Showing papers on "Enzyme assay published in 2018"

Use of Fluorogenic Model Substrates for Extracellular Enzyme Activity (EEA) Measurement of Bacteria

Abstract: "ectoenzymes" and the latter "extracellular" enzymes. Substrates for hydrolysis are generally proteins, carbohydrates, fats and organic P- or S- compounds. Mechanisms of decomposition of individual compounds within these groups may be studied by in vitro experiments. However, aquatic microbial ecologists require, in many cases, a more general measurement of the in situ hydrolytic capacity of the prevailing bacterial community. This has led to the adaptation of biochemical methods for determination of overall bacterial extracellular enzyme activities (peptidases, a- and P-glucosidases, chi- tinases, etc.) in natural waters (Table 1). These methods enable us to study the impact of extracellular enzyme activity (EEA) on bacterial substrate uptake, bacterial growth, and water chemistry. The quantitative estimates of total bacterial extracellular enzyme activity are completed by rapid and sensitive tests for the detection of enzymatic properties of bacterial isolates. The methods used for these purposes are based on the application of fluorogenic model substrates. These substrates have some characteristics in common: (1) they contain an artificial fluorescent molecule and one or more natural molecules (e.g., glucose, amino acids), linked by a specific binding (e.g., peptide binding, ester binding); fluorescence is observed after enzymatic splitting of the complex molecule (Figure 1); (2) the hydrolysis of model substrates is competitively inhibited by a variety of natural compounds with the same structural characteristics; (3) hydrolysis of model substrates follows first order enzyme kinetics; and (4) application of those model substrates allows enzyme activity measurements under natural (in situ) conditions within short incubation periods. The latter is highly im- portant for microbial ecologists, because the process of enzymatic hydrolysis is fully

Microbial enzyme-catalyzed processes in soils and their analysis.

Abstract: Currently, measuring enzyme activities in soils or other lignocellulose-based materials is technically feasible; this measurement is particularly suitable for evaluating soil processes of biopolymer (cellulose, hemicelluloses, lignin, chitin and others) degradation by microbes and for assessing cycling and mobilization of principal nutrients including nitrogen, phosphorus and sulfur. With some considerations, assay methods can provide reliable information on the concentration of enzymes in soil or the rates of enzyme-catalyzed processes. Enzyme analyses in recent studies demonstrated a high level of spatial variability of soil enzyme activity both in depth and in space. The vertical gradients of enzyme activities are most developed in forest soils. Furthermore, enzyme activity in soils is regulated by seasonally-dependent variables such as temperature, moisture and the input of fresh litter. While several enzymes are widely produced by different groups of soil microorganisms, some of them can be used as indicators of the presence or activity of specific microbial taxa.

Characterization of gossypol biosynthetic pathway.

Abstract: Gossypol and related sesquiterpene aldehydes in cotton function as defense compounds but are antinutritional in cottonseed products. By transcriptome comparison and coexpression analyses, we identified 146 candidates linked to gossypol biosynthesis. Analysis of metabolites accumulated in plants subjected to virus-induced gene silencing (VIGS) led to the identification of four enzymes and their supposed substrates. In vitro enzymatic assay and reconstitution in tobacco leaves elucidated a series of oxidative reactions of the gossypol biosynthesis pathway. The four functionally characterized enzymes, together with (+)-δ-cadinene synthase and the P450 involved in 7-hydroxy-(+)-δ-cadinene formation, convert farnesyl diphosphate (FPP) to hemigossypol, with two gaps left that each involves aromatization. Of six intermediates identified from the VIGS-treated leaves, 8-hydroxy-7-keto-δ-cadinene exerted a deleterious effect in dampening plant disease resistance if accumulated. Notably, CYP71BE79, the enzyme responsible for converting this phytotoxic intermediate, exhibited the highest catalytic activity among the five enzymes of the pathway assayed. In addition, despite their dispersed distribution in the cotton genome, all of the enzyme genes identified show a tight correlation of expression. Our data suggest that the enzymatic steps in the gossypol pathway are highly coordinated to ensure efficient substrate conversion.

Immobilization/Stabilization of Ficin Extract on Glutaraldehyde-Activated Agarose Beads. Variables That Control the Final Stability and Activity in Protein Hydrolyses

Abstract: Ficin extract has been immobilized on different 4% aminated-agarose beads. Using just ion exchange, immobilization yield was poor and expressed activity did not surpass 10% of the offered enzyme, with no significant effects on enzyme stability. The treatment with glutaraldehyde of this ionically exchanged enzyme produced an almost full enzyme inactivation. Using aminated supports activated with glutaraldehyde, immobilization was optimal at pH 7 (at pH 5 immobilization yield was 80%, while at pH 9, the immobilized enzyme became inactivated). At pH 7, full immobilization was accomplished maintaining 40% activity versus a small synthetic substrate and 30% versus casein. Ficin stabilization upon immobilization could be observed but it depended on the inactivation pH and the substrate employed, suggesting the complexity of the mechanism of inactivation of the immobilized enzyme. The maximum enzyme loading on the support was determined to be around 70 mg/g. The loading has no significant effect on the enzyme stability or enzyme activity using the synthetic substrate but it had a significant effect on the activity using casein; the biocatalysts activity greatly decreased using more than 30 mg/g, suggesting that the near presence of other immobilized enzyme molecules may generate some steric hindrances for the casein hydrolysis.

A comparative study of the binding properties, dipeptidyl peptidase-4 (DPP-4) inhibitory activity and glucose-lowering efficacy of the DPP-4 inhibitors alogliptin, linagliptin, saxagliptin, sitagliptin and vildagliptin in mice.

Abstract: Aims Since 2006, DPP-4 inhibitors have become established therapy for the treatment of type 2 diabetes. Despite sharing a common mechanism of action, considerable chemical diversity exists amongst members of the DPP-4 inhibitor class, raising the question as to whether structural differences may result in differentiated enzyme inhibition and antihyperglycaemic activity. Methods We have compared the binding properties of the most commonly used inhibitors and have investigated the relationship between their inhibitory potency at the level of the enzyme and their acute glucose-lowering efficacy. Results Firstly, using a combination of published crystal structures and in-house data, we demonstrated that the binding site utilized by all of the DPP-4 inhibitors assessed was the same as that used by neuropeptide Y, supporting the hypothesis that DPP-4 inhibitors are able to competitively inhibit endogenous substrates for the enzyme. Secondly, we ascertained that the enzymatic cleft of DPP-4 is a relatively large cavity which displays conformational flexibility to accommodate structurally diverse inhibitor molecules. Finally, we found that for all inhibitors, irrespective of their chemical structure, the inhibition of plasma DPP-4 enzyme activity correlates directly with acute plasma glucose lowering in mice. Conclusion The common binding site utilized by different DPP-4 inhibitors enables similar competitive inhibition of the cleavage of the endogenous DPP-4 substrates. Furthermore, despite chemical diversity and a range of binding potencies observed amongst the DPP-4 inhibitors, a direct relationship between enzyme inhibition in the plasma and glucose lowering is evident in mice for each member of the classes studied.

An Enzyme Cascade-Triggered Fluorogenic and Chromogenic Reaction Applied in Enzyme Activity Assay and Immunoassay

Abstract: An enzyme cascade-triggered reaction with novel signal generation mechanism is beneficial for the development and insight of the enzyme cascade, which is extensively used for signal transduction in potential applications. Inspired by the fluorogenic and chromogenic reaction between dopamine and resorcinol, and the specific catalytic properties of alkaline phosphatase (ALP) and tyrosinase, we designed and synthesized an unconventional substrate of ALP, named p-aminoethyl-phenyl phosphate disodium salt (PAPP). As expected, the ALP and tyrosinase-incubated PAPP solution exhibited pale yellow with intense blue fluorescence upon addition of resorcinol, owing to the ALP-catalyzed transformation of PAPP into an intermediate tyramine, and the tyrosinase-catalyzed hydroxylation of tyramine to dopamine, as well as the specific reaction between dopamine and resorcinol. Therefore, an enzyme cascade system has been developed herein based on the ALP and tyrosinase coupled enzymes-triggered fluorogenic and chromogenic r...

Relationship between Enzyme Activity(Urease-Catalase) and Nutrient Elementin Soil Use

Abstract: This study determined the relationship of urease and catalase enzyme activity and nutrient elements related to the use of soil. We identitified urease and catalase enzymes and calcium (Ca), magnesium (Mg), iron (Fe), zinc (Zn), copper (Cu), and manganese (Mn) as nutrient elements, and total nitrogen (N), phosphorus (P) useful for plants, and exchangeable potassium (K) were identified in soils used for different purposes (agriculture, forest, and pasture), and the relationships between urease and catalase enzymes and these nutrient elements were revealed. Soil samples were taken from 60 points as 2 aspects x3 areas x2 depths x5 recurrences and the analyses were conducted on each sample with 3 recurrences. Duncan’s test was carried out and the results were evaluated. The strongest relationships are identified between Mg and Fe (0.854), and Mg and Mn (0.867). The results of the study indicate that the depth factor has an effect only on catalase enzyme activity and Zn, soil use has an effect only on catalase enzyme activity and urease enzyme activity and Ca and Zn among micronutrient elements, and the aspect factor has an effect on all nutrient elements other than Zn – in contrast to other factors.

Investigating the Effect of Two-Point Surface Attachment on Enzyme Stability and Activity.

Abstract: Immobilization on solid supports provides an effective way to improve enzyme stability and simplify downstream processing for biotechnological applications, which has been widely used in research and in applications. However, surface immobilization may disrupt enzyme structure due to interactions between the enzyme and the supporting substrate, leading to a loss of the enzyme catalytic efficiency and stability. Here, we use a model enzyme, nitroreductase (NfsB), to demonstrate that engineered variants with two strategically positioned surface-tethering sites exhibit improved enzyme stability when covalently immobilized onto a surface. Tethering sites were designed based on molecular dynamics (MD) simulations, and enzyme variants containing cysteinyl residues at these positions were expressed, purified, and immobilized on maleimide-terminated self-assembled monolayer (SAM) surfaces. Sum frequency generation (SFG) vibrational spectroscopy and attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy were used to deduce the NfsB enzyme orientations, which were found to be consistent with those predicted from the MD simulations. Thermal stability analyses demonstrated that NfsB variants immobilized through two tethering sites exhibited generally improved thermal stability compared with enzymes tethered at only one position. For example, NfsB enzyme chemically immobilized via positions 423 and 111 exhibits at least 60% stability increase compared to chemically immobilized NfsB mutant via a single site. This research develops a generally applicable and systematic approach using a combination of simulation and experimental methods to rationally select protein immobilization sites for the optimization of surface-immobilized enzyme activity and stability.

Enzymatic CO2 reduction to formate by formate dehydrogenase from Candida boidinii coupling with direct electrochemical regeneration of NADH

Abstract: Enzymatic conversion of CO2 to formate was carried out in the cathodic cell of a two-chamber electrochemical apparatus where NAD+ was reduced on the surface of a Copper foam electrode. Formate dehydrogenase (FDH) was used as the biocatalyst in both free form and immobilized on the modified electrospun polystyrene nanofibers (EPSNF). The fabricated EPSNF were modified by a multistage procedure including acid treatment, silanization followed by activation with glutaraldehyde. The effects of regenerated NADH concentration and time of enzymatic reaction on the formate production in the both systems were studied. The results indicated that the EPSNF immobilized FDH had a desirable activity, long-term storage stability (41% after 20 days) and reusability after eight cycles of successive reactions (53% of the initial activity). Moreover, it was revealed that the increase of cofactor concentration at the early times of reaction was favorable to the formate production. However, an inhibitory effect was observed at higher concentrations of NADH, and the optimum values of 0.45 mM and 0.51 mM were obtained for the maximum enzyme activity by the free and immobilized enzymes respectively. The produced formate at the optimum cofactor concentration after 300 min was 0.61 mM and 0.31 mM for the free and immobilized enzyme systems. Finally, it can be concluded that the presented process is a promising approach to the enzymatic conversion of CO2.

S-nitrosylation/denitrosylation as a regulatory mechanism of salt stress sensing in sunflower seedlings.

Abstract: Nitric oxide (NO) and various reactive nitrogen species produced in cells in normal growth conditions, and their enhanced production under stress conditions are responsible for a variety of biochemical aberrations. The present findings demonstrate that sunflower seedling roots exhibit high sensitivity to salt stress in terms of nitrite accumulation. A significant reduction in S-nitrosoglutathione reductase (GSNOR) activity is evident in response to salt stress. Restoration of GSNOR activity with dithioerythritol shows that the enzyme is reversibly inhibited under conditions of 120 mM NaCl. Salt stress-mediated S-nitrosylation of cytosolic proteins was analyzed in roots and cotyledons using biotin-switch assay. LC-MS/MS analysis revealed opposite patterns of S-nitrosylation in seedling cotyledons and roots. Salt stress enhances S-nitrosylation of proteins in cotyledons, whereas roots exhibit denitrosylation of proteins. Highest number of proteins having undergone S-nitrosylation belonged to the category of carbohydrate metabolism followed by other metabolic proteins. Of the total 61 proteins observed to be regulated by S-nitrosylation, 17 are unique to cotyledons, 4 are unique to roots whereas 40 are common to both. Eighteen S-nitrosylated proteins are being reported for the first time in plant systems, including pectinesterase, phospholipase d-alpha and calmodulin. Further physiological analysis of glyceraldehyde-3-phosphate dehydrogenase and monodehydroascorbate reductase showed that salt stress leads to a reversible inhibition of both these enzymes in cotyledons. However, seedling roots exhibit enhanced enzyme activity under salinity stress. These observations implicate the role of S-nitrosylation and denitrosylation in NO signaling thereby regulating various enzyme activities under salinity stress in sunflower seedlings.

Pilot scale production of extracellular thermo‐alkali stable laccase from Pseudomonas sp. S2 using agro waste and its application in organophosphorous pesticides degradation

Abstract: Background Laccases are multicopper oxidases that are able to oxidize various aromatic or nonaromatic compounds owing to their multifarious applications. However till now only a few bacterial laccases are isolated and characterized. Hence there is an urgent need to study an extracellular thermo-alkalistable laccase. Results In present study, an extracellular thermo-alkali stable laccase was produced from Pseudomonas sp. S2 in 100 L bioreactor using agro waste (potato peel). The production was 17- fold higher than the control. The enzyme (S2LAC) was purified 12.16±1.6 fold to homogeneity with specific activity of 1089.70±16.8 Umg−1 and molecular mass of 38 kDa. The temperature and pH for maximum enzyme activity were 80°C and 9.0 respectively. The metal ions Na+, K+, Pb+2, Ca+2, Cu+2 and Co+2 enhanced enzyme activity. The purified enzyme showed maximum specificity to Pyrogallol > PPD > L-DOPA > Hydroquinone. The S2LAC was able to degrade organ-phosphorous pesticide including dichlorophos, chlorpyrifos, monocrotophos and profenovos upto 45.99±0.3%, 80.56±0.6%, 75.45±1.3%, 81.84±0.6% respectively in absence of any mediator. Conclusion S2LAC production using agro waste, stability and desirable property of degrading organophosphorus pesticides make attractive for industrial applications.

Methodological recommendations for optimizing assays of enzyme activities in soil samples

Abstract: Assays of enzyme activities in soil samples based on para-nitrophenol (pNP) spectrophotometry are a powerful tool in soil biochemistry. We evaluated potential sources of error and optimization strategies for soil enzyme assays across 12 diverse soils (6 USDA orders, 31–127 mg g−1 soil organic carbon [SOC]), using the activity of soil phosphomonoesterase (PHO) as an example. We hypothesized that dissolved organic matter (DOM) interference, pNP recovery, and substrate concentration would affect calculated enzyme activities, and that this would reflect the method of assay termination: 0.5 M NaOH + 0.5 M CaCl2 (Tabatabai, 1994), 0.2 M NaOH + 2.0 M CaCl2 (Schneider et al., 2000), 0.5 M NaOH + 2.0 M CaCl2 (this study), and 0.1 M Tris (pH 12) + 0.5 M CaCl2 (Klose et al., 2003). Terminations using 0.5 M NaOH increased pNP recovery compared to termination with 0.1 M Tris, but resulted in greater DOM interference (absorbance at 410 nm), which for terminations using NaOH but not Tris was positively correlated with total SOC (R2 = 0.45–0.38). Greatest DOM interference occurred for Andisols for termination with 0.5 M NaOH + 0.5 M CaCl2, which for two Andisols of intermediate SOC (97 and 68 mg g−1) was 1–2 orders of magnitude greater than other soils (346 and 246% overestimation of PHO activity). Increasing CaCl2 concentration (0.5 M–2.0 M) decreased DOM interference, but this effect was less pronounced than the effects of base type or concentration. Enzyme activity tended to be overestimated in assays terminated with NaOH due to DOM interference, and was more greatly underestimated in assays terminated with Tris buffer due to low recovery of pNP, which was soil-specific. Soil PHO Km values, which were not correlated with SOC, varied by soil (4.2–13.3 mM g−1 soil) demonstrating that substrate concentrations routinely employed (typically ≤ 10 mM g−1 soil) are likely insufficient to achieve recommended substrate conditions (5 × Km) for accurate measurement of PHO activity. This study illustrates the importance of a priori determination of soil enzyme Km to achieve conditions nearing substrate saturation, and recommends termination with 0.2 M NaOH + 2.0 M CaCl2, correction for pNP recovery, and correction for DOM absorbance at 410 nm to increase the accuracy of pNP-based enzyme assays in soils. Finally, to improve communication and thus comparison of measured enzyme activities among studies and assay methods (pNP vs 4-methylumbelliferone [MUF]), it is suggested that studies report the concentration of substrate for the final volume used in enzyme assays, report Km values on a soil mass basis, express enzyme activities on a molar pNP basis, and qualify enzyme activities, Km, and Vmax as ‘apparent’ if corrections for interferences are not performed.

The Inhibitory Effect of Flavonoid Aglycones on the Metabolic Activity of CYP3A4 Enzyme

Abstract: Flavonoids are natural compounds that have been extensively studied due to their positive effects on human health. There are over 4000 flavonoids found in higher plants and their beneficial effects have been shown in vitro as well as in vivo. However, data on their pharmacokinetics and influence on metabolic enzymes is scarce. The aim of this study was to focus on possible interactions between the 30 most commonly encountered flavonoid aglycones on the metabolic activity of CYP3A4 enzyme. 6β-hydroxylation of testosterone was used as marker reaction of CYP3A4 activity. Generated product was determined by HPLC coupled with diode array detector. Metabolism and time dependence, as well as direct inhibition, were tested to determine if inhibition was reversible and/or irreversible. Out of the 30 flavonoids tested, 7 significantly inhibited CYP3A4, most prominent being acacetin that inhibited 95% of enzyme activity at 1 µM concentration. Apigenin showed reversible inhibition, acacetin, and chrysin showed combined irreversible and reversible inhibition while chrysin dimethylether, isorhamnetin, pinocembrin, and tangeretin showed pure irreversible inhibition. These results alert on possible flavonoid–drug interactions on the level of CYP3A4.

Effect of high temperature stress on heat shock protein expression and antioxidant enzyme activity of two morphs of the mud crab Scylla paramamosain.

Abstract: The present study aimed to investigate the effect rapid temperature change from moderate temperature to high temperatures on heat shock protein (HSP) expression and antioxidant enzyme activities in mud crabs. Two mud crabs, one with one spine on the outer margin of the carpus of cheliped (Sp1) and another with two spines (Sp2), were acclimated at 25 °C and then transferred to a 33 °C environment, and HSP expression and antioxidant enzyme activity were assessed. HSP70 and HSP60 were markedly up-regulated in the gills and hepatopancreas of Sp1 and Sp2 after exposure to 35 °C. Exposure to 35 °C also significantly increased superoxide dismutase and catalase activity in the gills of Sp1 and Sp2, with transient changes in hepatopancreas. Apart from changes in antioxidant enzyme activities, HSPs were highly up-regulated after exposure to 37 °C, especially for HSP70. Gill HSP70 expression in Sp2 was 6.1 folds that of the control after 24 h of exposure to 37 °C, and 9.2 folds that of Sp1. Moreover, exposure to 37 °C further up-regulated HSP70 in the hepatopancreas of Sp1, compared to that in Sp2. Hence, HSPs play important roles in thermotolerance in S. paramamosain and Sp1 might have a stronger tolerance to hyperthermal stress than Sp2.