Showing papers on "Enzyme assay published in 2012"

Soil enzymology: classical and molecular approaches

Abstract: It is still problematic to use enzyme activities as indicators of soil functions because: (1) enzyme assays determine potential and not real enzyme activities; (2) the meaning of measured enzyme activities is not known; (3) the assumption that a single enzyme activity is an indicator of nutrient dynamics in soil neglects that the many enzyme activities are involved in such dynamic processes; (4) spatio-temporal variations in natural environments are not always considered when measuring enzyme activities; and (5) many direct and indirect effects make difficult the interpretation of the response of the enzyme activity to perturbations, changes in the soil management, changes in the plant cover of soil, etc. This is the first review discussing the links between enzyme-encoding genes and the relative enzyme activity of soil. By combining measurements of enzyme activity in soil with expression (transcriptomics and proteomics) of genes, encoding the relative enzymes may contribute to understanding the mode and timing of microbial communities’ responses to substrate availability and persistence and stabilization of enzymes in the soil.

Temperature sensitivity of soil enzyme kinetics under N-fertilization in two temperate forests

Abstract: Soil microbes produce extracellular enzymes that degrade carbon (C)-containing polymers in soil organic matter. Because extracellular enzyme activities may be sensitive to both increased nitrogen (N) and temperature change, we measured the effect of long-term N addition and short-term temperature variation on enzyme kinetics in soils from hardwood forests at Bear Brook, Maine, and Fernow Forest, West Virginia. We determined the Vmax and Km parameters for five hydrolytic enzymes: a-glucosidase, b-glucosidase, b-xylosidase, cellobiohydrolase, and N-acetyl-glucosaminidase. Temperature sensitivities of Vmax and Km were assessed within soil samples subjected to a range of temperatures. We hypothesized that (1) N additions would cause microbial C limitation, leading to higher enzyme Vmax values and lower Km values; and (2) both Vmax and Km would increase at higher temperatures. Finally, we tested whether or not temperature sensitivity of enzyme kinetics is mediated by N addition. Nitrogen addition significantly or marginally significantly increased Vmax values for all enzymes, particularly at Fernow. Nitrogen fertilization led to significantly lower Km values for all enzymes at Bear Brook, but variable Km responses at Fernow Forest. Both Vmax and Km were temperature sensitive, with Q10 values ranging from 1.64–2.27 for enzyme Vmax and 1.04–1.93 for enzyme Km. No enzyme showed a significant interaction between N and temperature sensitivity for Vmax, and only b-xylosidase showed a significant interaction between N and temperature sensitivity for Km. Our study is the first to experimentally demonstrate a positive relationship between Km and temperature for soil enzymes. Higher temperature sensitivities for Vmax relative to Km imply that substrate degradation will increase with temperature. In addition, the Vmax and Km responses to N indicate greater substrate degradation under N addition. Our results suggest that increasing temperatures and N availability in forests of the northeastern US will lead to increased hydrolytic enzyme activity, despite the positive temperature sensitivity of Km.

A theoretical model of C- and N-acquiring exoenzyme activities, which balances microbial demands during decomposition

Abstract: We developed an Extracellular EnZYme model (EEZY) of decomposition that produces two separate pools of C- and N-acquiring enzymes, that in turn hydrolyze two qualitatively different substrates, one containing only C (e.g., cellulose) and the other containing both C and N (e.g., chitin or protein). Hence, this model approximates the actions of commonly measured indicator enzymes s-1,4-glucosidase and s-1,4-N-acetylglucosaminidase (or leucine aminopeptidase) as they hydrolyze cellulose and chitin (or protein), respectively. EEZY provides an analytical solution to the allocation of these two enzymes, which in turn release C and N from the two substrates to maximize microbial growth. Model behaviors were both qualitatively and quantitatively consistent with patterns of litter decay generated by other decomposition models. However, EEZY demonstrated greater sensitivity to the C:N of individual substrate pools in addition to responding to factors directly affecting enzyme activity. Output approximated field observations of extracellular enzyme activities from studies of terrestrial soils, aquatic sediments, freshwater biofilm and plankton communities. Although EEZY is largely a theoretical model, simulated C- and N-acquiring enzyme activities approximated a 1:1 ratio, consistent with the bulk of these field observations, only when the N-containing substrate had a C:N ratio similar to commonly occurring substrates (e.g., proteins or chitin). This result supported the emerging view of the stoichiometry of extracellular enzyme activities from an environmental context, which suggests that a relatively narrow range of microbial C:N, carbon use efficiency and soil/sediment organic matter C:N across ecosystems explains the tendency towards this 1:1 ratio of enzyme activities associated with C- and N-acquisition. Sensitivity analyses indicated that simulated extracellular enzyme activity was most responsive to variations in carbon use efficiency of microorganisms, although kinetic characteristics of enzymes also had significant impacts. Thus EEZY provides a quantitative framework in which to interpret mechanisms underlying empirical patterns of extracellular enzyme activity.

Characterization of a NADH-Dependent Glutamate Dehydrogenase Mutant of Arabidopsis Demonstrates the Key Role of this Enzyme in Root Carbon and Nitrogen Metabolism

Abstract: The role of NADH-dependent glutamate dehydrogenase (GDH) was investigated by studying the physiological impact of a complete lack of enzyme activity in an Arabidopsis thaliana plant deficient in three genes encoding the enzyme. This study was conducted following the discovery that a third GDH gene is expressed in the mitochondria of the root companion cells, where all three active GDH enzyme proteins were shown to be present. A gdh1-2-3 triple mutant was constructed and exhibited major differences from the wild type in gene transcription and metabolite concentrations, and these differences appeared to originate in the roots. By placing the gdh triple mutant under continuous darkness for several days and comparing it to the wild type, the evidence strongly suggested that the main physiological function of NADH-GDH is to provide 2-oxoglutarate for the tricarboxylic acid cycle. The differences in key metabolites of the tricarboxylic acid cycle in the triple mutant versus the wild type indicated that, through metabolic processes operating mainly in roots, there was a strong impact on amino acid accumulation, in particular alanine, γ-aminobutyrate, and aspartate in both roots and leaves. These results are discussed in relation to the possible signaling and physiological functions of the enzyme at the interface of carbon and nitrogen metabolism.

Overexpression of Brassica juncea wild‐type and mutant HMG‐CoA synthase 1 in Arabidopsis up‐regulates genes in sterol biosynthesis and enhances sterol production and stress tolerance

Abstract: Brassica juncea 3-hydroxy-3-methylglutaryl-CoA synthase (HMGS) is encoded by four isogenes (BjHMGS1-BjHMGS4). In vitro enzyme assays had indicated that the recombinant BjHMGS1 H188N mutant lacked substrate inhibition by acetoacetyl-CoA (AcAc-CoA) and showed 8-fold decreased enzyme activity. The S359A mutant demonstrated 10-fold higher activity, while the H188N/S359A double mutant displayed a 10-fold increased enzyme activity and lacked inhibition by AcAc-CoA. Here, wild-type and mutant BjHMGS1 were overexpressed in Arabidopsis to examine their effects in planta. The expression of selected genes in isoprenoid biosynthesis, isoprenoid content, seed germination and stress tolerance was analysed in HMGS overexpressors (OEs). Those mRNAs encoding enzymes 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR), sterol methyltransferase 2 (SMT2), delta-24 sterol reductase (DWF1), C-22 sterol desaturase (CYP710A1) and brassinosteroid-6-oxidase 2 (BR6OX2) were up-regulated in HMGS-OEs. The total sterol content in leaves and seedlings of OE-wtBjHMGS1, OE-S359A and OE-H188N/S359A was significantly higher than OE-H188N. HMGS-OE seeds germinated earlier than wild-type and vector-transformed controls. HMGS-OEs further displayed reduced hydrogen peroxide (H(2) O(2) )-induced cell death and constitutive expression of salicylic acid (SA)-dependent pathogenesis-related genes (PR1, PR2 and PR5), resulting in an increased resistance to Botrytis cinerea, with OE-S359A showing the highest and OE-H188N the lowest tolerance. These results suggest that overexpression of HMGS up-regulates HMGR, SMT2, DWF1, CYP710A1 and BR6OX2, leading to enhanced sterol content and stress tolerance in Arabidopsis.

Biochemical properties of a novel and highly thermostable bacterial α-carbonic anhydrase from Sulfurihydrogenibium yellowstonense YO3AOP1.

Abstract: A new carbonic anhydrase (CA, EC 4.2.1.1) from the thermophilic bacterium Sulfurihydrogenibium yellowstonense YO3AOP1 was identified and characterized. The bacterial carbonic anhydrase gene was expressed in Escherichia coli yielding an active enzyme, which was purified in large amounts. The recombinant protein (SspCA) was found to belong to the α-CA class and displays esterase activity. The kinetic parameters were determined by using CO(2) and p-nitrophenylacetate (p-NpA) as substrates. The bacterial enzyme presented specific activity comparable to that of bovine carbonic anhydrase (bCA II) but it showed biochemical properties never observed for the mammalian enzyme. The thermophilic enzyme, in fact, was endowed with high thermostability and with unaltered residual activity after prolonged exposure to heat up to 100°C. SspCA and the bovine carbonic anhydrase (bCA II) were immobilized within a polyurethane (PU) foam. The immobilized bacterial enzyme was found to be active and stable at 100°C up to 50 h.

α-Amylase immobilization on the silica nanoparticles for cleaning performance towards starch soils in laundry detergents

Abstract: In this study, α-amylase was immobilized on silica nanoparticles and immobilized α-amylase was used in formulation of detergent powder for enhancing removal of starch soils. Detergent products contain very components which may affect the free enzyme activity and stability. Also various factors such as temperature, pH and humidity reduced enzyme activity and cleaning efficiency. Therefore the effect of enzyme immobilization on the removal of starch based soil was investigated on cotton fabrics as the model soil. The effect of temperature and humidity on stability of free and immobilized enzyme was compared. It was found that the immobilized enzyme increased cleaning efficiency toward starch soil removal on cotton fabrics, whereas free enzyme imposed a small effect on the enzymatic activity towards the same soil substrates. In addition, stability immobilized enzyme against temperature and humidity was much more than free enzyme.

Exogenous calcium alleviates the impact of cadmium-induced oxidative stress in Lens culinaris medic. Seedlings through modulation of antioxidant enzyme activities

Abstract: The effect of calcium (Ca) on lentil (Lens culinaris Medic.) seedlings exposed to cadmium (Cd) stress was studied by investigating plant growth and antioxidant enzyme activities. Plants were grown for 14 days in full-strength Hoagland nutrient media supplemented with Cd concentrations of 0, 10, 20, and 40 μM, and on corresponding medium supplied with 5 mM Ca(NO3)2 prior to Cd addition. Increasing Cd led to accumulation of metal and reduced the fresh weight of the shoots more strongly than that of the roots. Cd concentrations of 20 and 40 μM were selected to study its toxic effect on seedlings. Activities of superoxide dismutase, ascorbate peroxidase, catalase, dehydroascorbate reductase, and glutathione reductase decreased at much higher magnitude in the shoots than those observed in the roots under Cd exposure. Failure of antioxidant defense in scavenging of reactive oxygen species was evidenced by abnormal rise in H2O2, resulting in enhancement of lipid peroxidation and membrane electrolyte leakage as the marks of Cd-induced oxidative stress in lentil seedlings. Ca priming in the media significantly reduced the Cd accumulation and considerably alleviated the adverse impact of Cd treatment by modulating the antioxidant enzyme activity. Mitigation of Cd-induced stress by Ca application was strongly suggested by declining levels of H2O2 and consequent lowering of oxidative damage of membrane. Consequently, this enhanced fresh mass of plant parts as the sign of Ca-mediated normal growth in Cd-treated lentil seedlings.

Importance of β,β‐carotene 15,15′‐monooxygenase 1 (BCMO1) and β,β‐carotene 9′,10′‐dioxygenase 2 (BCDO2) in nutrition and health

Abstract: In humans, varying amounts of absorbed β-carotene are oxidatively cleaved by the enzyme β,β-carotene 15,15'-monooxygenase 1 (BCMO1) into two molecules of all-trans-retinal. The other carotenoid cleavage enzyme β,β-carotene 9',10'-dioxygenase (BCDO2) cleaves β-carotene at the 9',10' double bond forming β-apo-10'-carotenal and β-ionone. Although the contribution of BCDO2 to vitamin A formation has long been debated, BCMO1 is currently considered the key enzyme for retinoid metabolism. Furthermore, BCMO1 has limited enzyme activity towards carotenoids other than provitamin A carotenoids, whereas BCDO2 exhibits a broader specificity. Both enzymes are located at different sites within the cell, with BCMO1 being a cytosolic protein and BCDO2 being located in the mitochondria. Expression of BCMO1 in tissues other than the intestine has recently revealed its function for tissue-specific retinoid metabolism with importance in embryogenesis and lipid metabolism. On the other hand, biological activity of BCDO2 metabolites has been shown to be important in protecting against carotenoid-induced mitochondrial dysfunction. Single-nucleotide polymorphisms (SNPs) such as R267S and A379V in BCMO1 can partly explain inter-individual variations observed in carotenoid metabolism. Advancing knowledge about the physiological role of these two enzymes will contribute to understanding the importance of carotenoids in health and disease.

Toxicity of Citrate-Capped Silver Nanoparticles in Common Carp (Cyprinus carpio)

Abstract: Juvenile common carp (Cyprinus carpio) were used as a model to investigate acute toxicity and oxidative stress caused by silver nanoparticles (Ag-NPs). The fish were exposed to different concentrations of Ag-NPs for 48 h and 96 h. After exposure, antioxidant enzyme levels were measured, including glutathione-S-transferase (GST), superoxidase dismutase, and catalase (CAT). Other biochemical parameters and histological abnormalities in different tissues (i.e., the liver, gills, and brain) were also examined. The results showed that Ag-NPs agglomerated in freshwater used during the exposure experiments, with particle size remaining <100 nm. Ag-NPs had no lethal effect on fish after 4 days of exposure. Biochemical analysis showed that enzymatic activities in the brain of the fish exposed to 200 𝜇g/L of Ag-NPs were significantly reduced. Varied antioxidant enzyme activity was recorded in the liver and gills. Varied antioxidant enzyme activity was recorded for CAT in the liver and GST in the gills of the fish. However, the recovery rate of fish exposed to 200 𝜇g/L of Ag-NPs was slower than when lower particle concentrations were used. Other biochemical indices showed no significant difference, except for NH3 and blood urea nitrogen concentrations in fish exposed to 50 𝜇g/L of Ag-NPs. This study provides new evidence about the effects of nanoparticles on aquatic organisms.

Biphasic effect of copper on growth, proline, lipid peroxidation and antioxidant enzyme activities of wheat (Triticum aestivum cv. Hasaawi) at early growing stage.

Abstract: The biphasic effects of Cu +2 on the growth and some biochemical parameters of Hassawi wheat were studied. Hassawi wheat seeds were grown in various copper (Cu +2 ) levels (0, 2, 10, 20, 40, 80 and 100 mM) for 30 days. Cu +2 was applied on soil as Cu2SO4.5H2O. The results showed that Cu +2 concentration at 2 mM level promotes the growth and tested biochemical parameters of Hassawi wheat plants and can be considered as optimal dose. However, the studied parameters did not significantly change when Cu +2 applied more than the above mentioned concentration up to 10 mM, and thereafter the growth and biochemical parameters were significantly reduced, compared to untreated control plants. Furthermore, the activities of antioxidant enzymes such as, catalase, peroxidase, ascorbate peroxidase and superoxide dismutase were also increased. The results support the biphasic effect of copper on Hassawi wheat growth. The stimulatory effect of Cu +2 on the biosynthesis of free amino acids, proline and antioxidant enzyme activities could serve as important components of antioxidative defense mechanism against Cu 2+ toxicity.

A fluorescence-based supramolecular tandem assay for monitoring lysine methyltransferase activity in homogeneous solution.

Abstract: The demand for practical and convenient enzyme assays for histone lysine methyltransferases (HKMTs) emerges along with the rapid development of this young class of enzymes. A supramolecular reporter pair composed of p-sulfonatocalix[4]arene (CX4) and the fluorescent dye lucigenin (LCG) has been used to monitor enzymatic trimethylation of lysine residues in peptide substrates. The assay affords a switch-ON fluorescence response and operates in a continuous, real-time, and label-free fashion. The underlying working principle relies on the higher affinity of the macrocycle towards the trimethylated product of the enzymatic reaction as compared to the substrate, which allows the assay to be carried out in the product-selective mode. The final product incorporates a trimethylammonium moiety, a known high-affinity binding motif for CX4. Two substrates corresponding to the H3 N-terminal tail, namely, S2 (RTKQTARKSTGGKAP) and S6 (QTARKSTGGS), were selected as model compounds for methylation with the Neurospora crassa Dim-5 enzyme and investigated by the newly developed supramolecular tandem HKMTs assay. Only the longer substrate S2 underwent methylation in solution. The potential of the assay for inhibitor screening was demonstrated by means of inhibition studies with 1,10-phenanthroline to afford an inhibition constant of (70±20) μM.

Role of active site rigidity in activity: MD simulation and fluorescence study on a lipase mutant.

Abstract: Relationship between stability and activity of enzymes is maintained by underlying conformational flexibility. In thermophilic enzymes, a decrease in flexibility causes low enzyme activity while in less stable proteins such as mesophiles and psychrophiles, an increase in flexibility is associated with enhanced enzyme activity. Recently, we identified a mutant of a lipase whose stability and activity were enhanced simultaneously. In this work, we probed the conformational dynamics of the mutant and the wild type lipase, particularly flexibility of their active site using molecular dynamic simulations and time-resolved fluorescence techniques. In contrast to the earlier observations, our data show that active site of the mutant is more rigid than wild type enzyme. Further investigation suggests that this lipase needs minimal reorganization/flexibility of active site residues during its catalytic cycle. Molecular dynamic simulations suggest that catalytically competent active site geometry of the mutant is relatively more preserved than wild type lipase, which might have led to its higher enzyme activity. Our study implies that widely accepted positive correlation between conformation flexibility and enzyme activity need not be stringent and draws attention to the possibility that high enzyme activity can still be accomplished in a rigid active site and stable protein structures. This finding has a significant implication towards better understanding of involvement of dynamic motions in enzyme catalysis and enzyme engineering through mutations in active site.

Fine Tuning of the Lactate and Diacetyl Production through Promoter Engineering in Lactococcus lactis

Abstract: Lactococcus lactis is a well-studied bacterium widely used in dairy fermentation and capable of producing metabolites with organoleptic and nutritional characteristics. For fine tuning of the distribution of glycolytic flux at the pyruvate branch from lactate to diacetyl and balancing the production of the two metabolites under aerobic conditions, a constitutive promoter library was constructed by randomizing the promoter sequence of the H(2)O-forming NADH oxidase gene in L. lactis. The library consisted of 30 promoters covering a wide range of activities from 7,000 to 380,000 relative fluorescence units using a green fluorescent protein as reporter. Eleven typical promoters of the library were selected for the constitutive expression of the H(2)O-forming NADH oxidase gene in L. lactis, and the NADH oxidase activity increased from 9.43 to 58.17-fold of the wild-type strain in small steps of activity change under aerobic conditions. Meanwhile, the lactate yield decreased from 21.15 ± 0.08 mM to 9.94 ± 0.07 mM, and the corresponding diacetyl production increased from 1.07 ± 0.03 mM to 4.16 ± 0.06 mM with the intracellular NADH/NAD(+) ratios varying from 0.711 ± 0.005 to 0.383 ± 0.003. The results indicated that the reduced pyruvate to lactate flux was rerouted to the diacetyl with an almost linear flux variation via altered NADH/NAD(+) ratios. Therefore, we provided a novel strategy to precisely control the pyruvate distribution for fine tuning of the lactate and diacetyl production through promoter engineering in L. lactis. Interestingly, the increased H(2)O-forming NADH oxidase activity led to 76.95% lower H(2)O(2) concentration in the recombinant strain than that of the wild-type strain after 24 h of aerated cultivation. The viable cells were significantly elevated by four orders of magnitude within 28 days of storage at 4°C, suggesting that the increased enzyme activity could eliminate H(2)O(2) accumulation and prolong cell survival.

A highly efficient β-glucosidase from the buffalo rumen fungus Neocallimastix patriciarum W5

Abstract: Cellulose, which is the most abundant renewable biomass on earth, is a potential bio-resource of alternative energy. The hydrolysis of plant polysaccharides is catalyzed by microbial cellulases, including endo-β-1,4-glucanases, cellobiohydrolases, cellodextrinases, and β-glucosidases. Converting cellobiose by β-glucosidases is the key factor for reducing cellobiose inhibition and enhancing the efficiency of cellulolytic enzymes for cellulosic ethanol production. In this study, a cDNA encoding β-glucosidase was isolated from the buffalo rumen fungus Neocallimastix patriciarum W5 and is named NpaBGS. It has a length of 2,331 bp with an open reading frame coding for a protein of 776 amino acid residues, corresponding to a theoretical molecular mass of 85.1 kDa and isoelectric point of 4.4. Two GH3 catalytic domains were found at the N and C terminals of NpaBGS by sequence analysis. The cDNA was expressed in Pichia pastoris and after protein purification, the enzyme displayed a specific activity of 34.5 U/mg against cellobiose as the substrate. Enzymatic assays showed that NpaBGS was active on short cello-oligosaccharides from various substrates. A weak activity in carboxymethyl cellulose (CMC) digestion indicated that the enzyme might also have the function of an endoglucanase. The optimal activity was detected at 40°C and pH 5 ~ 6, showing that the enzyme prefers a weak acid condition. Moreover, its activity could be enhanced at 50°C by adding Mg2+ or Mn2+ ions. Interestingly, in simultaneous saccharification and fermentation (SSF) experiments using Saccharomyces cerevisiae BY4741 or Kluyveromyces marxianus KY3 as the fermentation yeast, NpaBGS showed advantages in cell growth, glucose production, and ethanol production over the commercial enzyme Novo 188. Moreover, we showed that the KY3 strain engineered with the NpaNGS gene can utilize 2 % dry napiergrass as the sole carbon source to produce 3.32 mg/ml ethanol when Celluclast 1.5 L was added to the SSF system. Our characterizations of the novel β-glucosidase NpaBGS revealed that it has a preference of weak acidity for optimal yeast fermentation and an optimal temperature of ~40°C. Since NpaBGS performs better than Novo 188 under the living conditions of fermentation yeasts, it has the potential to be a suitable enzyme for SSF.

Characterisation of a novel white laccase from the deuteromycete fungus Myrothecium verrucaria NF-05 and its decolourisation of dyes.

Abstract: A novel ‘white’ laccase was purified from the deuteromycete fungus, Myrothecium verrucaria NF-05, which was a high laccase-producing strain (40.2 U·ml−1 on the thirteenth day during fermentation). SDS-PAGE and native-PAGE revealed a single band with laccase activity corresponding to a molecular weight of approximately 66 kDa. The enzyme had three copper and one iron atoms per protein molecule determined by ICP-AES. Furthermore, both UV/visible and EPR spectroscopy remained silence, indicating the enzyme a novel laccase with new metal compositions of active centre and spectral properties. The N-terminal amino acid sequence of the purified protein was APQISPQYPM. Together with MALDI-TOF analysis, the protein revealed a high homology of the protein with that from reported M. verrucaria. The highest activity was detected at pH 4.0 and at 30°C. The enzyme activity was significantly enhanced by Na+, Mn2+, Cu2+ and Zn2+ while inhibited by DTT, NaN3 and halogen anions. The kinetic constant (Km) showed the enzyme was more affinitive to ABTS than other tested aromatic substrates. Twelve structurally different dyes could be effectively decolourised by the laccase within 10 min. The high production of the strain and novel properties of the laccase suggested its potential for biotechnological applications.