Showing papers on "Enzyme assay published in 2009"

The influence of time, storage temperature, and substrate age on potential soil enzyme activity in acidic forest soils using MUB-linked substrates and L-DOPA.

Abstract: The purpose of this experiment was to evaluate whether soil storage and processing methods significantly influence measurements of potential in situ enzyme activity in acidic forest soils. More specifically, the objectives were to determine if: (1) duration and temperature of soil storage; (2) duration of soil slurry in buffer; and (3) age of model substrates significantly influence the activity of six commonly measured soil extracellular enzymes using methylumbelliferone (MUB)-linked substrates and l-dihydroxyphenylalanine (l-DOPA). Soil collected and analyzed for enzyme activity within 2 h was considered the best measure of potential in situ enzyme activity and the benchmark for all statistical comparisons. Sub-samples of the same soil were stored at either 4 °C or −20 °C. In addition to the temperature manipulation, soils experienced two more experimental treatments. First, enzyme activity was analyzed 2, 7, 14, and 21 days after collection. Second, MUB-linked substrate was added immediately (i.e. <20 min) or 2 h after mixing soil with buffer. Enzyme activity of soil stored at 4 °C was not significantly different from soil stored at −20 °C. The duration of soil storage was minimal for β-glucosidase, β-xylosidase, and peroxidase activity. N-acetyl-glucosaminidase (NAGase), phosphatase, and phenol oxidase activity appeared to change the most when compared to fresh soils, but the direction of change varied. Likewise, the activities of these enzymes were most sensitive to extended time in buffer. Fluorometric MUB and MUB-linked substrates generally had a 3-day shelf life before they start to significantly suppress reported activities when kept at 4 °C. These findings suggest that the manner in which acidic forest soils are stored and processed are site and enzyme specific and should not initially be trivialized when conducting enzyme assays focusing on NAGase, phosphatase, and phenol oxidase. The activities of β-glucosidase, β-xylosidase, and peroxidase are insensitive to storage and processing methods.

Effects of Long-Term Nitrogen Addition on Microbial Enzyme Activity in Eight Forested and Grassland Sites: Implications for Litter and Soil Organic Matter Decomposition

Abstract: Long-term nitrogen (N) addition experiments have found positive, negative, and neutral effects of added N on rates of decomposition. A leading explanation for this variation is differential effects of N on the activity of microbially produced extracellular enzymes involved in decomposition. Specifically, it is hypothesized that adding N to N-limited ecosystems increases activity of cellulose degrading enzymes and decreases that of lignin degrading enzymes, and that shifts in enzyme activity in response to added N explain the decomposition response to N fertilization. We measured litter and soil organic matter (SOM) decomposition and microbial enzyme activity in a long-term N fertilization experiment at eight forested and grassland sites in central Minnesota, USA, to determine (1) variation among sites in enzyme activity, (2) variation in the response of enzymes, litter decomposition, and soil respiration to added N, and (3) whether changes in enzyme activity in response to added N explained variability among sites in the effect of N on litter and SOM decomposition. Site differences in pH, moisture, soil carbon, and microbial biomass explained much of the among-site variation in enzyme activity. Added N generally stimulated activities of cellulose degrading and N- and phosphorus-acquiring enzymes in litter and soil, but had no effect on lignin degrading enzyme activity. In contrast, added N generally had negative or neutral effects on litter and SOM decomposition in the same sites, with no correspondence between effects of N on enzyme activity and decomposition across sites.

Seasonal variation in enzyme activities and temperature sensitivities in Arctic tundra soils

Abstract: Arctic soils contain large amounts of organic matter due to very slow rates of detritus decomposition. The first step in decomposition results from the activity of extracellular enzymes produced by soil microbes. We hypothesized that potential enzyme activities are low relative to the large stocks of organic matter in Arctic tundra soils, and that enzyme activity is low at in situ temperatures. We measured the potential activity of six hydrolytic enzymes at 4 and 20 °C on four sampling dates in tussock, intertussock, shrub organic, and shrub mineral soils at Toolik Lake, Alaska. Potential activities of N-acetyl glucosaminidase, β-glucosidase, and peptidase tended to be greatest at the end of winter, suggesting that microbes produced enzymes while soils were frozen. In general, enzyme activities did not increase during the Arctic summer, suggesting that enzyme production is N-limited during the period when temperatures would otherwise drive higher enzyme activity in situ. We also detected seasonal variations in the temperature sensitivity (Q10) of soil enzymes. In general, soil enzyme pools were more sensitive to temperature at the end of the winter than during the summer. We modeled potential in situβ-glucosidase activities for tussock and shrub organic soils based on measured enzyme activities, temperature sensitivities, and daily soil temperature data. Modeled in situ enzyme activity in tussock soils increased briefly during the spring, then declined through the summer. In shrub soils, modeled enzyme activities increased through the spring thaw into early August, and then declined through the late summer and into winter. Overall, temperature is the strongest factor driving low in situ enzyme activities in the Arctic. However, enzyme activity was low during the summer, possibly due to N-limitation of enzyme production, which would constrain enzyme activity during the brief period when temperatures would otherwise drive higher rates of decomposition.

Computational structure-based redesign of enzyme activity.

Abstract: We report a computational, structure-based redesign of the phenylalanine adenylation domain of the nonribosomal peptide synthetase enzyme gramicidin S synthetase A (GrsA-PheA) for a set of noncognate substrates for which the wild-type enzyme has little or virtually no specificity. Experimental validation of a set of top-ranked computationally predicted enzyme mutants shows significant improvement in the specificity for the target substrates. We further present enhancements to the methodology for computational enzyme redesign that are experimentally shown to result in significant additional improvements in the target substrate specificity. The mutant with the highest activity for a noncognate substrate exhibits 1/6 of the wild-type enzyme/wild-type substrate activity, further confirming the feasibility of our computational approach. Our results suggest that structure-based protein design can identify active mutants different from those selected by evolution.

An optimized microplate assay system for quantitative evaluation of plant cell wall–degrading enzyme activity of fungal culture extracts

Abstract: Developing enzyme cocktails for cellulosic biomass hydrolysis complementary to current cellulase systems is a critical step needed for economically viable biofuels production. Recent genomic analysis indicates that some plant pathogenic fungi are likely a largely untapped resource in which to prospect for novel hydrolytic enzymes for biomass conversion. In order to develop high throughput screening assays for enzyme bioprospecting, a standardized microplate assay was developed for rapid analysis of polysaccharide hydrolysis by fungal extracts, incorporating biomass substrates. Fungi were grown for 10 days on cellulose- or switchgrass-containing media to produce enzyme extracts for analysis. Reducing sugar released from filter paper, Avicel, corn stalk, switchgrass, carboxymethylcellulose, and arabinoxylan was quantified using a miniaturized colorimetric assay based on 3,5-dinitrosalicylic acid. Significant interactions were identified among fungal species, growth media composition, assay substrate, and temperature. Within a small sampling of plant pathogenic fungi, some extracts had crude activities comparable to or greater than T. reesei, particularly when assayed at lower temperatures and on biomass substrates. This microplate assay system should prove useful for high-throughput bioprospecting for new sources of novel enzymes for biofuel production.

Comparative antioxidant, antiproliferative and phase II enzyme inducing potential of sorghum (Sorghum bicolor) varieties.

Abstract: Epidemiological evidence has linked consumption of sorghum with reduced incidences of gastrointestinal cancer, especially cancer of esophagus. No information is available on how sorghum may effect the chemoprotective properties. We investigated in vitro potential of eight sorghum varieties to induce phase II detoxifying enzymes using the NAD(P)H:quinone oxidoreductase (NQO) enzyme assay, and also inhibit proliferation of esophageal, OE33, and colon, HT-29, carcinoma cells using the PicoGreen and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays; these properties were compared to phenolic profile and antioxidant activity of the sorghum. Black sorghum extract high in 3-deoxyanthocyanins was the most potent NQO inducer, doubling NQO activity at 5.0 μg/mL and maximally inducing the enzyme activity by 3.0 times. White sorghum was a moderately strong inducer, maximally increasing NQO activity by 80%; tannin-containing sorghums were non-inducers. On the other hand, the tannin-containing sorghum extracts had strongest antiproliferative activity against both OE33 and HT-29 cells (IC50, 38–105 μg/mL); the white sorghum extract was the least potent (IC50, 389–>800 μg/mL). Antiproliferative activity correlated with antioxidant activity whereas NQO-inducer capacity did not. Sorghum extracts have strong chemoprotective potential which is partially independent of their antioxidant properties. They may thus be valuable health-promoting ingredients in whole-grain based products.

Intestinal function and gut microflora of broiler chickens as influenced by cereal grains and microbial enzyme supplementation

Abstract: Summary A study was conducted to investigate the effect of the key cereal grains and a microbial enzyme supplement on broiler chicken performance, gut microflora and intestinal function. Ingestion of the barley-based diet was associated with low 28-day body weight, decreased feed intake and high FCR. The supplemental enzyme increased feed intake and weight gain of the chickens on a wheat-based diet. The pH of the gizzard and caecal contents varied with the grain type. Enzyme supplementation reduced ileal viscosity, particularly in birds that received the diet based on wheat. The birds on the barley-based diet had lower ileal digestibility of dry matter, protein and energy than those given maize and sorghumbased diets. The ileal digestibility of starch was increased by enzyme supplementation. Enzyme supplementation increased the number of total anaerobic bacteria in the gizzard of birds fed on sorghum and increased lactobacilli in the gizzard of those fed both sorghum and wheat. The birds fed the sorghum-based diet had the lowest counts of caecal total anaerobic bacteria and lactobacilli. Jejunal villus height and villus:crypt ratio of birds fed the barley-based diet were the lowest when compared with those fed the other diets. Enzyme application induced an increase in villus height and villus:crypt ratio of birds on wheat, crypt depth on barley and a reduction in crypt depth of chickens on the sorghum-based diets. The highest activity of maltase and the lowest activity of sucrase were observed in tissue from birds fed on maize and sorghum-based diets respectively. The differences in the performance of broilers on cereal grains could be explained by changes in intestinal morphology, enzyme activities and gut microflora as well as nutrient digestibility. The improved performance by supplemental enzyme in wheat-fed chickens was associated with beneficial changes in intestinal morphology and digesta viscosity.

Isolation and characterization of novel α-amylase from marine Streptomyces sp. D1

Abstract: In this study, we have reported novel α-amylase enzyme from less extensively studied marine Streptomyces sp. D1. Enzyme production was determined by using media containing 2% sucrose, 0.35% peptone and 0.15% of malt extract. Optimum temperature for enzyme production and activity was found to be 45 °C and enzyme retained almost 50% of its activity at 85 °C. Enzyme activity was also retained in presence of commercially available detergent and oxidizing agents. The partially purified enzyme from strain D1 exhibited specific activity of 113.64 U/mg protein that corresponds to 2.8-fold purification. SDS-PAGE and zymogram activity staining showed a single band equal to molecular mass of 66 kDa. The reported enzyme may have wide spread application for detergent and pharmaceutical industry.

Lignocellulose-degrading enzyme production by white-rot Basidiomycetes isolated from the forests of Georgia

Abstract: The production of lignocellulolytic enzymes by eleven basidiomycetes species isolated from two ecosystems of Georgia was investigated for the first time under submerged (SF) and solid-state fermentation (SSF) of lignocellulosic by-products. Notable intergeneric and intrageneric differences were revealed with regard to the extent of hydrolase and oxidase activity. Several fungi produced laccase along with hydrolases in parallel with growth during the trophophase, showing that the synthesis of this enzyme is not connected with secondary metabolism. The lignocellulosic substrate type had the greatest impact on enzyme secretion. Some of the substrates significantly stimulated lignocellulolytic enzyme synthesis without supplementation of the culture medium with specific inducers. Exceptionally high carboxymethyl cellulase (CMCase, 122 U ml(-1)) and xylanase (195 U ml(-1)) activities were revealed in SF of mandarin peelings by Pseudotremella gibbosa IBB 22 and of residue after ethanol production (REP) by Fomes fomentarius IBB 38, respectively. The SSF of REP by T. pubescens IBB 11 ensured the highest level of laccase activity (24,690 U l(-1)), whereas the SSF of wheat bran and SF of mandarin peels provided the highest manganese peroxidase activity (570-620 U l(-1)) of Trichaptum biforme IBB 117. Moreover, the variation of lignocellulosic growth substrate provides an opportunity to obtain enzyme preparations containing different ratios of individual enzymes.

Screening of Selected Food and Medicinal Plant Extracts for Pancreatic Lipase Inhibition

Abstract: Lipids are important components in human nutrition; however, their increased intake contributes to the development of obesity and can lead to multiple long-term complications. Pancreatic lipase (triacylglycerol acylhydrolase, EC 3.1.1.3) is a key enzyme for the absorption of dietary triglycerides. Interference with fat hydrolysis results in the reduced utilization of ingested lipids, therefore inhibition of lipases decreases fat absorption. Extracts from 106 species of medicinal plants, vegetables and fruits were screened for potential lipase inhibitory activity. p-Nitrophenylpalmitate and 5-bromo-4-chloro-3-indoxylpalmitate were used as substrates in an in vitro test with crude porcine pancreatic lipase. Bearberry (Arctostaphylos uva-ursi), garden pea (Pisum sativum), Norway spruce (Picea abies) and large-leaved lime (Tilia platyphyllos) extracts were the most active. Additionally, the activity of selected extracts with removed polyphenols was measured. Extracts of bearberry, garden pea and large-leaved lime are a promising source for developing functional foods or isolating active compounds.

Polymer-assisted iron oxide magnetic nanoparticle immobilized keratinase.

Abstract: Nanotechnology holds the prospect for avant-garde changes to improve the performance of materials in various sectors. The domain of enzyme biotechnology is no exception. Immobilization of industrially important enzymes onto nanomaterials, with improved performance, would pave the way to myriad application-based commercialization. Keratinase produced by Bacillus subtilis was immobilized onto poly(ethylene glycol)-supported Fe3O4 superparamagnetic nanoparticles. The optimization process showed that the highest enzyme activity was noted when immobilized onto cyanamide-activated PEG-assisted MNP prepared under conditions of 25??C and pH 7.2 of the reaction mixture before addition of H2O2 (3%? w/w), 2%?(w/v) PEG6000 and 0.062:1 molar ratio of PEG to FeCl2?4H2O. Further statistical optimization using response surface methodology yielded an R2 value that could explain more than 94% of the sample variations. Along with the magnetization studies, the immobilization of the enzyme onto the PEG-assisted MNP was characterized by UV, XRD, FTIR and TEM. The immobilization process had resulted in an almost fourfold increase in the enzyme activity over the free enzyme. Furthermore, the immobilized enzyme exhibited a significant thermostability, storage stability and recyclability. The leather-industry-oriented application of the immobilized enzyme was tested for the dehairing of goat-skin.

Inhibition studies of bovine xanthine oxidase by luteolin, silibinin, quercetin, and curcumin.

Abstract: Xanthine oxidoreductase (XOR) is a molybdenum-containing enzyme that under physiological conditions catalyzes the final two steps in purine catabolism, ultimately generating uric acid for excretion. Here we have investigated four naturally occurring compounds that have been reported to be inhibitors of XOR in order to examine the nature of their inhibition utilizing in vitro steady-state kinetic studies. We find that luteolin and quercetin are competitive inhibitors and that silibinin is a mixed-type inhibitor of the enzyme in vitro, and, unlike allopurinol, the inhibition is not time-dependent. These three natural products also decrease the production of superoxide by the enzyme. In contrast, and contrary to previous reports in the literature based on in vivo and other nonmechanistic studies, we find that curcumin did not inhibit the activity of purified XO nor its superoxide production in vitro.

Activation and conformational changes of mushroom polyphenoloxidase by high pressure microfluidization treatment.

Abstract: Activation and conformational changes of mushroom polyphenoloxidase (PPO) after high pressure microfluidization treatment were observed by means of UV spectrophotometer, far-UV circular dichroism, fluorescence emission spectra, UV absorption spectra and sulphydryl groups detection. The results indicated that, treated under pressures of 90 MPa, 110 MPa, 130 MPa and 150 MPa one pass, and 150 MPa 1, 2 and 3 passes, respectively, mushroom PPO exhibited an increase in activity. The circular dichroism (CD) analysis demonstrated that some of secondary structures such as α-helix were destroyed. There were some indices that the increase of relative activity was accompanied by a decrease in α-helix content. The fluorescence emission spectra analysis indicated that Trp and Tyr residues in mushroom PPO were more or less exposed to solvent, and the result was in good agreement with that of UV absorption spectra analysis. The sulphydryl groups detection showed that the sulphydryl groups content on the surface of mushroom PPO was increased. Industrial relevance Results of the present study show that high pressure microfluidization can not be used to inactivate mushroom PPO. However, it provides a new way for enzyme preparation, such as enzyme modification, with higher activity. High pressure microfluidization can be easily operated, with shorter treatment time and higher food safety compared with chemical methods.

Multiple serum enzyme analyses in chronic alcoholics.

Abstract: . Multiple serum enzyme analyses in 100 chronic alcoholics with a history of heavy drinking revealed that most serum enzyme activities were increased in some of these patients. Increased serum creatine kinase activity, characteristic of muscle injury, was found in 43 patients. Since cardiac damage could be excluded by other enzyme tests, skeletal muscle damage seems to be common in alcoholic patients. Elevated serum ornithine carbamoyl transferase (OCT), which is specific for liver damage, was found in 56 patients. Serum GOT, commonly used for detection of liver damage, seems in alcoholics to be partly released from skeletal muscles and should thus be replaced by the more specific OCT, for which there is a simple method. In alcoholics the increased serum activity of the intramitochondrial enzyme, glutamate dehydrogenase, suggests a rather severe liver damage in nearly half of the patients. Serum guanine deaminase (guanase) was pathological less often than the other “liver enzymes”. Heavy increased values were encountered often in serum γ-glutamyltranspeptidase but leucine aminopeptidase and alkaline phosphatase were less often elevated.

Calcium Alginate: A Support Material for Immobilization of Proteases from Newly Isolated Strain of Bacillus subtilis KIBGE-HAS

Abstract: Partially purified neutral protease was entrapped in calcium alginate beads and characterized using casein as a substrate. Temperature and pH maxima of the enzyme showed no changes before and after immobilization and remained stable at 50°C and 7.5 respectively. However substrate concentration for maximum enzyme activity of immobilized enzyme was shifted from 0.45% to 0.8%. Reaction time for immobilized enzyme assay was also increased by 5 minutes with reference to soluble enzyme i.e., from 15 to 20 minutes. Enzyme activity was decreased when the concentration of alginate was increased above 2%. Immobilized enzyme retained its activity for longer time and can be reused upto three times. The storage stability of entrapped protease at 4°C was found upto 10 days, while at 30°C the enzyme lost its activity within three days.

Growth, lipid peroxidation and antioxidant enzyme activities as a selection criterion for the salt tolerance of maize cultivars grown under salinity stress.

Abstract: Different responses of three maize (Zea mays L) cultivars SC 129, SC 13 and SC 155 (differently susceptible to salinity stress) in pot experiment were investigated The rate of oxidative injury on growth, lipid peroxidation and activities of antioxidant enzymes in relation to NaCl-stress were tested The cv SC 129 tolerated NaCl up to 100 mM and cv SC13 up to 50 mM, whereas cv SC 155 was markedly affected even at the lowest salinity level used Moreover, the cv SC 155 died at a concentration higher than 200 mM NaCl Concomitantly, the activity of antioxidant enzymes catalase, peroxidase, ascorbate, peroxidase and superoxide dismutase in the salt-tolerant cultivars (cv SC 129 & cv SC 13) increased markedly during salinity stress, while they were mostly decreased by salinity stress in the salt sensitive cultivar (cv SC 155) Consequently, this led to a marked difference in the behavior of lipid peroxidation in the three maize cultivars We concluded that the activities of antioxidant enzymes and lipid peroxidation were associated with the dry mass production and consequently with the salt tolerance of the three maize cultivars

Highly Thermostable Xylanase of the Thermophilic Fungus Talaromyces thermophilus: Purification and Characterization

Abstract: A thermostable xylanase from a newly isolated thermophilic fungus Talaromyces thermophilus was purified and characterized. The enzyme was purified to homogeneity by ammonium sulfate precipitation, diethylaminoethyl cellulose anion exchange chromatography, P-100 gel filtration, and Mono Q chromatography with a 23-fold increase in specific activity and 17.5% recovery. The molecular weight of the xylanase was estimated to be 25 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration. The enzyme was highly active over a wide range of pH from 4.0 to 10.0. The relative activities at pH5.0, 9.0, and 10.0 were about 80%, 85.0%, and 60% of that at pH7.5, respectively. The optimum temperature of the purified enzyme was 75 degrees C. The enzyme showed high thermal stability at 50 degrees C (7 days) and the half-life of the xylanase at 100 degrees C was 60 min. The enzyme was free from cellulase activity. K (m) and V (max) values at 50 degrees C of the purified enzyme for birchwood xylan were 22.51 mg/ml and 1.235 micromol min(-1) mg(-1), respectively. The enzyme was activated by Ag(+), Co(2+), and Cu(2+); on the other hand, Hg(2+), Ba(2+), and Mn(2+) inhibited the enzyme. The present study is among the first works to examine and describe a secreted, cellulase-free, and highly thermostable xylanase from the T. thermophilus fungus whose application as a pre-bleaching aid is of apparent importance for pulp and paper industries.