scispace - formally typeset
Search or ask a question

Showing papers on "Enzyme assay published in 2017"


Journal ArticleDOI
TL;DR: A novel regulatory target, glucose-6-phosphate dehydrogenase (G6PD) from the pentose phosphate pathway (PPP), is presented in boosting microalgal lipid accumulation by enhancing NADPH supply and demonstrate that G6PD is a promising target for metabolic engineering.

127 citations


Journal ArticleDOI
TL;DR: Investigating the comparative responses to Si and Se in relation to antioxidant enzyme system and the glutathione-ascorbate cycle in flowering Chinese cabbage plants under Cd stress reveals that Se-mediated alleviation of Cd toxicity is due to increasing antioxidant enzyme activities and the GSH-AsA cycle efficiency.

124 citations


Journal ArticleDOI
TL;DR: In the present study, Trichoderma reesei cellulase was covalently immobilized on chitosan-coated magnetic nanoparticles using glutaraldehyde as a coupling agent to reduce the enzyme consumption during lignocellulosic material saccharification for bioethanol production.
Abstract: In the present study, Trichoderma reesei cellulase was covalently immobilized on chitosan-coated magnetic nanoparticles using glutaraldehyde as a coupling agent. The average diameter of magnetic nanoparticles before and after enzyme immobilization was about 8 and 10 nm, respectively. The immobilized enzyme retained about 37 % of its initial activity, and also showed better thermal and storage stability than free enzyme. Immobilized cellulase retained about 80 % of its activity after 15 cycles of carboxymethylcellulose hydrolysis and was easily separated with the application of an external magnetic field. However, in this reaction, K m was increased eight times. The immobilized enzyme was able to hydrolyze lignocellulosic material from Agave atrovirens leaves with yield close to the amount detected with free enzyme and it was re-used in vegetal material conversion up to four cycles with 50 % of activity decrease. This provides an opportunity to reduce the enzyme consumption during lignocellulosic material saccharification for bioethanol production.

123 citations


Journal ArticleDOI
TL;DR: Hepatic ZIP8 reclaims Mn from bile and regulates whole-body Mn homeostasis, thereby modulating the activity of Mn-dependent enzymes, thus linking SLC39A8 variants with other physiological traits.
Abstract: Genetic variants at the solute carrier family 39 member 8 (SLC39A8) gene locus are associated with the regulation of whole-blood manganese (Mn) and multiple physiological traits. SLC39A8 encodes ZIP8, a divalent metal ion transporter best known for zinc transport. Here, we hypothesized that ZIP8 regulates Mn homeostasis and Mn-dependent enzymes to influence metabolism. We generated Slc39a8-inducible global-knockout (ZIP8-iKO) and liver-specific-knockout (ZIP8-LSKO) mice and observed markedly decreased Mn levels in multiple organs and whole blood of both mouse models. By contrast, liver-specific overexpression of human ZIP8 (adeno-associated virus-ZIP8 [AAV-ZIP8]) resulted in increased tissue and whole blood Mn levels. ZIP8 expression was localized to the hepatocyte canalicular membrane, and bile Mn levels were increased in ZIP8-LSKO and decreased in AAV-ZIP8 mice. ZIP8-LSKO mice also displayed decreased liver and kidney activity of the Mn-dependent enzyme arginase. Both ZIP8-iKO and ZIP8-LSKO mice had defective protein N-glycosylation, and humans homozygous for the minor allele at the lead SLC39A8 variant showed hypogalactosylation, consistent with decreased activity of another Mn-dependent enzyme, β-1,4-galactosyltransferase. In summary, hepatic ZIP8 reclaims Mn from bile and regulates whole-body Mn homeostasis, thereby modulating the activity of Mn-dependent enzymes. This work provides a mechanistic basis for the association of SLC39A8 with whole-blood Mn, potentially linking SLC39A8 variants with other physiological traits.

119 citations


Journal ArticleDOI
TL;DR: The results show that the detergent properties of some reaction products increase the enzyme release from the hydrophobic support, and this problem increased if the concentration of the reactants increased.

103 citations


Journal ArticleDOI
TL;DR: The cytochrome P450 2A6 (CYP2A6) enzyme metabolizes several clinically relevant substrates, including nicotine—the primary psychoactive component in cigarette smoke—and its expression and activity is impacted by non-genetic factors, including induction or inhibition by pharmacological, endogenous, and dietary substances.
Abstract: The cytochrome P450 2A6 (CYP2A6) enzyme metabolizes several clinically relevant substrates, including nicotine—the primary psychoactive component in cigarette smoke. The gene that encodes the CYP2A6 enzyme is highly polymorphic, resulting in extensive interindividual variation in CYP2A6 enzyme activity and the rate of metabolism of nicotine and other CYP2A6 substrates including cotinine, tegafur, letrozole, efavirenz, valproic acid, pilocarpine, artemisinin, artesunate, SM-12502, caffeine, and tyrosol. CYP2A6 expression and activity are also impacted by non-genetic factors, including induction or inhibition by pharmacological, endogenous, and dietary substances, as well as age-related changes, or interactions with other hepatic enzymes, co-enzymes, and co-factors. As variation in CYP2A6 activity is associated with smoking behavior, smoking cessation, tobacco-related lung cancer risk, and with altered metabolism and resulting clinical responses for several therapeutics, CYP2A6 expression and enzyme activity is an important clinical consideration. This review will discuss sources of variation in CYP2A6 enzyme activity, with a focus on the impact of CYP2A6 genetic variation on metabolism of the CYP2A6 substrates.

99 citations


Journal ArticleDOI
TL;DR: The physical and chemical effects of ultrasound on enzyme have been correlated with thermodynamic parameters (enthalpy and entropy) and various techniques used for investigating the conformation changes in enzyme after sonication have been highlighted.
Abstract: Over the last decade, ultrasound technique has emerged as the potential technology which shows large applications in food and biotechnology processes. Earlier, ultrasound has been employed as a method of enzyme inactivation but recently, it has been found that ultrasound does not inactivate all enzymes, particularly, under mild conditions. It has been shown that the use of ultrasonic treatment at appropriate frequencies and intensity levels can lead to enhanced enzyme activity due to favourable conformational changes in protein molecules without altering its structural integrity. The present review article gives an overview of influence of ultrasound irradiation parameters (intensity, duty cycle and frequency) and enzyme related factors (enzyme concentration, temperature and pH) on the catalytic activity of enzyme during ultrasound treatment. Also, it includes the effect of ultrasound on thermal kinetic parameters and Michaelis-Menten kinetic parameters (km and Vmax) of enzymes. Further, in this review, the physical and chemical effects of ultrasound on enzyme have been correlated with thermodynamic parameters (enthalpy and entropy). Various techniques used for investigating the conformation changes in enzyme after sonication have been highlighted. At the end, different techniques of immobilization for ultrasound treated enzyme have been summarized.

86 citations


Journal ArticleDOI
TL;DR: It is concluded that the spatio-temporal pattern of rhizosphere enzymatic hotspots is mainly affected by plant growth, and high temperature sensitivity at the root-soil interface for the tested enzymes revealed that warming will lead to faster nutrient mobilization in therhizosphere than in root-free soil.
Abstract: The rhizosphere is the most dynamic hotspot of microbial activity in the soil. Despite these dynamics, the spatial pattern of many rhizosphere properties may remain stable because they are continuously reproduced in the changing environment. Low substrate concentration can strongly reduce the rate response of an enzymatic reaction subjected to increased temperature and is recognized as a canceling effect on enzyme temperature sensitivity. Carbon input from rhizodeposits affects C availability in the rhizosphere, and thus the enzyme activities responsible for organic matter decomposition, and their temperature sensitivities, upset the dynamics and stability of biochemical processes in the rhizosphere. However, it is unclear whether a canceling effect occurs in the rhizosphere. We studied temperature effects on chitinase and phosphatase during rice (Oryza sativa L.) growth at 18 and 25 °C. The spatial distribution of enzyme activities was imaged using soil zymography and showed that the overall activities of these enzymes increased with temperature but decreased with rice growth. The temporal dynamics of hotspot areas were enzyme-specific. During growing days 14–30, hotspot areas decreased from 2-2.5% to 0.3–0.5% for chitinase, but increased from 2% to 6–7% for phosphatase. The distribution pattern of both enzymes shifted from being dispersed throughout the soil to being associated with the roots. For the first time, we showed the extent of rhizosphere enzyme activity in paddy soil and demonstrated that it is temporally stationary and independent of temperature. However, the temperature sensitivity of enzyme activities declined radically (Q10∼1.3–1.4) at the root surface compared to that of bulk soil (Q10 ∼1). We conclude that the spatio-temporal pattern of rhizosphere enzymatic hotspots is mainly affected by plant growth. High temperature sensitivity (Q10 > 1) at the root-soil interface for the tested enzymes revealed that warming will lead to faster nutrient mobilization in the rhizosphere than in root-free soil.

83 citations


Journal ArticleDOI
13 Jun 2017-PLOS ONE
TL;DR: At the early stage of low light treatment, the chlorophyll b content increased to improve photosynthesis, and when theLow light treatment was extended, antioxidant enzyme activity and the activity of anthocyanin biosynthesis enzymes were inhibited, causing the purple pak-choi seedlings to fade from purple to green.
Abstract: Anthocyanins are secondary metabolites that contribute to red, blue, and purple colors in plants and are affected by light, but the effects of low light on the physiological responses of purple pak-choi plant leaves are still unclear. In this study, purple pak-choi seedlings were exposed to low light by shading with white gauze and black shading in a phytotron. The responses in terms of photosynthetic properties, carbohydrate metabolism, antioxidant enzyme activity, anthocyanin biosynthetic enzyme activity, and the relative chlorophyll and anthocyanin content of leaves were measured. The results showed that chlorophyll b, intracellular CO2 content, stomatal conductance and antioxidant activities of guaiacol peroxidase, catalase and superoxide dismutase transiently increased in the shade treatments at 5 d. The malondialdehyde content also increased under low light stress, which damages plant cells. With the extension of shading time (at 15 d), the relative chlorophyll a, anthocyanin and soluble protein contents, net photosynthetic rate, transpiration rate, stomata conductance, antioxidant enzyme activities, and activities of four anthocyanin biosynthetic enzymes decreased significantly. Thus, at the early stage of low light treatment, the chlorophyll b content increased to improve photosynthesis. When the low light treatment was extended, antioxidant enzyme activity and the activity of anthocyanin biosynthesis enzymes were inhibited, causing the purple pak-choi seedlings to fade from purple to green. This study provides valuable information for further deciphering genetic mechanisms and improving agronomic traits in purple pak-choi under optimal light requirements.

78 citations


Journal ArticleDOI
31 Aug 2017-ACS Nano
TL;DR: This study presents a method to encapsulate enzymes in a DNA cage that could transform its conformation depending on the pH, allowing reversible control of the accessibility of the enzyme to the surrounding environment.
Abstract: Reversible regulation of enzyme activity by chemical and physical stimuli is often achieved by incorporating stimuli-responsive domains in the enzyme of interest. However, this method is suitable for a limited number of enzymes with well-defined structural and conformational changes. In this study, we present a method to encapsulate enzymes in a DNA cage that could transform its conformation depending on the pH, allowing reversible control of the accessibility of the enzyme to the surrounding environment. This enabled us to regulate various properties of the enzyme, such as its resistance to protease-dependent degradation, binding affinity to the corresponding antibody, and most importantly, enzyme activity. Considering that the size and pH responsiveness of the DNA cage can be easily adjusted by the DNA length and sequence, our method provides a broad-impact platform for controlling enzyme functions without modifying the enzyme of interest.

71 citations


Journal ArticleDOI
TL;DR: The optimization of fermentation conditions for α-amylase production from thermophilic bacterial strain Bacillus sp.
Abstract: Amylase is an industrially important enzyme and applied in many industrial processes such as saccharification of starchy materials, food, pharmaceutical, detergent, and textile industries. This research work deals with the optimization of fermentation conditions for α-amylase production from thermophilic bacterial strain Bacillus sp. BCC 01-50 and characterization of crude amylase. The time profile of bacterial growth and amylase production was investigated in synthetic medium and maximum enzyme titer was observed after 60 h. In addition, effects of different carbon sources were tested as a substrate for amylase production and molasses was found to be the best. Various organic and inorganic compounds, potassium nitrate, ammonium chloride, sodium nitrate, urea, yeast extract, tryptone, beef extract, and peptone, were used and beef extract was found to be the best among the nitrogen sources used. Temperature, pH, agitation speed, and size of inoculum were also optimized. Highest enzyme activity was obtained when the strain was cultured in molasses medium for 60 h in shaking incubator (150 rpm) at 50°C and pH 8. Crude amylase showed maximal activity at pH 9 and 65°C. Enzyme remained stable in alkaline pH range 9-10 and 60–70°C. Crude amylase showed great potential for its application in detergent industry and saccharification of starchy materials.

Journal ArticleDOI
TL;DR: The flux of metabolites in the living cell depend on enzyme activities, and the scientific community needs to measure the kinetics of all enzymes in a systematic way to move further.
Abstract: The flux of metabolites in the living cell depend on enzyme activities. Recently, many metabolic phenotypes have been explained by computer models that incorporate enzyme activity data. To move further, the scientific community needs to measure the kinetics of all enzymes in a systematic way.

Journal ArticleDOI
TL;DR: The apparent in vivo catalytic efficiency of TEM1-β-lactamase inside living cells is found to be lower than in vitro, with significant cell-to-cell variability, indicating that in vitro data cannot be simply extrapolated to the crowded in vivo environment.

Journal ArticleDOI
TL;DR: In general, PEI treatment was the most effective in increasing enzyme stability, while glutaraldehyde had a milder stabilization effect, and crowded enzyme preparations were intermolecular crosslinked using Glu plus PEI, but some molecules still remained non-crosslinked.

Journal ArticleDOI
TL;DR: The strategy permitted to avoid enzyme desorption, very likely by physical intermolecular crosslinking improving enzyme stability, while still maintaining the reversibility of the immobilization.

Journal ArticleDOI
Tingting Zhang1, Qianqian Lu1, Chunlei Su1, Yaru Yang1, Dan Hu1, Qinsong Xu1 
TL;DR: Results suggest that L. minor plants were able to cope with Hg toxicity through the activation of various mechanisms involving enzymatic and non-enzymatic antioxidants, up-regulation of proline, and induction of Hsp70.

Journal ArticleDOI
TL;DR: Findings on the relationship between in vivo and in vitro enzyme catalysis are discussed and it is shown how proteomics can be used to characterize the efficiency of enzyme utilization across conditions.

Journal Article
TL;DR: Amylase from apple exhibited high efficiency for the removal of chocolate stains in combination with commercial detergent (Tide) and the wash performance analysis of Chocolate stains on cotton fabric showed an increase in reflectance with detergent and enzyme as compared to detergent only.
Abstract: Amylases are widely distributed in microbial, plant and animal kingdoms. They degrade starch and related polymers to yield products characteristic of individual amylolytic enzymes. Alpha amylase has been derived from several fungi, yeasts, bacteria and actinomycetes, however, enzymes from fungal and bacterial sources have dominated applications in industrial sectors. The pH dependence of amylase activity was assayed in a pH range of 4-11, using the standard reaction mixture. The optimum temperature for maximal Amylase activity was found to be 30-35 0C, after which the enzyme activity dropped. The addition of metals like Ca2+ and Mn2+ and Mg2+ increased the enzyme activity while a significant inhibitory effect on the protease activity was observed with Hg2+. Amylase from apple exhibited high efficiency for the removal of chocolate stains in combination with commercial detergent (Tide). The wash performance analysis of chocolate stains on cotton fabric showed an increase in reflectance with detergent and enzyme as compared to detergent only.

Journal ArticleDOI
TL;DR: The immobilization of the enzyme β-galactosidase at pH 7 at low ionic strength on pre-activated glutaraldehyde supports seems to be the most adequate in terms of activity, stability and immobilization rate.
Abstract: The enzyme β-galactosidase from Aspergillus oryzae has been immobilized in aminated (MANAE)-agarose beads via glutaraldehyde chemistry using different strategies. The immobilization on MANAE-supports was first assayed at different pH values (this gave different stabilities to the immobilized enzymes) and further modified with glutaraldehyde. Dramatic drops in activity were found, even using 0.1% (v/v) glutaraldehyde. The use of a support with lower activation permitted to get a final activity of 30%, but stability was almost identical to that of the just adsorbed enzyme. Next, the immobilization on pre-activated glutaraldehyde beads was assayed at pH 5, 7 and 9. At pH 7, full, rapid immobilization and a high expressed enzyme activity were accomplished. At pH 9, some decrease in enzyme activity was observed. Direct covalent immobilization of the enzyme was very slow; even reducing the volume of enzyme/support ratio, the yield was not complete after 24 h. The stability of the biocatalyst using pre-activated supports was about 4–6 folds more stable than that of the enzyme immobilized via ion exchange at pH 5, with small differences among them. Thus, the immobilization of the enzyme at pH 7 at low ionic strength on pre-activated glutaraldehyde supports seems to be the most adequate in terms of activity, stability and immobilization rate.

Journal ArticleDOI
TL;DR: The thermostable β-glucosidase enzyme from B. subtilis RA10 would facilitate efficient saccharification of cellulosic biomass into fermentable sugar and be recovered and reused to reduce the cost of overall bioethanol production process.
Abstract: Saccharification is the most crucial and cost-intensive process in second generation biofuel production. The deficiency of β-glucosidase in commercial enzyme leads to incomplete biomass hydrolysis. The decomposition of biomass at high temperature environments leads us to isolate thermotolerant microbes with β-glucosidase production potential. A total of 11 isolates were obtained from compost and cow dung samples that were able to grow at 50 °C. On the basis of qualitative and quantitative estimation of β-glucosidase enzyme production, Bacillus subtilis RA10 was selected for further studies. The medium components and growth conditions were optimized and β-glucosidase enzyme production was enhanced up to 19.8-fold. The β-glucosidase from B. subtilis RA10 retained 78% of activity at 80 °C temperature and 68.32% of enzyme activity was stable even at 50 °C after 48 h of incubation. The supplementation of β-glucosidase from B. subtilis RA10 into commercial cellulase enzyme resulted in 1.34-fold higher glucose release. Furthermore, β-glucosidase was also functionally elucidated by cloning and overexpression of full length GH1 family β-glucosidase gene from B. subtilis RA10. The purified protein was characterized as thermostable β-glucosidase enzyme. The thermostable β-glucosidase enzyme from B. subtilis RA10 would facilitate efficient saccharification of cellulosic biomass into fermentable sugar. Consequently, after saccharification, thermostable β-glucosidase enzyme would be recovered and reused to reduce the cost of overall bioethanol production process.

Journal ArticleDOI
TL;DR: An acidic thermostable protease was extracellularly produced either in shake flask or in stirred tank bioreactor by an Aspergillus foetidus strain isolated from the Brazilian savanna soil using different nitrogen sources and it was confirmed that the purified acid protease belongs to the aspartic protease type.

Journal ArticleDOI
TL;DR: An extracellular thermostable alkaline serine protease enzyme from Aeribacillus pallidus C10 was purified and it was determined that the enzyme had remained stable at the range of pH 7.0–10.0 and preserved more than 80% of its activity at a broad temperature range.
Abstract: An extracellular thermostable alkaline serine protease enzyme from Aeribacillus pallidus C10 (GenBank No: KC333049), was purified 4.85 and 17. 32-fold with a yield of 26.9 and 19.56%, respectively, through DE52 anion exchange and Probond affinity chromatography. The molecular mass of the enzyme was determined through sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), with approximately 38.35 kDa. The enzyme exhibited optimum activity at pH 9 and at temperature 60 °C. It was determined that the enzyme had remained stable at the range of pH 7.0–10.0, and that it had preserved more than 80% of its activity at a broad temperature range (20–80 °C). The enzyme activity was found to retain more than 70% and 55% in the presence of organic solvents and commercial detergents, respectively. In addition, it was observed that the enzyme activity had increased in the presence of 5% SDS. KM and Vmax values were calculated as 0.197 mg/mL and 7.29 μmol.mL−1.min−1, respectively.

Journal ArticleDOI
TL;DR: It was observed that two copies of the tHMG1 gene on a dual gene expression cassette improvedSqualene synthesis in laboratory strain by 16.8-fold in comparison to single-copy expression, and it was demonstrated that overexpression of full-length POS5 has improved squalenes synthesis in cytosol.
Abstract: Although overexpression of the tHMG1 gene is a well-known strategy for terpene synthesis in Saccharomyces cerevisiae, the optimal level for tHMG1p has not been established. In the present study, it was observed that two copies of the tHMG1 gene on a dual gene expression cassette improved squalene synthesis in laboratory strain by 16.8-fold in comparison to single-copy expression. It was also observed that tHMG1p is limited by its cofactor (NADPH), as the overexpression of NADPH regenerating genes’, viz., ZWF1 and POS5 (full length and without mitochondrial presequence), has led to its increased enzyme activity. Further, it was demonstrated that overexpression of full-length POS5 has improved squalene synthesis in cytosol. Finally, when tHMG1 and full-length POS5 were co-overexpressed there was a net 27.5-fold increase in squalene when compared to control strain. These results suggest novel strategies to increase squalene accumulation in S. cerevisiae.

Journal ArticleDOI
TL;DR: This is the first study identifying and confirming the role of CYP2A6 enzyme in AFB1 bioactivation in AA broiler liver (in vivo), and the hepatoprotective role of curcumin via inhibiting CYP 2A6 expression and enzyme activity.
Abstract: Cytochrome P450 enzymes are often responsible for the toxic and carcinogenic effects of toxicants, such as aflatoxin B1 (AFB1). The human hepatic CYP2A6 enzyme mediates the oxidative metabolism of several procarcinogens. In this study, we characterized a partial sequence of CYP2A6 gene from Arbor Acres (AA) broiler and studied its role in AFB1 bioactivation. Moreover, the effect of curcumin on CYP2A6 is illustrated. Six groups of AA broiler were treated for 28 day including the control group (fed only basal diet), curcumin alone-treated group (450 mg/kg feed), the group fed AFB1-contaminated feed (5 mg/kg feed) plus the low (150 mg), medium (300 mg) or high (450 mg) of curcumin, and the group fed AFB1-contaminated diet alone (5 mg/kg feed). After the end of treatment period, liver samples were collected for different analyses. The results revealed that the histopathological examination showed clear signs of liver toxicity in AA broliers in AFB1-fed group, but curcumin-supplementation in feed prevented partially AFB1-induced liver toxicity. Liver and body weights were recorded to study the AFB1 harmful effects. We noted an obvious increase in liver weight and decrease in body weight in AFB1-fed group. But, the administration of curcumin partially ameliorated the increase in liver weight and decrease in body weight in a dose-dependent manner. The results (RT-PCR and Elisa) revealed that mRNA and protein expression level enhanced in AFB1-fed group. Consistently, CYP2A6 enzyme activity also increased in AFB1-fed group, suggesting that AA broiler CYP2A6 actively involved in bioactivation of AFB1. However, curcumin treatment inhibited CYP2A6 at mRNA and protein levels in AFB1 treated AA broiler in a dose-dependent manner. Maximum inhibition of liver CYP2A6 enzyme activity in AA broiler has been achieved at a dose of 450 mg/kg curcumin. This is the first study identifying and confirming the role of CYP2A6 enzyme in AFB1 bioactivation in AA broiler liver (in-vivo), and the hepatoprotective role of curcumin via inhibiting CYP2A6 expression and enzyme activity. The study contributed to identify an important CYP enzyme involved in AFB1 bioactivation in broilers and thus could pave the way for the prevention of the harmful effects of AFB1 in broilers.

Journal ArticleDOI
TL;DR: Results demonstrate that the enzyme has heat stability at higher temperatures and the possibility of being used to construct biosensors and other analytical methods in various fields of science.

Journal ArticleDOI
TL;DR: In this paper, a mesocosm study was performed to examine short-term responses of selected soil enzyme activities to chlorpyrifos (Lorsban ® 4E).

Journal ArticleDOI
TL;DR: It is suggested that roots act as barrier of arsenic mediated by a prominent sulfur metabolism which is characterized by the biosynthesis of high amount of PCs.

Journal ArticleDOI
TL;DR: It is concluded that soil warming (at least up to 30 °C) increases hotspot areas of enzyme activity and the maximum reaction rate (Vmax) in the root-detritusphere, which leads to faster substrate exhaustion and shortens the duration of hot moments.
Abstract: Temperature effects on enzyme kinetics and on the spatial distribution of microbial hotspots are important because they are crucial to soil organic matter decomposition. We used soil zymography (in situ method for the two dimensional quantification of enzyme activities) to study the spatial distributions of enzymes responsible for P (phosphatase), C (cellobiohydrolase) and N (leucine-aminopeptidase) cycles in the rhizosphere (living roots of maize) and root-detritusphere (7 and 14 days after cutting shoots). Soil zymography was coupled with enzyme kinetics to test temperature effects (10, 20, 30 and 40 °C) on the dynamics and localization of these three enzymes in the root-detritusphere. The percentage area of enzyme activity hotspots was 1.9–7.9 times larger and their extension was broader in the root-detritusphere compared to rhizosphere. From 10 to 30 °C, the hotspot areas enlarged by a factor of 2–24 and Vmax increased by 1.5–6.6 times; both, however, decreased at 40 °C. For the first time, we found a close positive correlation between Vmax and the areas of enzyme activity hotspots, indicating that maximum reaction rate is coupled with hotspot formation. The substrate turnover time at 30 °C were 1.7–6.7-fold faster than at 10 °C. The Km of cellobiohydrolase and phosphatase significantly increased at 30 and 40 °C, indicating low affinity between enzyme and substrate at warm temperatures. We conclude that soil warming (at least up to 30 °C) increases hotspot areas of enzyme activity and the maximum reaction rate (Vmax) in the root-detritusphere. This, in turn, leads to faster substrate exhaustion and shortens the duration of hot moments.

Journal ArticleDOI
TL;DR: It is shown that low micromolar levels of copper compounds such as Cu(II)Urea2, Cu( II)Ser2 and CuCl2 can interfere with the MTT assay making improper the detection of formazan product of MTT reduction.

Journal ArticleDOI
TL;DR: High levels of IL 1-octyl-3-methylimidazolium tetrafluoroborate ([Omim]BF4) could have significantly toxic effects on soil enzyme activities and the diversity of the microbial communities.