Showing papers on "Enzyme assay published in 2019"

The effects of hesperidin on sodium arsenite-induced different organ toxicity in rats on metabolic enzymes as antidiabetic and anticholinergics potentials: A biochemical approach.

Abstract: In our work, it was purposed to investigate the effects of sodium arsenite (SA) and hesperidin (HSP) administered to rats on some metabolic enzymes including carbonic anhydrase (CA), aldose reductase (AR), paraoxonase-1 (PON1), α-glycosidase (α-Gly), butyrylcholine esterase (BChE), acetylcholine esterase (AChE) enzymes activities in the brain, heart, liver, testis, and kidney tissues of rats. CA activities were significantly decreased in testis, liver, and heart tissues of rats given HSP, SA, SA+HSP-100, and SA+HSP-200 compared to control (p < 0.05). In liver tissue, AChE and BChE enzymes activities were significantly reduced given in all groups. In all tissues, α-Gly activity was reduced given in all groups. In the current study, aldose reductase enzyme activity was reduced significantly in testis, brain, and heart tissues of all groups compared to standard (p < 0.05). PON1 enzyme activity was increased significantly in kidney and liver tissues of rats HSP groups and decreased SA groups compared to control. PRACTICAL APPLICATIONS: α-Glycosidase is the key enzyme involved in the digestion of the carbohydrate. Another enzyme α-amylase hydrolyzes the α-linked polysaccharide derivatives into oligosaccharide molecules, and α-glycosidase enzymes, which are characterized in small intestine, catalyze the final stage in the digestive mechanism of carbohydrate molecule to release absorbable monosaccharides like glucose. Conforming to the cholinergic hypothesis, impairment of the cholinergic pathways plays a good role in the development of neurodegenerative diseases like depression, schizophrenia, Alzheimer's disease (AD) problems with the regulation of traumatic brain injury and sleep. The AD is the main reason for dementia disease, and mild to moderate cases are generally treated with AChE inhibitors. Human CA inhibitor compounds are clinically used for more than 70 years as antiglaucoma and diuretics drugs.

Enzymes inhibitors from natural sources with antidiabetic activity: A review.

Abstract: Natural products have been extensively investigated for antidiabetic therapy. Many of the natural products have direct or indirect effect in diabetes pathways as enzyme inhibitors. The most involved mechanisms are inhibition of intestinal alpha-glucosidase and alpha-amylase, lens aldose reductase, oxidative stress protection, inhibition of formation of advanced glycation end products, inhibition of aldose reductase, lowering plasma glucose levels, altering enzyme activity of hexokinases and glucose-6-phosphate, synthesizing and releasing of insulin, postprandial hyperglycemia inhibition, stimulation of GLUT-4, decreasing activity of G6P, lowering the level of skeletal hexokinases, etc. The following medicinal plants products or extracts showed promising effects as enzyme inhibitors: Abelmoschus moschatus, Alangium salvifolium, Azadirachta indica, Bidens pilosa, Boerhaavia diffusa, Capsicum frutescens, Cassia alata, Eclipta alba, Embellica officinalis, Ficus carica, Gentiana Olivier, Glycyrrhiza glabra, Gymnema sylvestre, Hordeum vulgare, Ipomoea aquatica, Juniperus communis, Mangifera indica, Momordica charantia, Ocimum sanctum, Punica granatum, and Zingiber officinale. Some of the group of phytochemicals isolated with enzyme inhibition activities are Alkaloids, sesquiterpene and saponins, polysaccharides, flavonoids, dietary fibers, ferulic acid, tannins, limonene, and oleuropeoside. This review will provide very useful material to enhance the efficiency of rational antidiabetic drug design.

Application of WST-8 based colorimetric NAD(P)H detection for quantitative dehydrogenase assays

Abstract: The reduction of tetrazolium salts by NAD(P)H to formazan product has been widely used to determine the metabolic activity of cells, and as an indicator of cell viability. However, the application of a WST-8 based assay for the quantitative measurement of dehydrogenase enzyme activity has not been described before. In this study, we reported the application of an assay based on the tetrazolium salt WST-8 for the quantitative measurement of dehydrogenase activity. The assay is performed in a microplate format, where a single endpoint is measured at 450 nm. The optimized dehydrogenase-WST-8 assay conditions, the limit of detection (LOD), accuracy, and precision for measuring NAD(P)H, were demonstrated. The sensitivity of the WST-8 assay for detecting NAD(P)H was 5-fold greater than the spectrophotometric measurement of NAD(P)H absorption at 340 nm (LOD of 0.3 nmole vs 1.7 nmole, respectively). In the dehydrogenase assay, the colorimetric WST-8 method exhibits excellent assay reproducibility with a Z’ factor of 0.9. The WST-8 assay was also used to determine dehydrogenase activity in biological samples, and for screening the substrate of uncharacterized short-chain dehydrogenase/oxidoreductase from Burkholderia pseudomallei. The results suggest that the WST-8 assay is a sensitive and rapid method for determining NAD(P)H concentration and dehydrogenase enzyme activity, which can be further applied for the high-throughput screening of dehydrogenases.

Enzyme activity during germination of different cereals: A review

Abstract: The germination process increases enzymatic activity However, this does not occur in the same way in all cereals It depends on the type of enzyme, the cereal, and the conditions of germination D

Purification and characterization of the carbonic anhydrase enzyme from horse mackerel (Trachurus trachurus) muscle and the impact of some metal ions and pesticides on enzyme activity

Abstract: In this paper, the total carbonic anhydrase (CA) enzyme was purified from horse mackerel (Trachurus trachurus) muscle with a specific activity of 23,063.93 EU/mg, purification fold of 551.08, total activity of 1522.22 EU/mL and a yield of 18.50% using sulfanilamide affinity column chromatography. For obtaining the subunit molecular mass and enzyme purity, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for this part was performed and a single band was clearly recorded. The molecular mass of this enzyme was found approximately 35 kDa. The optimum temperature and pH values were obtained from Arrhenius plot. In addition, the inhibitory effects of different heavy metal ions (Fe2+, Cu2+, Co2+, Pb2+ Hg2+ and As3+) and some pesticides (thiram, clofentezine, propineb, deltamethrin, azoxystrobin and thiophanate) on horse mackerel (Trachurus trachurus) muscle tissue CA enzyme activities were investigated by utilizing esterase assay activity. The used metal ions and pesticides had IC50 values in the range of 0.21–13.84 mM and 3.78–70.58 mM, respectively.

Off-on switching of enzyme activity by near-infrared light-induced photothermal phase transition of nanohybrids.

Abstract: The off-on manipulation of enzyme activity is a challenging task. We report a new strategy for reversible off-on control of enzyme activity by near-infrared light. Enzymes acting on macromolecular substrates are embedded with an ultrasmall platinum nanoparticle and decorated with thermoresponsive copolymers, which exhibit upper critical solution temperature (UCST) behavior. The polymer-enzyme nanohybrids form microscale aggregates in solution below the UCST to prevent macromolecular substrates from approaching the enzymes and thus inhibit the enzyme activity, and they disassemble above the UCST to reactivate the enzyme. Upon near-infrared irradiation, platinum nanoparticles inside the enzymes generate heat through a photothermal effect to cause phase transition of the copolymers. Therefore, we can reversibly switch off and on the activities of three enzymes acting on polysaccharide, protein, and plasmid. The enzyme activities are increased by up to 61-fold after laser irradiation. This study provides a facile and efficient method for off-on control of enzyme activity.

Amelioration of cadmium stress in Withania somnifera by ROS management: active participation of primary and secondary metabolism

Abstract: Cadmium (Cd) is considered as a non-essential heavy metal with substantial toxicity on environment. Withania somnifera, a reputed therapeutic herb exhibits vast pharmacological activities due to the presence of steroidal lactones-withanolides. The present study deals with reactive oxygen species (ROS) management through primary and secondary metabolism as adaptive response, on exogenous Cd exposure of W. somnifera. Increased invertase enzyme activity resulted in higher reducing sugars in plant under Cd stress to provide additional carbon source and NADH or NADPH. Higher activities of proline metabolic pathway enzymes such as ornithine aminotransferase (OAT) and pyrroline-5-corboxylate synthase (P5CS) resulted in elevated proline accumulation. The considerable participation of phenylpropanoid metabolism also found dominantly upregulated. Increased enzyme activities of phenylalanine ammonia lyase (PAL), shikimate dehydrogenase (SKDH), glucose-6-phosphate dehydrogenase (G6PDH), and cinnamyl alcohol dehydrogenase (CADH) resulted in phenolics and flavonoids accumulation under higher Cd stress. Upregulation of glutathione-S-transferase (GST) activity conferred its role in Cd chelation. In addition, glutamate oxaloacetate transaminase (GOT), amyl esterase (EST) and diaphorase (DIA) established their participation in Cd tolerance mechanism. Thus, present study elucidated major role of proline and phenylpropanoid metabolism in providing the osmoticum and antioxidants to the plants under Cd stress. The information may be helpful in developing stress resistant plants by targeting these pathways using conventional and molecular approaches.

Knockout of OsPRP1, a gene encoding proline-rich protein, confers enhanced cold sensitivity in rice (Oryza sativa L.) at the seedling stage

Abstract: Proline-rich proteins (PRPs) play multiple physiological and biochemical roles in plant growth and stress response. In this study, we reported that the knockout of OsPRP1 induced cold sensitivity in rice. Mutant plants were generated by CRISPR/Cas9 technology to investigate the role of OsPRP1 in cold stress and 26 mutant plants were obtained in T0 generation with the mutation rate of 85% including 15% bi-allelic, 53.3% homozygous, and 16.7% heterozygous and 16 T-DNA-free lines in T1 generation. The conserved amino acid sequence was changed and the expression level of OsPRP1 was reduced in mutant plants. The OsPRP1 mutant plants displayed more sensitivity to cold stress and showed low survival rate with decreased root biomass than wild-type (WT) and homozygous mutant line with large fragment deletion was more sensitive to low temperature. Mutant lines accumulated less antioxidant enzyme activity and lower levels of proline, chlorophyll, abscisic acid (ABA), and ascorbic acid (AsA) content relative to WT under low-temperature stress. The changes of antioxidant enzymes were examined in the leaves and roots with exogenous salicylic acid (SA) treatment which resulted in increased activity of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) under cold stress, while enzyme antioxidant activity was lower in untreated seedlings which showed that exogenous SA pretreatment could alleviate the low-temperature stress in rice. Furthermore, the expression of three genes encoding antioxidant enzyme activities (SOD4, POX1, and OsCAT3) was significantly down-regulated in the mutant lines as compared to WT. These results suggested that OsPRP1 enhances cold tolerance by modulating antioxidants and maintaining cross talk through signaling pathways. Therefore, OsPRP1 gene could be exploited for improving cold tolerance in rice and CRISPR/Cas9 technology is helpful to study the function of a gene by analyzing the phenotypes of knockout mutants generated.

Is the enzymatic hydrolysis of soil organic phosphorus compounds limited by enzyme or substrate availability

Abstract: The mineralization of soil organic phosphorus (P) is catalysed by extracellular phosphatases that hydrolyse different non-phytate phosphomonoesters (e.g., sugar phosphates and nucleotides), inositol phosphates (e.g., phytic acids), and phosphodiesters (e.g., nucleic acids, phospholipids). The availabilities of both the organic P compound and the respective phosphatase enzyme jointly determine whether the hydrolysis reaction is substrate limited (enzyme availability > substrate availability) or enzyme limited (enzyme availability < substrate availability), with potential consequences for overall organic P composition and accumulation in soil. Our objective was to evaluate whether the hydrolysis of various organic P compounds in soil is limited by availability of substrate or enzyme. To this end, we combined the principles of enzyme activity assays and enzyme addition assays. Ten soils with contrasting properties and origins received either model organic P substrate, specific phosphatases or a combination of both, added either to a soil:water suspension or a soil:water filtrate. Soil indigenous activity of phosphatases was low in all filtrates, confirming that enzymes were mostly associated with the solid fraction of the soil. In soil suspensions, the rapid hydrolysis of added non-phytate phosphomonoester substrate by soil indigenous enzymes indicated high availability of non-phytase phosphomonoesterase enzymes. In combination with the low availability of non-phytate phosphomonoester substrate in soil suspensions, determined by adding enzymes to the suspensions, this indicated a substrate limitation for the hydrolysis of non-phytate phosphomonoesters in soil. In contrast, enzyme-limitation was found for phytate and phosphodiesters. A review of the available literature on the production, stabilisation and hydrolytic activity of each of the three analysed organic P substrates and the respective enzymes supported our findings. We therefore suggest that not only the production and stabilisation but also differences in available phosphatase enzymes strongly determine the occurrence of specific organic P compounds in soil.

Lignin peroxidase isoenzyme: a novel approach to biodegrade the toxic synthetic polymer waste.

Abstract: Fungal metabolites are playing an immense role in developing various sustainable waste treatment processes. The present study aimed at production and characterization of fungal lignin peroxidase (EC 1.11.1.14) with a potential to degrade Polyvinyl Chloride. Optimization studies revealed that the maximum enzyme production occurred at a temperature 25°C, pH 5 in the 4th week of the incubation period with fungal strain. Enzyme assay was performed to find out the dominating enzyme in the culture broth. The molecular weight of the enzyme was found to be 46 kDa. Partially purified lignin peroxidase from Phanerocheate chrysosporium was used for the degradation of PVC films. A significant reduction in the weight of PVC film was observed (31%) in shake flask experiment. FTIR spectra of the enzyme-treated plastic film revealed structural changes in the chemical composition, indicating a specific peak at 2943 cm−1 that corresponded to alkenyl C–H stretch. Moreover, deterioration on the surface of PVC films w...

The enzyme activity of histone deacetylase 8 is modulated by a redox-switch.

Abstract: Enzymes from the histone deacetylase (HDAC) family are highly regulated by different mechanisms. However, only very limited knowledge exists about the regulation of HDAC8, an established target in multiple types of cancer. A previous dedicated study of HDAC class I enzymes identified no redox-sensitive cysteinyl thiol in HDAC8. This is in contrast to the observation that HDAC8 preparations show different enzyme activities depending on the addition of reducing agents. In the light of the importance of HDAC8 in tumorigenesis a possible regulation by redox signaling was investigated using biochemical and biophysical methods combined with site directed mutagenesis. The occurrence of a characteristic disulfide bond under oxidizing conditions is associated with a complete but reversible loss of enzyme activity. Cysteines 102 and 153 are the integral components of the redox-switch. A possible regulation of HDAC8 by redox signal transduction is suggested by the observed relationship between inhibition of reactive oxygen species generating NOX and concomitant increased HDAC8 activity in neuroblastoma tumor cells. The slow kinetics for direct oxidation of HDAC8 by hydrogen peroxide suggests that transmitters of oxidative equivalents are required to transfer the H2O2 signal to HDAC8.

Pectinase Production from Schizophyllum commune Through Central Composite Design Using Citrus Waste and Its Immobilization for Industrial Exploitation

Abstract: Pectinase is an important group of industrial enzymes. Pectinase manufacturing occupies about 10% of the overall enzyme production world over. The aim of this study is to produce pectin lyase from Schizophyllum commune using the mosambi (sweet lime) fruit peels as substrate in solid state fermentation. The cultural parameters optimized through response surface methodology showed maximum pectin lyase production of 480.45 U/mL at initial medium pH of 6, incubation temperature of 35 °C, time period of 1 day, substrate concentration of 15 g and 3 mL of inoculum size. A purification fold of 3.08 with 355 U/mg specific activity and 4.16% yield was obtained after purification. Enzyme immobilization was done by entrapment with sodium alginate and adsorption with chitosan. Chitosan immobilized enzyme exhibited best thermal stability in the range of 45–55 °C and pH 8.0–9.0. Enzyme activity was stimulated in the presence of Ca2+ and Mg2+ while EDTA inhibited the enzyme activity. Chitosan immobilized pectin lyase was stable up to six cycles of reuse. The pH and thermal stability of S. commune pectin lyase makes it an important enzyme for industrial use. The results showed that pectin lyase produced from S. commune has significant potential for applications in the detergent and fruit juice industry. The enzyme produced from citrus agro waste via the proposed optimized biotechnological process can be explored for multiple industrial applications.

ε-Polylysine Inhibits Shewanella putrefaciens with Membrane Disruption and Cell Damage

Abstract: e-Polylysine (e-PL) was studied for the growth inhibition of Shewanella putrefaciens (S. putrefaciens). The minimal inhibitory concentration (MIC) of e-PL against S. putrefaciens was measured by the broth dilution method, while the membrane permeability and metabolism of S. putrefaciens were assessed after e-PL treatment. Additionally, growth curves, the content of alkaline phosphatase (AKP), the electrical conductivity (EC), the UV absorbance and scanning electron microscope (SEM) data were used to study cellular morphology. The impact of e-PL on cell metabolism was also investigated by different methods, such as enzyme activity (peroxidase [POD], catalase [CAT], succinodehydrogenase [SDH] and malic dehydrogenase [MDH]) and cell metabolic activity. The results showed that the MIC of e-PL against S. putrefaciens was 1.0 mg/mL. When S. putrefaciens was treated with e-PL, the growth of the bacteria was inhibited and the AKP content, electrical conductivity and UV absorbance were increased, which demonstrated that e-PL could damage the cell structure. The enzyme activities of POD, CAT, SDH, and MDH in the bacterial solution with e-PL were decreased compared to those in the ordinary bacterial solution. As the concentration of e-PL was increased, the enzyme activity decreased further. The respiratory activity of S. putrefaciens was also inhibited by e-PL. The results suggest that e-PL acts on the cell membrane of S. putrefaciens, thereby increasing membrane permeability and inhibiting enzyme activity in relation to respiratory metabolism and cell metabolism. This leads to inhibition of cell growth, and eventually cell death.

Combi-CLEAs of glucose oxidase and catalase for conversion of glucose to gluconic acid eliminating the hydrogen peroxide to maintain enzyme activity in a bubble column reactor

Abstract: In this study combined cross-linked aggregates of catalase from bovine liver and glucose-oxidase from Aspergillus niger were prepared, and the effects of the precipitant and crosslinking agents, as well as the use of bovine serum albumin (BSA) as a feeder protein, on enzyme immobilization yield and thermal stability of both enzymes, were evaluated. Combi- crosslinking of enzyme aggregates (CLEAs) prepared using dimethoxyethane as precipitant, 25 mM glutaraldehyde and BSA/enzymes mass ratio of 5.45 (w/w), exhibited the highest enzyme activities and stabilities at 40 °C, pH 6.0, and 250 rpm for 5 h. The stability of both immobilized enzymes was fairly similar, eliminating one of the problems of enzyme coimmobilization. Combi-CLEAs were used in gluconic acid (GA) production in a bubble column reactor operated at 40 °C, pH 6.0 and 10 vvm of aeration, using 26 g L−1 glucose as the substrate. Results showed conversion of around 96% and a reaction course very similar to the same process using free enzymes. The operational half-life was 34 h, determined from kinetic profiles and the first order inactivation model. Combi-CLEAs of glucose-oxidase and catalase were shown to be a robust biocatalyst for applications in the production of gluconic acid from glucose.

A High-Throughput Mass Spectrometric Enzyme Activity Assay Enabling the Discovery of Cytochrome P450 Biocatalysts

Abstract: Assaying for enzymatic activity is a persistent bottleneck in biocatalyst and drug development. Existing high-throughput assays for enzyme activity tend to be applicable only to a narrow range of biochemical transformations, whereas universal enzyme characterization methods usually require chromatography to determine substrate turnover, greatly diminishing throughput. We present an enzyme activity assay that allows the high-throughput mass-spectrometric detection of enzyme activity in complex matrices without the need for a chromatographic step. This technology, which we call probing enzymes with click-assisted NIMS (PECAN), can detect the activity of medically and biocatalytically significant cytochrome P450s in cell lysate, microsomes, and bacteria. Using this approach, a cytochrome P450BM3 mutant library was successfully screened for the ability to catalyze the oxidation of the sesquiterpene valencene.

LOX inhibition downregulates MMP-2 and MMP-9 in gastric cancer tissues and cells.

Abstract: Objective: The objective of this study was to analyze the effects of lysyl oxidase (LOX) on the expression and enzyme activity of the matrix metalloproteinases 2 (MMP-2) and 9 (MMP-9) and to study its preliminary effect mechanisms. Methods: We collected fresh cancer specimens from 49 gastric cancer patients who underwent surgery. Immunohistochemistry was used to quantitate the protein expression levels of LOX and MMP-9 in gastric cancer tissues and to analyze their correlation. Also, six-week old nude mice were divided into a control group and a LOX inhibition group. SGC-7901 gastric cancer cells were inoculated subcutaneously into the backs of the two groups of these mice to construct a gastric cancer-bearing nude mouse model. In the LOX inhibition group, β-aminopropionitrile (BAPN) was used to inhibit LOX. Western blotting was used to quantitate the relative expression levels of MMP-2 and MMP-9 in mouse tumor tissues, and gelatin zymography was used to quantitate their enzyme activity levels. In addition, BGC-823 gastric cancer cells were cultured, then 0.1 mM, 0.2 mM, and 0.3 mM BAPN and 2.5 nM, 5 nM, and 10 nM LOX were added to treat BGC-823 cells. ELISA and gelatin zymography were used to quantitate the protein concentrations and changes in enzyme activity of MMP-2 and MMP-9 in the culture supernatant. Western blotting was used to quantitate the relative expression levels of platelet derived growth factor receptor (PDGFR) in the BGC-823 gastric cancer cells after LOX inhibition and exogenous LOX addition. Results: In the tissues from the gastric cancer patients, the relative expression levels of LOX and MMP-9 were positively correlated (r = 0.326, P < 0.05). Compared with the control group, the tumor tissues from mice in the LOX inhibition group had reduced relative expression levels and enzyme activities of MMP-2 and MMP-9 (P < 0.05). After LOX were inhibited with different concentrations of BAPN in BGC-823 gastric cancer cells, the protein concentrations and enzyme activity levels of MMP-2 and MMP-9 in the culture supernatants were decreased (P < 0.05). In addition, the relative expression level of PDGFR in gastric cancer was decreased when BAPN concentrations increased, showing a negative dose-dependent manner (rPDGFR-α = -0.964, rPDGFR-β = -0.988, P < 0.05). After exogenous LOX treating BGC-823 cells, the concentrations and enzyme activity levels of MMP-2 and MMP-9 in the cell supernatant were increased (P < 0.05). Further, the relative expression of PDGFR in gastric cancer cells was increased with the increase of exogenous LOX, showing a positive dose-dependent manner (rPDGFR-α=0.952, rPDGFR-β=0.953, P<0.05). Conclusions: LOX inhibition can inhibit the expression and enzyme activity of MMP-2 and MMP-9 in gastric cancer tissues and cells, and the probable mechanism is through its effects on the PDGF-PDGFR signaling pathway.

The effect of cold atmospheric plasma on diabetes-induced enzyme glycation, oxidative stress, and inflammation; in vitro and in vivo.

Abstract: Cold atmospheric plasma (CAP) is known as the versatile tool in different biological, and medical applications. In this study, we investigated the effect of cold plasma on diabetes via in vitro and in vivo assessments. We performed the in vitro assay to evaluate the impact of CAP on glycated glutathione peroxidase (GPx) through enzyme activity measurement as a function index and far- and near-UV circular dichroism (CD) and fluorescence analysis as structure indices. The result of in vitro assessment showed that the exposure of glycated GPx to plasma causes a considerable increase in enzyme activity up to 30%. Also, the evaluation of far- and near-UV CD and fluorescence analysis indicated a modification in the protein structure. According to obtained result from in vitro assessment, in vivo assay evaluated the effect of CAP on diabetic mice through analyzing of blood glucose level (BGL), advanced glycation end products (AGEs), antioxidant activity, oxidative stress biomarkers such as malondialdehyde (MDA), advanced oxidation protein products (AOPP), and oxidized low-density lipoprotein (oxLDL), and inflammation factors including tumor necrosis factor (TNF-α), interleukin-1 (IL-1), and interleukin-6 (IL-6). The result of in vivo experiment also showed a 20% increase in antioxidant activity. Also, the reduction in AGEs, oxidative stress biomarkers, and inflammatory cytokines concentrations was observed. The result of this study revealed that CAP could be useful in diabetes treatment and can be utilized as a complementary method for diabetes therapy.