Showing papers on "Enzyme assay published in 2015"
TL;DR: The results suggest that transgenic lines were able to cope better with salt stress than untransformed controls by protecting photosynthetic and antioxidant enzyme activities.
172 citations
TL;DR: The unique property of solvent-thermostable-alkalophilic, nature proves the potential candidature of this isolate for current mainstream biomass conversion into fuel and other industrial process.
Abstract: The rising concerns about the scarcity of fossil fuels, the emission of green house gasses and air pollution by incomplete combustion of fossil fuel have also resulted in an increasing focus on the use of cellulases to perform enzymatic hydrolysis of the lignocellulosic materials for the generation of bioethanol. The aim of this study was to isolate a potential thermo-solvent tolerant cellulase producing bacterium from natural resources, and then applied for purification and characterization. The purified enzyme was to be accessible for the bioethanol production as well as industrial exploitation (discuss in our next study). It is the first instance when thermo-solvent tolerant cellulase producing bacterium was isolated from soil sample. The culture was identified as Bacillus vallismortis RG-07 by 16S rDNA sequence analysis. Bacillus vallismortis RG-07 reported maximum cellulase production from sugarcane baggase (4105 U ml−1) used as agro-waste carbon source. The cellulase enzyme produced by the Bacillus sp. was purified by (NH4)2SO4 precipitation, ion exchange and gel filtration chromatography, with overall recovery of 28.8%. The molecular weight of purified cellulase was 80 kDa as revealed by SDS-PAGE and activity gel analysis. The optimum temperature and pH for enzyme activity was determined as 65°C and 7.0 and it retained 95 and 75% of activity even at 95°C, and 9.0 respectively. The enzyme activity was enhanced in the presence of organic solvents (30%) n-dodecane, iso-octane, n-decane, xylene, toluene, n-haxane, n-butanol, and cyclohexane, after prolonged incubation (7 days). The enzyme activity was also stimulated by Ca2+, mercaptoethanol, Tween-60, and Sodium hypochloride whereas strongly inhibited by Hg. Kinetic analysis of purified enzyme showed the Km and Vmax to be 1.923 mg ml−1 and 769.230 μg ml−1 min−1, respectively. The unique property of solvent-thermostable-alkalophilic, nature proves the potential candidature of this isolate for current mainstream biomass conversion into fuel and other industrial process.
165 citations
TL;DR: Three studies evaluating the usefulness of serum angiotensin-converting enzyme assays for supporting a diagnosis of active sarcoidosis found increased concentrations in 75.3 per cent of the patients with active, untreated disease had increased serum concentrations of angiotensing enzyme.
Abstract: We have expanded our studies evaluating the usefulness of serum angiotensin-converting enzyme assays for supporting a diagnosis of active sarcoidosis. Increased concentrations (>35 units/ml, >2 SD above the mean for normal control subjects) were found in 57.8 per cent of 391 patients with sarcoidosis; 23.3 per cent of these patients were either receiving corticosteroid medication or had a diagnosis of inactive or resolved disease and had normal serum angiotensin-converting enzyme activity. Thus, 75.3 per cent of the patients with active, untreated disease had increased serum concentrations of angiotensin-converting enzyme. Serum concentrations of angiotensin-converting enzyme tended to be higher in blacks with sarcoidosis than in whites with sarcoidosis. Pediatric control subjects had higher concentrations of angiotensin-converting enzyme than adults; values greater than 50 units/ml were required for the concentration to be considered to be significantly increased. Patients placed on steroid therapy had s...
162 citations
TL;DR: Chl-a and Chl-b contents in safflower leaves were negatively affected by salinity, whereas the carotenoid contents remained unchanged with NaCl concentrations, suggesting ion homeostasis.
136 citations
TL;DR: While octyl-agarose released enzyme molecules after incubation at high temperatures or in the presence of organic solvents and detergents, the covalently immobilized enzyme remained attached to the support even after boiling the enzyme in SDS, eliminating the risks of product contamination.
Abstract: A new heterofunctional support, octyl-glyoxyl agarose, is proposed in this study. The supports were prepared by simple periodate oxidation of the commercial octyl-agarose, introducing 25 μmol of glyoxyl groups per wet gram of support. This support was assayed with three different lipases (those from Candida antarctica (form B), Thermomyces lanuginosus (TLL) or Rhizomucor miehei) and the artificial phospholipase Lecitase Ultra. Used at pH 7, the new support maintained as first immobilization step the lipase interfacial activation. Thus, it was possible to have the purification and immobilization of the enzyme in one step. Moreover, stabilization of the open form of the lipase was achieved. The covalent enzyme/support bonds cannot be obtained if the immobilized enzyme was not incubated at alkaline pH value. This incubation at pH 10 of the previously immobilized enzymes produced a smaller decrease in enzyme activity when compared to the direct immobilization of the enzymes on glyoxyl-agarose at pH 10, because the immobilization via interfacial activation promoted a stabilization of the lipases. Except in the case of TLL (covalent attachment involved 70% of the enzyme molecules), covalent immobilization yield was over 80%. The non-covalent attached enzyme molecules were discarded by washings with detergent solutions and the new biocatalysts were compared to the octyl-agarose immobilized enzymes. While the stability in thermal and organic solvents inactivations was increased for Lecitase Ultra, CALB and RML, TLL improved its stability in organic media but its thermal stability decreased after covalent attachment of the interfacially activated enzyme. This stabilization resulted in octyl-glyoxyl-lipase preparations which presented higher activity in the presence of organic solvents. Finally, while octyl-agarose released enzyme molecules after incubation at high temperatures or in the presence of organic solvents and detergents, the covalently immobilized enzyme remained attached to the support even after boiling the enzyme in SDS, eliminating the risks of product contamination.
122 citations
TL;DR: Gene screening results showed that in several cases, fungi exhibited enzyme activity without the presence of the corresponding gene and vice versa, indicating that the use of genome-derived information for the prediction of potential enzyme production has substantial limitations and cannot replace functional screening of fungal cultures.
106 citations
TL;DR: It is found that a novel aldehyde dehydrogenase, GabD4 from Cupriavidus necator, possessed the highest enzyme activity toward 3‐HPA reported to date, and can be a key enzyme for the development of industrial 3‐HP‐producing microbial strains.
Abstract: 3-Hydroxypropionic acid (3-HP) can be produced in microorganisms as a versatile platform chemical. However, owing to the toxicity of the intermediate product 3-hydroxypropionaldehyde (3-HPA), the minimization of 3-HPA accumulation is critical for enhancing the productivity of 3-HP. In this study, we identified a novel aldehyde dehydrogenase, GabD4 from Cupriavidus necator, and found that it possessed the highest enzyme activity toward 3-HPA reported to date. To augment the activity of GabD4, several variants were obtained by site-directed and saturation mutagenesis based on homology modeling. Escherichia coli transformed with the mutant GabD4_E209Q/E269Q showed the highest enzyme activity, which was 1.4-fold higher than that of wild type GabD4, and produced up to 71.9 g L−1 of 3-HP with a productivity of 1.8 g L−1 h−1. To the best of our knowledge, these are the highest 3-HP titer and productivity values among those reported in the literature. Additionally, our study demonstrates that GabD4 can be a key enzyme for the development of industrial 3-HP-producing microbial strains, and provides further insight into the mechanism of aldehyde dehydrogenase activity. Biotechnol. Bioeng. 2015;112: 356–364. © 2014 Wiley Periodicals, Inc.
97 citations
TL;DR: In this article, the effectivity of different potassium salts on growth and certain components of nitrogen metabolism and antioxidant system in oat and their possible role in amelioration of water stress was analyzed.
Abstract: Potassium is actively involved in many functions such as enzyme activation, osmotic adjustment and uptake of deleterious ions like Na. Present report analyses the effectivity of different potassium salts on growth and certain components of nitrogen metabolism and antioxidant system in oat and their possible role in amelioration of water stress. Potassium induced enhancement in the activities of nitrate reductase and aminotransferases was evident indicating a positive role of potassium in nitrogen metabolism. Potassium supplementation enhanced activities of antioxidant enzymes (superoxide dismutase, catalase and ascorbate peroxidase) and contents of total phenols and tannins, probably strengthening both the enzymatic as well as non enzymatic antioxidant system. Free amino acids, proline and free sugars also exhibited the same trend in treated plants ensuring better plant growth.
95 citations
TL;DR: In this article, the authors reported the suppression of autophagosome clearance and activation of NADPH oxidase (Nox)2 in both high fat-fed murine hearts and palmitate-treated H9C2 cardiomyocytes (CMs).
93 citations
TL;DR: All the extracts inhibited ACE, but ACE inhibition was thought to be improved by the increased number of terminal units in the seed samples, and the effects of enzymes on the mDP and %G of the extracts were related to their enzymatic activity.
84 citations
TL;DR: The alkaline incubation produced an increase in enzyme activity with some substrates, and some of the DVS-CALB preparations exhibited a higher specific activity than the octyl-preparations, suggesting the different orientation of the enzyme.
Abstract: The lipase B from C. antarctica (CALB) has been immobilized on divinylsulfone (DVS) activated agarose beads under different conditions (pH 5–10). In the presence of 0.3% Triton X-100, the immobilization rate was rapid at pH 10 and the slowest one was at pH 5. Incubation at pH 10 for 72 h of the immobilized enzymes before blocking of the support with ethylenediamine permitted improvement of the enzyme stability. Enzyme features (activity, stability, specificity versus different substrates, effect of the pH on enzyme properties) were quite different on the different CALB preparations, suggesting the different orientation of the enzyme. The alkaline incubation produced an increase in enzyme activity with some substrates, and some of the DVS-CALB preparations exhibited a higher specific activity than the octyl-preparations. The indirect fluorescence spectrum of the different immobilized preparations confirmed that different structures of the CALB molecules were generated after immobilization.
TL;DR: This work presents a robust method for separating recalcitrant oligosaccharides from high solid loading hydrolysate in gramme quantities and helps determine whether the commercial enzyme mixtures lack the enzyme activities required to completely de-polymerize the plant cell wall.
Abstract: Accumulation of recalcitrant oligosaccharides during high-solids loading enzymatic hydrolysis of cellulosic biomass reduces biofuel yields and increases processing costs for a cellulosic biorefinery. Recalcitrant oligosaccharides in AFEX-pretreated corn stover hydrolysate accumulate to the extent of about 18–25 % of the total soluble sugars in the hydrolysate and 12–18 % of the total polysaccharides in the inlet biomass (untreated), equivalent to a yield loss of about 7–9 kg of monomeric sugars per 100 kg of inlet dry biomass (untreated). These oligosaccharides represent a yield loss and also inhibit commercial hydrolytic enzymes, with both being serious bottlenecks for economical biofuel production from cellulosic biomass. Very little is understood about the nature of these oligomers and why they are recalcitrant to commercial enzymes. This work presents a robust method for separating recalcitrant oligosaccharides from high solid loading hydrolysate in gramme quantities. Composition analysis, recalcitrance study and enzyme inhibition study were performed to understand their chemical nature.
Oligosaccharide accumulation occurs during high solid loading enzymatic hydrolysis of corn stover (CS) irrespective of using different pretreated corn stover (dilute acid: DA, ionic liquids: IL, and ammonia fibre expansion: AFEX). The methodology for large-scale separation of recalcitrant oligosaccharides from 25 % solids-loading AFEX-corn stover hydrolysate using charcoal fractionation and size exclusion chromatography is reported for the first time. Oligosaccharides with higher degree of polymerization (DP) were recalcitrant towards commercial enzyme mixtures [Ctec2, Htec2 and Multifect pectinase (MP)] compared to lower DP oligosaccharides. Enzyme inhibition studies using processed substrates (Avicel and xylan) showed that low DP oligosaccharides also inhibit commercial enzymes. Addition of monomeric sugars to oligosaccharides increases the inhibitory effects of oligosaccharides on commercial enzymes. The carbohydrate composition of the recalcitrant oligosaccharides, ratios of different DP oligomers and their distribution profiles were determined. Recalcitrance and enzyme inhibition studies help determine whether the commercial enzyme mixtures lack the enzyme activities required to completely de-polymerize the plant cell wall. Such studies clarify the reasons for oligosaccharide accumulation and contribute to strategies by which oligosaccharides can be converted into fermentable sugars and provide higher biofuel yields with less enzyme.
TL;DR: The antihypertensive effect of Nigella sativa oil appears to be mediated by a reduction in cardiac oxidative stress and angiotensin-converting enzyme activity, an increase in cardiac heme oxygenase-1 activity and a prevention of plasma nitric oxide loss.
TL;DR: Both enzymes showed a potential in the removal of caffeic acid, SPS and CPW, and immobilized SBP had the highest oxidation performance.
TL;DR: Experimental evidence is presented that the aromatic amino acids W324 and H579 play critical roles in the stabilization of intermediate carbocations and the potential of these residues to serve as the catalytic base facilitating the terminal deprotonation reaction is discussed.
Abstract: Crystal structural data for (4S)-limonene synthase [(4S)-LS] of spearmint (Mentha spicata L.) were used to infer which amino acid residues are in close proximity to the substrate and carbocation intermediates of the enzymatic reaction. Alanine-scanning mutagenesis of 48 amino acids combined with enzyme fidelity analysis [percentage of (−)-limonene produced] indicated which residues are most likely to constitute the active site. Mutation of residues W324 and H579 caused a significant drop in enzyme activity and formation of products (myrcene, linalool, and terpineol) characteristic of a premature termination of the reaction. A double mutant (W324A/H579A) had no detectable enzyme activity, indicating that either substrate binding or the terminating reaction was impaired. Exchanges to other aromatic residues (W324H, W324F, W324Y, H579F, H579Y, and H579W) resulted in enzyme catalysts with significantly reduced activity. Sequence comparisons across the angiosperm lineage provided evidence that W324 is a conserved residue, whereas the position equivalent to H579 is occupied by aromatic residues (H, F, or Y). These results are consistent with a critical role of W324 and H579 in the stabilization of carbocation intermediates. The potential of these residues to serve as the catalytic base facilitating the terminal deprotonation reaction is discussed.
TL;DR: Recycling of the entire insoluble solids fraction was sufficient for recycling of adhered enzymes together with biomass, indicative of an effective method to increase enzyme productivity.
Abstract: Development of efficient methods for production of renewable fuels from lignocellulosic biomass is necessary to maximize yields and reduce operating costs. One of the main challenges to industrial application of the lignocellulosic conversion process is the high costs of cellulolytic enzymes. Recycling of enzymes may present a potential solution to alleviate this problem. In the present study enzymes associated with the insoluble fraction were recycled after enzymatic hydrolysis of pretreated sugarcane bagasse, utilizing different processing conditions, enzyme loadings, and solid loadings. It was found that the enzyme blend from Chrysoporthe cubensis and Penicillium pinophilum was efficient for enzymatic hydrolysis and that a significant portion of enzyme activity could be recovered upon recycling of the insoluble fraction. Enzyme productivity values (g glucose/mg enzyme protein) over all recycle periods were 2.4 and 3.7 for application of 15 and 30 FPU/g of glucan, representing an increase in excess of ten times that obtained in a batch process with the same enzyme blend and an even greater increase compared to commercial cellulase enzymes. Contrary to what may be expected, increasing lignin concentrations throughout the recycle period did not negatively influence hydrolysis efficiency, but conversion efficiencies continuously improved. Recycling of the entire insoluble solids fraction was sufficient for recycling of adhered enzymes together with biomass, indicative of an effective method to increase enzyme productivity.
TL;DR: Interestingly, the Ca2+ did not improve its enzymatic activity, optimal temperature, or thermostability of the enzyme, indicating that the TpAA was Ca2-independent, which may have potential applications in starch-to-ethanol conversion process.
Abstract: Alpha-amylase is a very important enzyme in the starch conversion process. Most of the α-amylases are calcium-dependent and exhibit poor performance in the simultaneous saccharification and fermentation process of industrial bioethanol production that uses starch as feedstock. In this study, an extracellular amylolytic enzyme was purified from the culture broth of newly isolated Talaromyces pinophilus strain 1-95. The purified amylolytic enzyme, with an apparent molecular weight of 58 kDa on SDS-PAGE, hydrolyzed maltopentaose, maltohexaose, and maltoheptaose into mainly maltose and maltotriose and minor amount of glucose, confirming the endo-acting mode of the enzyme, and hence, was named Talaromyces pinophilus α-amylase (TpAA). TpAA was most active at pH 4.0–5.0 (with the temperature held at 37°C) and 55°C (at pH 5.0), and stable within the pH range of 5.0–9.5 (at 4°C) and below 45°C (at pH 5.0). Interestingly, the Ca2+ did not improve its enzymatic activity, optimal temperature, or thermostability of the enzyme, indicating that the TpAA was Ca2+-independent. TpAA displayed higher enzyme activity toward malto-oligosaccharides and dextrin than other previously reported α-amylases. This highly active Ca2+-independent α-amylase may have potential applications in starch-to-ethanol conversion process.
TL;DR: Investigation of the effect of high water temperature on the regulation of fatty acid metabolism in juvenile Atlantic salmon fed different dietary ARA/EPA ratios showed that fatty acid metabolic utilisation, and likely also their dietary requirements for optimal performance, can be affected by changes in their relative levels and by environmental temperature in Atlantic salmon.
Abstract: Salmons raised in aquaculture farms around the world are increasingly subjected to sub-optimal environmental conditions, such as high water temperatures during summer seasons. Aerobic scope increases and lipid metabolism changes are known plasticity responses of fish for a better acclimation to high water temperature. The present study aimed at investigating the effect of high water temperature on the regulation of fatty acid metabolism in juvenile Atlantic salmon fed different dietary ARA/EPA ratios (arachidonic acid, 20:4n-6/ eicosapentaenoic acid, 20:5n-3), with particular focus on apparent in vivo enzyme activities and gene expression of lipid metabolism pathways. Three experimental diets were formulated to be identical, except for the ratio EPA/ARA, and fed to triplicate groups of Atlantic salmon (Salmo salar) kept either at 10°C or 20°C. Results showed that fatty acid metabolic utilisation, and likely also their dietary requirements for optimal performance, can be affected by changes in their relative levels and by environmental temperature in Atlantic salmon. Thus, the increase in temperature, independently from dietary treatment, had a significant effect on the β-oxidation of a fatty acid including EPA, as observed by the apparent in vivo enzyme activity and mRNA expression of pparα -transcription factor in lipid metabolism, including β-oxidation genes- and cpt1 -key enzyme responsible for the movement of LC-PUFA from the cytosol into the mitochondria for β-oxidation-, were both increased at the higher water temperature. An interesting interaction was observed in the transcription and in vivo enzyme activity of Δ5fad–time-limiting enzyme in the biosynthesis pathway of EPA and ARA. Such, at lower temperature, the highest mRNA expression and enzyme activity was recorded in fish with limited supply of dietary EPA, whereas at higher temperature these were recorded in fish with limited ARA supply. In consideration that fish at higher water temperature recorded a significantly increased feed intake, these results clearly suggested that at high, sub-optimal water temperature, fish metabolism attempted to increment its overall ARA status -the most bioactive LC-PUFA participating in the inflammatory response- by modulating the metabolic fate of dietary ARA (expressed as % of net intake), reducing its β-oxidation and favouring synthesis and deposition. This correlates also with results from other recent studies showing that both immune- and stress- responses in fish are up regulated in fish held at high temperatures. This is a novel and fundamental information that warrants industry and scientific attention, in consideration of the imminent increase in water temperatures, continuous expansion of aquaculture operations, resources utilisation in aquafeed and much needed seasonal/adaptive nutritional strategies.
TL;DR: The study shows the efficacy, durability, and sustainability of immobilized catalytic system which could be efficiently used for various juice processing operations.
Abstract: Purified fungal xylanase was entrapped in alginate beads. Its further cross-linking using glutaraldehyde resulted in large enzyme aggregates which may function as both a catalyst and a support material for numerous substrate molecules. Enzyme cross-linking presented a negative impact on enzyme leaching during repeated washings and recovery of enzyme activity was substantial after twelve cycles of usage. The entrapment followed by cross-linking doubled the total bound activity and also greatly improved the enzyme stability at extreme chemical environment. The wide pH stability, better thermo- and storage stability, lowered Km value, and protection from some metal ions are salient achievements of present immobilization. The study shows the efficacy, durability, and sustainability of immobilized catalytic system which could be efficiently used for various juice processing operations.
TL;DR: There was a significant inverse correlation between residual enzyme ACAD activity and phenotypic severity of ACAD9-deficient patients, providing evidence that in cells where it is strongly expressed, ACad9 plays a physiological role in fatty acid oxidation, which contributes to the severity of the phenotype in AC AD9- deficient patients.
Abstract: Acyl-CoA dehydrogenase 9 (ACAD9) is an assembly factor for mitochondrial respiratory chain Complex I (CI), and ACAD9 mutations are recognized as a frequent cause of CI deficiency. ACAD9 also retains enzyme ACAD activity for long-chain fatty acids in vitro, but the biological relevance of this function remains controversial partly because of the tissue specificity of ACAD9 expression: high in liver and neurons and minimal in skin fibroblasts. In this study, we hypothesized that this enzymatic ACAD activity is required for full fatty acid oxidation capacity in cells expressing high levels of ACAD9 and that loss of this function is important in determining phenotype in ACAD9-deficient patients. First, we confirmed that HEK293 cells express ACAD9 abundantly. Then, we showed that ACAD9 knockout in HEK293 cells affected long-chain fatty acid oxidation along with Cl, both of which were rescued by wild type ACAD9. Further, we evaluated whether the loss of ACAD9 enzymatic fatty acid oxidation affects clinical severity in patients with ACAD9 mutations. The effects on ACAD activity of 16 ACAD9 mutations identified in 24 patients were evaluated using a prokaryotic expression system. We showed that there was a significant inverse correlation between residual enzyme ACAD activity and phenotypic severity of ACAD9-deficient patients. These results provide evidence that in cells where it is strongly expressed, ACAD9 plays a physiological role in fatty acid oxidation, which contributes to the severity of the phenotype in ACAD9-deficient patients. Accordingly, treatment of ACAD9 patients should aim at counteracting both CI and fatty acid oxidation dysfunctions.
TL;DR: In this paper, a hierarchical composite material with a hierarchical flower-like structure and extremely high enzyme activity was synthesized, which was confirmed by Fourier transform infrared spectroscopy, and X-ray diffraction.
Abstract: We have synthesized a novel composite material with a hierarchical flower-like structure and extremely high enzyme activity. The spherical, hierarchical flower-like structure with numerous small flowers was formed through self-assembly using papain as the organic component and Cu3(PO4)2·3H2O as the inorganic component. This hybrid-nanoflower structure was confirmed by Fourier transform infrared spectroscopy, and X-ray diffraction. The enzyme activity of papain embedded in the hybrid nanoflowers was further evaluated using BAEE as a substrate. Compared with free papain in solution, the hybrid materials exhibited extremely high enzyme activity (∼4510%). A study on the relationship of enzyme weight percentage and pattern structure with enzyme activity revealed that enzyme activity was mainly affected by the material structure. These results demonstrate that this hierarchical structure can effectively increase enzyme activity.
TL;DR: The method developed in this study will enable us to carry out a parallelized, multiplex digital ELISA, and demonstrate a dual-color digital enzyme assay with a ALP/4-MUP and β-galactosidase (β-gal)/resorufin-β-d-Galactopyranoside combination.
Abstract: Digitalization of fluorogenic enzymatic assays through the use of femtoliter chamber array technology is an emerging approach to realizing highly quantitative bioassays with single-molecule sensitivity. However, only a few digital fluorogenic enzyme assays have been reported, and the variations of the digital enzyme assays are basically limited to fluorescein- and resorufin-based fluorogenic assays. This limitation hampers the realization of a multiplex digital enzyme assay such as a digital enzyme-linked immunosorbent assay (ELISA). In this study, after optimization of buffer conditions, we achieved a single-molecule digital enzyme alkaline phosphatase (ALP) assay with a cumarin-based fluorogenic substrate, 4-methylunbelliferyl phosphate (4-MUP). When ALP molecules were encapsulated in a 44-femtoliter chamber array at a low ratio of less than 1 molecule per chamber, each chamber showed a discrete fluorescence signal in an all-or-none manner, allowing the digital counting of the number of active enzyme molecules. The fraction of fluorescent chambers linearly decreased with the enzyme concentration, obeying the Poisson distribution as expected. We also demonstrated a dual-color digital enzyme assay with a ALP/4-MUP and β-galactosidase (β-gal)/resorufin-β-D-galactopyranoside combination. The activities of single ALP and β-gal molecules were clearly detected simultaneously. The method developed in this study will enable us to carry out a parallelized, multiplex digital ELISA.
TL;DR: The enzymes from MRL-TL could degrade various aliphatic polyesters; therefore, it might be applied for bioremediation in the polyesters-contaminated environments.
TL;DR: In this article, the authors investigated the enzymatic properties of commercial polygalacturonase (PG) under ultrasound treatment, including enzyme activity, kinetic and thermodynamic properties and temperature stability.
Abstract: Polygalacturonase (PG) is one of the most commonly used enzymes during fruit and vegetable processing in the food industry. Ultrasound has the potential to enhance enzyme activity, modify the PG enzyme and enlarge its application range. This study investigated the enzymatic properties of commercial PG under ultrasound treatment, including enzyme activity, kinetic and thermodynamic properties and temperature stability. These properties were investigated with the aid of a chemical reaction kinetics model, Michaelis–Menten equation, Arrhenius equation, Eyring transition state theory and biphasic inactivation kinetics model. PG structures were also studied using fluorescence spectroscopy and circular dichroism (CD) spectroscopy. The maximum activity of PG was observed at 4.5 W ml−1 intensity and ultrasound duration of 15 min, under which the enzyme activity increased by 20.98% over the control. Results of degradation kinetics and thermodynamics of hydrolysis reactions catalysed by PG certified that ultrasound treatment could make PG exhibit higher reaction ability, which was evidenced from the increased rate constants and reduced thermodynamic parameters. Meanwhile, after ultrasound treatment, the value of Vmax in the enzymatic reaction increased, whereas Km decreased as compared with the control. These results demonstrated that the substrate was converted into the product at a higher rate and efficiency, and the enzyme displayed better affinity to the substrate. Ultrasound improved the temperature stability of PG and prolonged its lifetime without affecting its optimum temperature. Fluorescence spectra and far-UV CD spectra revealed that ultrasound treatment irreversibly decreased the amount of tryptophan on the PG surface but increased the β-sheet in PG secondary conformation, possibly by the exposure of more active sites.
TL;DR: A substantial improvement in the performance of the immobilized enzymes with reference to the free enzyme was obtained, and the relative activities of immobilized enzyme are superior than free enzyme over the broader pH and temperature ranges.
Abstract: Enzymes play an essential role in catalyzing various reactions. However, their instability upon repetitive/prolonged use, elevated temperature, acidic or alkaline pH remains an area of concern. α-Amylase, a widely used enzyme in food industries for starch hydrolysis, was covalently immobilized on the surface of two developed matrices, amino-functionalized silica-coated magnetite nanoparticles (AFSMNPs) alone and covered with chitosan. The synthesis steps and characterizations of NPs were examined by FT-IR, VSM, and SEM. Modified nanoparticles with average diameters of 20–80 nm were obtained. Enzyme immobilization efficiencies of 89 and 74 were obtained for AFSMNPs and chitosan-coated AFSMNPs, respectively. The optimum pH obtained was 6.5 and 8.0 for the enzyme immobilized on AFSMNPs and chitosan-coated AFSMNPs, respectively. Optimum temperature for the immobilized enzyme shifted toward higher temperatures. Considerable enhancements in thermal stabilities were observed for the immobilized enzyme at elevated temperatures up to 80 °C. A frequent use experiment demonstrated that the immobilized enzyme retained 74 and 85 % of its original activity even after 20 times of repeated use in AFSMNPs and chitosan-coated AFSMNPs, respectively. Storage stability demonstrated that free enzyme lost its activity completely within 30 days. But, immobilized enzyme on AFSMNPs and chitosan-coated AFSMNPs preserved 65.73 and 78.63 % of its initial activity, respectively, after 80 days of incubation. In conclusion, a substantial improvement in the performance of the immobilized enzyme with reference to the free enzyme was obtained. Furthermore, the relative activities of immobilized enzyme are superior than free enzyme over the broader pH and temperature ranges.
TL;DR: A small molecule inhibitor of C3larvin enzymatic activity was discovered called M3, and to the authors' knowledge, is the first inhibitor of transferase activity of the C3 toxin family.
TL;DR: The results indicate the potential suitability of the enzyme for industrial application in the production of cellulosic bioethanol and Tween 20 was found to have a beneficial effect on enzyme activity and thermal stability.
Abstract: A novel endoglucanase encoding gene was cloned from Alicyclobacillus vulcanalis and expressed in E. coli. The deduced amino acid sequence showed highest identity with α-l-arabinofuranosidase-like proteins from glycoside hydrolase family 51. The recombinant enzyme was purified by affinity chromatography and characterised in terms of its potential suitability for lignocellulose hydrolysis at high temperature in the production of bioethanol. The purified enzyme displayed maximum activity at 80 °C and pH 3.6–4.5. Tween 20 was found to have a beneficial effect on enzyme activity and thermal stability. When incubated in the presence of 0.1 % Tween 20, the enzyme retained full activity after 72 h at 70 °C and 78 % of original activity after 72 h at 75 °C. Maximum activity was observed on carboxymethyl cellulose, and the purified enzyme also hydrolysed lichenan, barley β-glucan and xylan. The purified enzyme decreased the viscosity of carboxymethyl cellulose when assessed at 70–85 °C and was capable of releasing reducing sugars from acid-pretreated straw at 70 and 75 °C. The results indicate the potential suitability of the enzyme for industrial application in the production of cellulosic bioethanol.
TL;DR: The present review summarizes the literature on CE-based enzyme assays published between January 2010 and April 2015 and refers to immobilized enzyme reactors as well as microfluidic devices applied to the study of enzymatic activity.
Abstract: Capillary electrophoresis (CE) has become a flexible and accurate, high-efficiency analytical separation technique in many areas requiring only minute amounts of sample and chemicals. Thus, CE has also been recognized as a suitable technique to study enzymatic reactions including the determination of Michaelis–Menten kinetic data or the identification and characterization of inhibitors. The most often applied CE-based enzyme assay modes can be divided into two categories: (1) pre-capillary assays where incubations are performed offline followed by CE analysis of substrate(s) and/or product(s) and (2) in-capillary assays in which the enzymatic reaction and analyte separation are performed in the same capillary. In case of the in-capillary assays, the enzyme may be immobilized or in solution. The latter is also referred to as electrophoretically mediated microanalysis (EMMA), while in the case of immobilized enzyme the term immobilized enzyme reactor (IMER) is used. The present review summarizes the literature on CE-based enzyme assays published between January 2010 and April 2015. Immobilized enzyme reactors as well as microfluidic devices applied to the study of enzymatic activity will also be briefly addressed.
TL;DR: The production, purification, and characterization of an extracellular protease released by Rhodotorula mucilaginosa L7 were evaluated and may lead to potential biotechnological applications that require enzyme activity and stability under acidic conditions and/or high salt concentrations.