail. At protein level, metal exposure modified the electrophoretic profiles of fungal secretome and affected the diversity of secreted proteins. Two laccase isoenzymes, LacA and LacB, identified by mass spectrometry were differentially glycosylated according to the metal exposure. The amount of secreted LacA and LacB was strongly correlated with the stimulation of laccase activity by Cu, Cd and metal cocktail. These modifications of extracellular enzymatic system suggest that fungal oxidases could be used as biomarkers of metal exposure. Copyright © 2010 Elsevier Ltd. All rights reserved.

Enzymatic degradation of endomorphins.

PubMed

Janecka, Anna; Staniszewska, Renata; Gach, Katarzyna; Fichna, Jakub

2008-11-01

Centrally acting plant opiates, such as morphine, are the most frequently used analgesics for the relief of severe pain, even though their undesired side effects are serious limitation to their usefulness. The search for new therapeutics that could replace morphine has been mainly focused on the development of peptide analogs or peptidomimetics with high selectivity for one receptor type and high bioavailability, that is good blood-brain barrier permeability and enzymatic stability. Drugs, in order to be effective, must be able to reach the target tissue and to remain metabolically stable to produce the desired effects. The study of naturally occurring peptides provides a rational and powerful approach in the design of peptide therapeutics. Endogenous opioid peptides, endomorphin-1 and endomorphin-2, are two potent and highly selective mu-opioid receptor agonists, discovered only a decade ago, which display potent analgesic activity. However, extensive studies on the possible use of endomorphins as analgesics instead of morphine met with failure due to their instability. This review deals with the recent investigations that allowed determine degradation pathways of endomorphins in vitro and in vivo and propose modifications that will lead to more stable analogs.

Non-enzymatic browning and flavour kinetics of vacuum dried onion slices

NASA Astrophysics Data System (ADS)

Mitra, Jayeeta; Shrivastava, Shanker L.; Rao, Pavuluri S.

2015-01-01

Onion slices were dehydrated under vacuum to produce good quality dried ready-to-use onion slices. Colour development due to non-enzymatic browning and flavour loss in terms of thiosulphinate concentration was determined, along with moisture content and rehydration ratio. Kinetics of non-enzymatic browning and thiosulphinate loss during drying was analysed. Colour change due to non-enzymatic browning was found to be much lower in the case of vacuum dried onion, and improved flavour retention was observed as compared to hot air dried onion slices. The optical index values for non-enzymatic browning varied from 18.41 to 38.68 for untreated onion slices and from 16.73 to 36.51 for treated slices, whereas thiosulphinate concentration in the case of untreated onion slices was within the range of 2.96-3.92 μmol g-1 for dried sample and 3.71-4.43 μmol g-1 for the treated onion slices. Rehydration ratio was also increased, which may be attributed to a better porous structure attained due to vacuum drying. The treatment applied was found very suitable in controlling non-enzymatic browning and flavour loss during drying, besides increasing rehydration ratio. Hence, high quality dried ready- to-use onion slices were prepared.

Prothrombin activation on the activated platelet surface optimizes expression of procoagulant activity

PubMed Central

Wood, Jeremy P.; Silveira, Jay R.; Maille, Nicole M.; Haynes, Laura M.

2011-01-01

Effective hemostasis relies on the timely formation of α-thrombin via prothrombinase, a Ca2+-dependent complex of factors Va and Xa assembled on the activated platelet surface, which cleaves prothrombin at Arg271 and Arg320. Whereas initial cleavage at Arg271 generates the inactive intermediate prethrombin-2, initial cleavage at Arg320 generates the enzymatically active intermediate meizothrombin. To determine which of these intermediates is formed when prothrombin is processed on the activated platelet surface, the cleavage of prothrombin, and prothrombin mutants lacking either one of the cleavage sites, was monitored on the surface of either thrombin- or collagen-activated platelets. Regardless of the agonist used, prothrombin was initially cleaved at Arg271 generating prethrombin-2, with α-thrombin formation quickly after via cleavage at Arg320. The pathway used was independent of the source of factor Va (plasma- or platelet-derived) and was unaffected by soluble components of the platelet releasate. When both cleavage sites are presented within the same substrate molecule, Arg271 effectively competes against Arg320 (with an apparent IC50 = 0.3μM), such that more than 90% to 95% of the initial cleavage occurs at Arg271. We hypothesize that use of the prethrombin-2 pathway serves to optimize the procoagulant activity expressed by activated platelets, by limiting the anticoagulant functions of the alternate intermediate, meizothrombin. PMID:21131592

Prothrombin activation on the activated platelet surface optimizes expression of procoagulant activity.

PubMed

Wood, Jeremy P; Silveira, Jay R; Maille, Nicole M; Haynes, Laura M; Tracy, Paula B

2011-02-03

Effective hemostasis relies on the timely formation of α-thrombin via prothrombinase, a Ca(2+)-dependent complex of factors Va and Xa assembled on the activated platelet surface, which cleaves prothrombin at Arg271 and Arg320. Whereas initial cleavage at Arg271 generates the inactive intermediate prethrombin-2, initial cleavage at Arg320 generates the enzymatically active intermediate meizothrombin. To determine which of these intermediates is formed when prothrombin is processed on the activated platelet surface, the cleavage of prothrombin, and prothrombin mutants lacking either one of the cleavage sites, was monitored on the surface of either thrombin- or collagen-activated platelets. Regardless of the agonist used, prothrombin was initially cleaved at Arg271 generating prethrombin-2, with α-thrombin formation quickly after via cleavage at Arg320. The pathway used was independent of the source of factor Va (plasma- or platelet-derived) and was unaffected by soluble components of the platelet releasate. When both cleavage sites are presented within the same substrate molecule, Arg271 effectively competes against Arg320 (with an apparent IC(50) = 0.3μM), such that more than 90% to 95% of the initial cleavage occurs at Arg271. We hypothesize that use of the prethrombin-2 pathway serves to optimize the procoagulant activity expressed by activated platelets, by limiting the anticoagulant functions of the alternate intermediate, meizothrombin.

Effect of ascorbic acid postharvest treatment on enzymatic browning, phenolics and antioxidant capacity of stored mung bean sprouts.

PubMed

Sikora, Małgorzata; Świeca, Michał

2018-01-15

Enzymatic browning limits the postharvest life of minimally processed foods, thus the study selected the optimal inhibitors of polyphenol oxidase (PPO) and evaluated their effect on enzymatic browning, phenolics and antioxidant capacity of stored mung bean sprouts. The sprouts treated with 2mM and 20mM ascorbic acid had a lowered PPO activity; compared to the control by 51% and 60%, respectively. The inhibition was reflected in a significant decrease in enzymatic browning. The sprouts treated with 20mM ascorbic acid had 22% and 23% higher phenolic content after 3 and 7days of storage, respectively. Both storage and ascorbic acid treatment increased potential bioaccessibility of phenolics. Generally, there was no effect of the treatments on the antioxidant capacity; however, a significant increase in the reducing potential was determined for the sprouts washed with 20mM ascorbic acid. In conclusion, ascorbic acid treatments may improve consumer quality of stored sprouts. Copyright © 2017 Elsevier Ltd. All rights reserved.

Enzymatic carotenoid cleavage in star fruit (Averrhoa carambola).

PubMed

Fleischmann, Peter; Watanabe, Naoharu; Winterhalter, Peter

2003-05-01

This paper presents the first description of an enzyme fraction exhibiting carotenoid cleavage activity isolated from fruit skin of Averrhoa carambola. Partial purification of the enzyme could be achieved by acetone precipitation, ultrafiltration (300 kDa, 50 kDa), isoelectric focusing (pH 3-10) and sodium dodecyl sulfate polyacrylamide gel electrophoresis (7.5%). In this way, an enzymatically active protein fraction was obtained, consisting of four proteins in the molecular weight range of between 12 and 90 kDa. Using beta-carotene as substrate, the enzyme activity was detected spectrophotometrically at 505 nm. The main reaction product, detected by GC analysis, was beta-ionone. This proves that the isolated enzymes are closely related to aroma metabolism and release of star fruit. The time constant of the reaction was 16.6 min, the Michaelis Constant K(m)=3.6 micromol 1(-1) and the maximum velocity V(max)=10.5 x 10(-3) micromol l(-1) s(-1) mg((Protein))(-1). The optimum temperature was 45 degrees C.

Use of ultrasonic energy in the enzymatic treatment of cotton fabric

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

Yachmenev, V.G.; Blanchard, E.J.; Lambert, A.H.

Application of enzymes in the textile industry is becoming increasingly popular because of mild processing conditions and the capability for replacing harsh organic/inorganic chemicals. The combination of ultrasound with conventional enzymatic treatment of cotton offers significant advantages such as less consumption of expensive enzymes, shorter processing time, less fiber damage, and better uniformity of enzymatic treatment. Laboratory research has shown that introduction of ultrasonic energy during enzymatic treatment resulted in significant improvement in the performance of cellulase enzyme (CELLUSOFT L). It was established that ultrasound does not inactivate the complex structure of the enzyme molecules and weight loss of cottonmore » fabric sonicated and treated with cellulase enzyme increased up to 25--35%. The experimental data indicate that the maximum benefit provided by sonification occurs at relatively low enzyme concentrations. Ultrasonic energy significantly intensified the enzymatic treatment of the cotton fabrics but did not contribute to a decrease in tensile strength of the cotton textiles.« less

Reducing sugar loss in enzymatic hydrolysis of ethylenediamine pretreated corn stover.

PubMed

Li, Wen-Chao; Li, Xia; Qin, Lei; Zhu, Jia-Qing; Han, Xiao; Li, Bing-Zhi; Yuan, Ying-Jin

2017-01-01

In this study, the effect of ethylenediamine (EDA) on enzymatic hydrolysis with different cellulosic substrates and the approaches to reduce sugar loss in enzymatic hydrolysis were investigated. During enzymatic hydrolysis, xylose yield reduced 21.2%, 18.1% and 13.0% with 7.5mL/L EDA for AFEX pretreated corn stover (CS), washed EDA pretreated CS and CS cellulose. FTIR and GPC analysis demonstrated EDA reacted with sugar and produced high molecular weight (MW) compounds. EDA was prone to react with xylose other than glucose. H 2 O 2 and Na 2 SO 3 cannot prevent sugar loss in glucose/xylose-EDA mixture, although they inhibited the browning and high MW compounds formation. By decreasing temperature to 30°C, the loss of xylose yield reduced to only 3.8%, 3.6% and 4.2% with 7.5mL/L EDA in the enzymatic hydrolysis of AFEX pretreated CS, washed EDA pretreated CS and CS cellulose. Copyright © 2016 Elsevier Ltd. All rights reserved.

Activity theory as a tool to address the problem of chemistry's lack of relevance in secondary school chemical education

NASA Astrophysics Data System (ADS)

van Aalsvoort, Joke

In a previous article, the problem of chemistry's lack of relevance in secondary chemical education was analysed using logical positivism as a tool. This article starts with the hypothesis that the problem can be addressed by means of activity theory, one of the important theories within the sociocultural school. The reason for this expectation is that, while logical positivism creates a divide between science and society, activity theory offers a model of society in which science and society are related. With the use of this model, a new course for grade nine has been constructed. This results in a confirmation of the hypothesis, at least at a theoretical level. A comparison with the Salters' approach is made in order to demonstrate the relative merits of a mediated way of dealing with the problem of the lack of relevance of chemistry in chemical education.

Effect of enzymatic hydrolysis on native starch granule structure.

PubMed

Blazek, Jaroslav; Gilbert, Elliot Paul

2010-12-13

Enzymatic digestion of six starches of different botanical origin was studied in real time by in situ time-resolved small-angle neutron scattering (SANS) and complemented by the analysis of native and digested material by X-ray diffraction, differential scanning calorimetry, small-angle X-ray scattering, and scanning electron microscopy with the aim of following changes in starch granule nanostructure during enzymatic digestion. This range of techniques enables coverage over five orders of length-scale, as is necessary for this hierarchically structured material. Starches studied varied in their digestibility and displayed structural differences in the course of enzymatic digestion. The use of time-resolved SANS showed that solvent-drying of digested residues does not induce any structural artifacts on the length scale followed by small-angle scattering. In the course of digestion, the lamellar peak intensity gradually decreased and low-q scattering increased. These trends were more substantial for A-type than for B-type starches. These observations were explained by preferential digestion of the amorphous growth rings. Hydrolysis of the semicrystalline growth rings was explained on the basis of a liquid-crystalline model for starch considering differences between A-type and B-type starches in the length and rigidity of amylopectin spacers and branches. As evidenced by differing morphologies of enzymatic attack among varieties, the existence of granular pores and channels and physical penetrability of the amorphous growth ring affect the accessibility of the enzyme to the substrate. The combined effects of the granule microstructure and the nanostructure of the growth rings influence the opportunity of the enzyme to access its substrate; as a consequence, these structures determine the enzymatic digestibility of granular starches more than the absolute physical densities of the amorphous growth rings and amorphous and crystalline regions of the semicrystalline

Enzymatic degradation of cell wall and related plant polysaccharides.

PubMed

Ward, O P; Moo-Young, M

1989-01-01

Polysaccharides such as starch, cellulose and other glucans, pectins, xylans, mannans, and fructans are present as major structural and storage materials in plants. These constituents may be degraded and modified by endogenous enzymes during plant growth and development. In plant pathogenesis by microorganisms, extracellular enzymes secreted by infected strains play a major role in plant tissue degradation and invasion of the host. Many of these polysaccharide-degrading enzymes are also produced by microorganisms widely used in industrial enzyme production. Most commerical enzyme preparations contain an array of secondary activities in addition to the one or two principal components which have standardized activities. In the processing of unpurified carbohydrate materials such as cereals, fruits, and tubers, these secondary enzyme activities offer major potential for improving process efficiency. Use of more defined combinations of industrial polysaccharases should allow final control of existing enzyme processes and should also lead to the development of novel enzymatic applications.

Microwave pretreatment of paramylon enhances the enzymatic production of soluble β-1,3-glucans with immunostimulatory activity.

PubMed

Gissibl, Alexander; Care, Andrew; Parker, Lindsay M; Iqbal, Sameera; Hobba, Graham; Nevalainen, Helena; Sunna, Anwar

2018-09-15

A hydrothermal microwave pretreatment was established to facilitate the enzymatic production of soluble bioactive β-1,3-glucans from the recalcitrant substrate paramylon. The efficacy of this pretreatment was monitored with a newly developed direct Congo Red dye-based assay over a range of temperatures. Microwave pretreatment at 170 °C for 2 min resulted in a significantly enhanced enzymatic hydrolysis of paramylon. The action of endo-β-1,3- and exo- β-1,3-glucanases on the microwave-pretreated paramylon produced soluble β-1,3-glucans with degrees of polymerisation (DP) ranging from 2-59 and 2-7, respectively. In comparison, acid-mediated hydrolysis of untreated paramylon resulted in β-1,3-glucans with a DP range of 2-38. The hydrolysates were assayed on their immunostimulatory effect on murine macrophages by measuring the production of the inflammation-linked marker tumour necrosis factor alpha (TNFα) using immunofluorescence. All of the tested hydrolysis products were shown to induce TNFα production, with the most significant immunostimulatory effect observed with the hydrolysate from the exo-β-1,3-glucanase treatment. Copyright © 2018 Elsevier Ltd. All rights reserved.

Using temperature-responsive zwitterionic surfactant to enhance the enzymatic hydrolysis of lignocelluloses and recover cellulase by cooling.

PubMed