Showing papers on "Michaelis–Menten kinetics published in 2012"

The Dual Arrhenius and Michaelis–Menten kinetics model for decomposition of soil organic matter at hourly to seasonal time scales

Abstract: Decomposition of soil carbon stocks is one of the largest potential biotic feedbacks to climate change. Models of decomposition of soil organic matter and of soil respiration rely on empirical functions that relate variation in temperature and soil water content to rates of microbial metabolism using soil-C substrates. Here, we describe a unifying modeling framework to combine the effects of temperature, soil water content, and soluble substrate supply on decomposition of soluble soil-C substrates using simple functions based on process concepts. The model's backbone is the Michaelis–Menten equation, which describes the relationship between reaction velocity and soluble organic-C and O2 substrate concentrations at an enzyme's reactive site, which are determined by diffusivity functions based on soil water content. Temperature sensitivity is simulated by allowing the maximum velocity of the reaction (Vmax) to vary according to Arrhenius function. The Dual Arrhenius and Michaelis–Menten kinetics (DAMM) model core was able to predict effectively observations from of laboratory enzyme assays of β-glucosidase and phenol-oxidase across a range of substrate concentrations and incubation temperatures. The model also functioned as well or better than purely empirical models for simulating hourly and seasonal soil respiration data from a trenched plot in a deciduous forest at the Harvard Forest, in northeastern United States. The DAMM model demonstrates that enzymatic processes can be intrinsically temperature sensitive, but environmental constrains of substrate supply under soil moisture extremes can prevent that response to temperature from being observed. We discuss how DAMM could serve as a core module that is informed by other modules regarding microbial dynamics and supply of soluble-C substrates from plant inputs and from desorption of physically stabilized soil-C pools. Most importantly, it presents a way forward from purely empirical representation of temperature and moisture responses and integrates temperature-sensitive enzymatic processes with constraints of substrate supply.

Initial Rate Enzyme Kinetics

Abstract: I Nomenclature, Definitions, and Evolution of the Kinetic Mechanism- A Nomenclature- B Evolution of Initial Rate Kinetics- References- II Derivation of Initial Velocity Rate Equations- A Definitions and Derivations- 1 Steady-State- 2 Initial Velocity- 3 The Maximal Velocity and Michaelis Constant- 4 Reverse Reaction Parameters and Rate Constants- B The Equilibrium Assumption- C Derivation of Complex Steady-State Rate Equations- D Derivation of the Rate Equation Using the Rapid Equilibrium Assumption- 1 The Random Bi Bi Mechanism- 2 The Ordered Bi Bi Mechanism- E Derivation of Initial Rate Equations Using a Combination of Equilibrium and Steady-State Assumptions- F Derivation of Steady-State Rate Equations by Using the Digital Computer- References- III Experimental Protocol and Plotting of Kinetic Data- A General Considerations- B Analysis of Radioactive Substrates and Determination of Radiopurity- C pH Effects- D Substrate Concentration- E Studies of Forward and Reverse Reactions- F Studies of Nucleotide Dependent Enzymic Reactions- G The Kinetic Assay- 1 The Continuous Assay- 2 The Stop-Time Assay- H Plotting Methods- I Graphical Procedures- J The Point of Convergence of Sequential Double Reciprocal Plots as a Criterion of Kinetic Mechanism- K Protocol and Data Plotting for Three Substrate Systems- L Graphical Methods for Differentiating between Steady-State and Equilibrium Ordered Bi Bi Mechanisms- References- IV Use of Competitive Substrate Analogs and Alternative Substrates for Studying Kinetic Mechanisms- A Competitive Inhibition- B Partial Competitive Inhibition- C Noncompetitive Inhibition- D Ucompetitive Inhibition- E Nonlinear Enzyme Inhibition- F The Use of Substrate Analogs for Studying Kinetic Mechanisms- 1 Bireactant Enzymic Systems- 2 Terreactant Systems- 3 Kinetic Studies of Adenylosuccinate Synthetase Using Dead End Inhibitors- G Cleland's Rules for Dead End Inhibition- H The Stereochemical Nature of Enzyme and Substrate Interaction- I Kinetics of Enzyme Specificity- J The Kinetics of Transition State Analogs- References- V Product, Substrate, and Alternative Substrate Inhibition- A Product Inhibition Experiments- 1 Experimental Protocol- 2 One Substrate Systems- 3 Two Substrate Systems- 4 Abortive Ternary Complex Formation- 5 Calculation of Rate Constants from Product Inhibition Experiments- 6 Noncompetitive Product Effects- B Substrate Inhibition- 1 A Simple Model for Substrate Inhibition- 2 Two Substrate Systems- C Alternative Substrate Inhibition- 1 Alternative Substrates Acting as Inhibitors Only- 2 Bireactant Systems- 3 Terreactant Systems- D Alternative Product Inhibition- E Multisite Ping Pong Mechanisms- F Enzymes with Identical Substrate-Product Pairs- References- VI Isotope Exchange- A Abortive Complex Formation- B Derivation of Rate Equations- 1 The Equilibrium Case: Ping Pong Bi Bi- 2 The Steady-State Case: Ordered Bi Bi (Theorell-Chance)- 3 Random Bi Bi- 4 Theorell-Chance Mechanism- C Substrate Synergism- D Calculation of Kinetic Parameters- 1 The Ping Pong Bi Bi Mechanism- 2 The Random Bi Bi Mechanism (Rapid Equilibrium)- E Experimental Protocol- F Isotope-Trapping- References- VII Isomerization Mechanisms and the ? and Haldane Relationships- A The ? Relationships- B The Haldane Relationships- 1 Ordered Bi Bi- 2 Rapid Equilibrium Random Bi Bi- 3 Steady-State Random Bi Bi- C Isomerization Mechanisms- References- VIII The Effect of Temperature and pH on Enzyme Activity- A Effect of pH on Enzyme Kinetics- 1 pH Functions- 2 The Effect of pH on Unireactant Models- 3 Evaluation of Ionization Constants- 4 Bisubstrate Systems- 5 Cooperative Proton Binding- 6 Identification of Amino Acid Residues from Studies of pH Kinetics- 7 Some Limitations in the Study of pH Kinetics- 8 Choosing a Buffer for Kinetic Experiments- 9 The pH Kinetics of the Fumarase Reaction- B The Effect of Temperature on Enzyme Catalyzed Reactions- 1 Collision Theory and the Arrhenius Equation- 2 Transition-State Theory- 3 Significance of Activation Enthalpy and Activation Entropy- 4 Application of Transition-State Theory to the ?-Chymotrypsin Reaction- References- IX Cooperativity and Allostery- A Cooperativity- 1 The Hill Equation- 2 The Adair Equation- 3 The Scatchard Plot- B Molecular Models- 1 The Monod Model- 2 The Adair-Koshland Model- 3 Subunit-Subunit Polymerization- 4 Protein Isomerization- C Kinetic Models- 1 Kinetic Models Involving Subunit-Subunit Interaction- 2 Kinetic Models Involving Alternative Pathways of Substrate Addition and Enzyme Isomerization- D Allostery- 1 Nonsigmoidal Systems- 2 The Monod Model- 3 The Adair-Koshland Model- 4 Enzyme Isomerization Mechanisms- 5 Kinetic Models- E Product Effects- References- Appendix I Rate Equations, Determinants, and Haldane Expressions for Some Common Kinetic Mechanisms- Appendix II A Computer Program for Deriving Enzyme Rate Equations- Appendix III Plotting and Statistical Analysis of Kinetic Data Using the OMNITAB Program

Enzyme Activity in the Crowded Milieu

Abstract: The cytosol of a cell is a concentrated milieu of a variety of different molecules, including small molecules (salts and metabolites) and macromolecules such as nucleic acids, polysaccharides, proteins and large macromolecular complexes. Macromolecular crowding in the cytosolic environment is proposed to influence various properties of proteins, including substrate binding affinity and enzymatic activity. Here we chose to use the synthetic crowding agent Ficoll, which is commonly used to mimic cytosolic crowding conditions to study the crowding effect on the catalytic properties of glycolytic enzymes, namely phosphoglycerate kinase, glyceraldehyde 3-phosphate dehydrogenase, and acylphosphatase. We determined the kinetic parameters of these enzymes in the absence and in the presence of the crowding agent. We found that the Michaelis constant, Km, and the catalytic turnover number, kcat, of these enzymes are not perturbed by the presence of the crowding agent Ficoll. Our results support earlier findings which suggested that the Michaelis constant of certain enzymes evolved in consonance with the substrate concentration in the cell to allow effective enzyme function in bidirectional pathways. This conclusion is further supported by the analysis of nine other enzymes for which the Km values in the presence and absence of crowding agents have been measured.

Catalytic activity and stability of glucose oxidase/horseradish peroxidase co-confined in macroporous silica foam

Abstract: Investigation of the catalytic activity and stability of enzymes in confined nano/microspace provides valuable contributions to the fundamental understanding of biological reactions taking place on a mesoscopic scale within confined spaces. In this paper, macroporous silica foam (MSF) is used as a nanoreactor to co-confine glucose oxidase (GOD) and horseradish peroxidase (HRP). Then, the enzymatic cascade reactions, which act in tandem inside nanoreactors, for oxidation of glucose and 3,3',5,5'-tetramethylbenzidine (TMB) were studied. The catalytic kinetic parameters of apparent Michaelis constant (K(m)(app)) and maximum rate (V(max)) were obtained from Lineweaver-Burk plot by UV-vis spectrometry. Results showed that the catalytic activity of the co-confined enzymes is reduced compared to that of free enzymes in solution at room temperature. The stabilities of co-confined enzymes in denaturing agents, such as guanidinium chloride (GdmCl) and urea, were higher than those of free enzymes in solution. When employing a co-confined bienzyme system as a biosensor for the detection of glucose, a wider linear range of glucose was obtained for the co-confined bienzyme system than for free enzymes in solution.

Immobilization of Aspergillus oryzae β-Galactosidase onto Duolite A568 Resin via Simple Adsorption Mechanism

Abstract: In this study, a rapid, simple and economic method of enzyme immobilization was developed to hydrolyze lactose. Duolite A568 resin was used for the immobilization of β-galactosidase via simple adsorption mechanism. The effects of immobilization parameters such as time, pH, and temperature were studied. Immobilization parameters for maximum enzyme activity were estimated at 35 °C temperature, pH 4.5, 5 mg/mL enzyme concentration, and approximately 60 min immobilization time. A significant amount of enzyme was immobilized with high catalytic activity. Enzyme immobilization procedure explained in this study slightly affected the enzyme kinetic. The value of Michaelis constant K m for immobilized enzyme was significantly larger, indicating decreased affinity by the enzyme for its substrate. It was observed that both free and immobilized enzyme showed maximum activity at 65 °C reaction temperature. Immobilized β-galactosidase was significantly more active at all temperatures as compared to its free form. However, optimal pH of immobilized enzyme was slightly affected by immobilization procedure. The optimum pH of immobilized enzyme was shifted up 0.5 unit to a more alkaline value of 6.0 compared to the free enzyme.

A perturbation solution of Michaelis–Menten kinetics in a “total” framework

Abstract: In this paper we expand the equations governing Michaelis–Menten kinetics in a total quasi-steady state setting, finding the first order uniform expansions. Our results improve previous approximations and work well especially in presence of an enzyme excess.

Unravelling the suicide inactivation of tyrosinase: A discrimination between mechanisms

Abstract: The suicide inactivation kinetics of tyrosinase acting on 3-isopropyl-6-methylcatechol, 3-tert-butyl-6-methylcatechol and 3,6-difluorocatechol was studied. All three substrates act as suicide substrates despite the fact that their 3 and 6 positions are occupied, confirming the mechanism proposed in Biochem. J. (2008) 416, 431–440. Although the most active substrate for the suicide inactivation was 3,6-difluorocatechol, its efficiency was much lower than that of the catechol used as reference. Its r value, the number of turnovers made by one mol of enzyme before its inactivation, is the highest described in the bibliography, highlighting the great difference between the catalytic and inactivation constants. A study of the effect of the pH on the enzymatic activity of tyrosinase showed that both 3-isopropyl-6-methylcatechol and 3-tert-butyl-6-methylcatechol behave as typical substrates of tyrosinase, while 3,6-difluorocatechol behaves differently. The remarkable behavior of 3,6-difluorocatechol when reacts with tyrosinase may be due to the fact that its two hydroxyl groups have very low pK values as a result of the strong electron-withdrawing effect of the fluorine atoms in the ortho positions, so that, at pH 7.0, the substrate would be mainly negatively charged. The apparent Michaelis constant shows a minimum value at pH 6.0, but increases at both high and low pH. However, the values of the catalytic constant and maximum apparent inactivation constant do not vary with the pH, so that the r remains practically constant. Under anaerobic conditions, 3,6-difluorocatechol acts as an irreversible inhibitor of the deoxy- and met-tyrosinase forms.

Evaluation of Models for Spinach Respiratory Metabolism Under Low Oxygen Atmospheres

Abstract: Anaerobic respiration is a major problem that causes the deterioration of fresh produce packaged under low O2 atmospheres. The problem becomes more severe and causes high losses in the packages handling at ambient conditions, especially in developing countries. In designing modified atmosphere packaging, the risk of anaerobic development greatly depends upon the accuracy of respiration rate prediction; therefore, the respiration rate model for a particular produce has to be identified. In this study, different atmospheric storage conditions in a closed system were realized to examine the adaptability of respiration rate models for spinach storage under low O2 at an expected ambient temperature of 25 °C. Six models were applied and it was found that, for aerobic conditions, the respiration rate could be described with a constant respiratory quotient by three models, viz., (a) Michaelis–Menten model without inhibition, (b) Michaelis–Menten model with uncompetitive inhibition, and (c) Langmuir adsorption model, whereas three other models, viz. (d) Michaelis–Menten model with competitive inhibition, (e) Michaelis–Menten model with noncompetitive inhibition, and (f) Michaelis–Menten with mixed inhibition could not be fitted. Among the three successful models, the Michaelis–Menten with uncompetitive inhibition was found to be the most suitable model for practical applications in developing countries where cold-chain systems are lacking. This model can be applied for the prediction of gas composition and optimize the packages, particularly to ensure the aerobic respiration.

Kinetics of enzymatic deacetylation of chitosan

Abstract: The current paper reports on an investigation of the kinetics of chitosan deacetylation by chitin deacetylase isolated from Absidia orchidis vel coerulea. The reaction rate was correlated with the concentration of GlcNHAc units of the polymer. It is shown that the process follows the Michaelis–Menten mechanism. Modification of the Michaelis–Menten equation by introducing the activity of the enzyme instead of its concentration was tested and found to give a better approximation to the experimental data than the original Michaelis–Menten model. Parameters for both the original and the modified Michaelis–Menten equations are also proposed.

Kinetics of fungal extracellular alpha-amylase from Fusarium solani immobilized in calcium alginate beads.

Abstract: Extracellular alpha-amylase mass produced by Fusarium solani using mango kernel as substrate was immobilized in calcium alginate beads through entrapment technique. Maximum enzyme immobilization efficiency was achieved in 2 mm size beads formed by 6.5% (w/v) of sodium alginate in 2% (w/v) calcium chloride. The catalytic properties of the immobilized alpha-amylase were compared with that of free enzyme (soluble). The activity yield of the immobilized enzyme was 81% of the free enzyme. The immobilized enzyme showed optimum activityat pH 4.5-6.0 and temperature 40 degrees C, in contrast to the free enzyme at 5.5 and 30 degrees C, respectively. Thermal stability of the immobilized enzyme was found to be more than the free enzyme over a longer time interval. The immobilized enzyme retained activity upto 20% of optimum even after 180 min. While the free enzyme lost its 80% activity after 60 min and lost total activity down to zero by 120 min. The kinetic constants, viz., K(M) (Michaelis constant), V(max) and activation energy were affected by immobilization. However, the immobilized alpha-amylase in calcium alginate beads supports its long-term storage which has immense industrial applications.

Immobilization/stabilization of acid urease on Eupergit® supports

Abstract: The adsorption capacity and immobilization rate of two Eupergit® supports for acid urease was studied by varying the ionic strength and enzyme preparation concentration in the immobilizing solution at pH 7. Eupergit® C250 L yielded a series of derivatives with enzyme loadings (YP/B) ranging from 48 to 171 mg of bovine serum albumin equivalent (BSAE) per gram of dry support (ds). Use of drastic postimmobilization conditions at pH 9 for 3–9 days yielded a slight decrease (8–14%) in the initial activity of immobilized enzymes and a limited increase in the stabilization factor (1.1–1.5), as assessed by accelerated aging tests at 65°C. Further storage tests at 4°C in the wet state showed that the activity of several derivatives either stabilized or not was practically constant for as long as 547 days. Both free enzyme and immobilized acid urease derivatives exhibited a kinetic pattern of the Michaelis–Menten type. Using the Eadie–Hofstee diagram, the specific ammonia formation rate constant for free (kcat) or immobilized (k′cat) enzyme resulted to be little affected by immobilization (kcat ≈ k′cat ≈ 18.86 ± 0.34 IU/mg BSAE), whereas the apparent Michaelis constant for immobilized enzymes exhibited a statistically significant increase at P < 0.05 from the intrinsic value (2.55 ± 0.14 mM) for free enzyme to 5.38 ± 0.87 mM as YP/B increased to 171 mg BSAE/g ds. By estimating the observable Thiele modulus (ϕobs), the activity of the biocatalyst with the greatest enzyme loading at the lowest urea concentrations tested (0.833 mM) was reduced by a factor of about 2 due to internal diffusional limitations. By operating in the pseudofirst-order regime with immobilized derivatives at YP/B about 126 mg BSAE/g ds, their activity after grinding was no more limited by intraparticle diffusion and approached the value for free enzyme. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012

Substrate-dependent kinetics in tyrosinase-based biosensing: amperometry vs . spectrophotometry

Abstract: Despite the broad use of enzymes in electroanalytical biosensors, the influence of enzyme kinetics on the function of prototype sensors is often overlooked or neglected. In the present study, we employ amperometry as an alternative or complementary method to study the kinetics of tyrosinase, whose catalytic activity results in o-quinone products. We further compare our results for four monophenolic substrates with those obtained from ultraviolet–visible spectrophotometry and show that the results from both assays are in good agreement.We also observe large variations in the enzyme kinetics for different monophenolic substrates depending on the R-group at the para position. To further study this effect, we investigate the stability of quinone products in the enzymatic assay. This information can in principle be utilized to discriminate between different phenolic species by monitoring the reaction rate

Quantitative kinetics of proteolytic enzymes determined by a surface concentration-based assay using peptide arrays.

Abstract: Peptide arrays have emerged as a key technology for drug discovery, diagnosis, and cell biology. Despite the promise of these arrays, applications of peptide arrays to quantitative analysis of enzyme kinetics have been limited due to the difficulty in obtaining quantitative information of enzymatic reaction products. In this study, we developed a new approach for the quantitative kinetics analysis of proteases using fluorescence-conjugated peptide arrays, a surface concentration-based assay with solid-phase peptide standards using dry-off measurements, and compared it with an applied concentration-based assay. For fabrication of the peptide arrays, substrate peptides of cMMP-3, caspase-3, caspase-9, and calpain-1 were functionalized with TAMRA and cysteine, and were immobilized onto amine-functionalized arrays using a heterobifunctional linker, N-[γ-maleimidobutyloxy]succinimide ester. The proteolytic activities of the four enzymes were quantitatively analyzed by calculating changes induced by enzymatic reactions in the concentrations of peptides bound to array surfaces. In addition, this assay was successfully applied for calculating the Michaelis constant (Km,surf) for the four enzymes. Thus, this new assay has a strong potential for use in the quantitative evaluation of proteases, and for drug discovery through kinetics studies including the determination of Km and Vmax.

Spectroscopy and kinetics of tyrosinase catalyzed trans-resveratrol oxidation.

Abstract: The spectroscopy and kinetics of the tyrosinase catalyzed trans-resveratrol oxidation were investigated by measuring both UV–vis absorption spectra over the 200–500 nm range and Raman spectra over the 600–1800 cm–1 region. Room temperature UV–vis absorption spectra, as a function of time, showed the presence of two isosbestic points located at λ1 = 270 nm and λ2 = 345.5 nm delimiting two different regions: the reactant region around 300 nm, where the absorption decreased with time, and the product region over the low wavelength (λ 390 nm) wavelength zone in which the absorption increased with time until, in both cases, constant values were achieved. A first-order kinetics was deduced with a rate coefficient of k1 = (0.10 ± 0.001) min–1, which turned out to be independent of substrate concentration over the 50–5 μM range; a feature that was rationalized by invoking the limiting case of the Michaelis–Menten scheme appropriate for substrate concentration much lower than the respective Michaelis constant. The observation of the distinct resonance enhanced Raman lines, specifically those peaking at 830 cm–1, 753 cm–1, and 642 cm–1 together with their time evolution, permitted us to gain insight into some crucial features and steps of the catalytic reaction. Namely, that the formation of the so-called trans-resveratrol and tyrosinase SP complex with its O–O bridge plays a crucial role in the first steps of this enzymatic reaction and that the hydroxylation of the ortho C–H bond of the trans-resveratrol OH group occurs after O–O bond cleavage in the tyrosinase active site. The present study makes clear that a class of potential inhibitors of tyrosinase can be found in compounds able to bind the two Cu (II) ions of the enzyme bidentate form.

Interaction of Ferrocene Mediators with Gluconobacter oxydans Immobilized Whole Cells and Membrane Fractions in Oxidation of Ethanol

Abstract: Gluconobacter oxydans whole cells and membrane fractions in combination with ferrocene mediators were used to study oxidation of ethanol. The efficiency of mediator?enzyme interaction was assessed by the ratio of maximum current to the apparent Michaelis constant (Imax/KM) for saturating mediator concentrations. The bioelectrocatalytic processes were found to be more efficient with membrane fractions. The highest Imax/KM value of 120 and 3200 mu A g/mol for, respectively, cells and fractions was obtained for ferrocene carboxylic acid. In test measurements of biological oxygen demand for rye distillers grains, the values obtained by a biosensor based on Gluconobacter membrane fractions and ferrocene were found to correlate with the reference data. (Less)

Effect of ionic liquids activation on laccase from Trametes versicolor: Enzymatic stability and activity

Abstract: The stability and activity of laccase from Trametes versicolor in two water-soluble ionic liquids (ILs), namely 1-butyl-3-methylimidazolium methyl sulfate, [bmim][MeSO4] and 1,3-dimethylimidazolium methyl sulfate, [mmim][MeSO4], were investigated in this study. Thermal inactivation of laccase was characterized in the presence of these both ILs and as expected first-order kinetics was followed. Inactivation rate constant (k), half-life time (t1/2), and energy of activation (Ea) were determined. Kinetics of 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) oxidation by laccase in the presence of these ILs was studied and Michaelis–Menten parameters were calculated. There is no enzymatic inactivation since the maximum reaction rate remained constant for IL concentrations up to 25%, and surprisingly, it was found that laccase was activated for concentrations of 35% of ILs, since the reaction rate increased 1.7 times.

Promotion effect by ε-poly-L-lysine on the enzymatic reaction of glucose oxidase with ferricyanide ion as an oxidant.

Abstract: An enzymatic reaction rate of glucose oxidase (GOD) using ferricyanide ion ([Fe(CN)(6)](3-)) as an oxidant significantly increased by the addition of e-poly-L-lysine (e-PL). The bimolecular rate constant between GOD and [Fe(CN)(6)](3-) in the presence of e-PL reached about 10000-fold relative to what it was measured without the e-PL, and the Michaelis constant decreased. The reaction rate reached a maximum at around pH 6, where e-PL and GOD possess highly positive and negative charges, respectively. The increment of the reaction rate by e-PL can be attributed to the electrostatic association of the polycationic e-PL with the negatively charged GOD to form a polyion complex soluble in the aqueous medium. The adduction of the cationic polymer may relieve the electrostatic repulsion between GOD and [Fe(CN)(6)](3-), so that the electron transfer effectively occurs between them.

Purification and properties of a phytate-degrading enzyme produced by Enterobacter sakazakii ASUIA279

Abstract: An extracellular phytate-degrading enzyme produced by Enterobacter sakazakii ASUIA279 was purified to homogeneity using FPLC anion exchange chromatography and gel filtration. The enzyme was purified about 66-fold with a recovery of 27%. Its molecular mass was estimated to be 43 kDa by SDS-PAGE. The Michaelis constant (KM) and turnover number (kcat ) for sodium phytate at pH 5.0 and 50°C were calculated from the Lineweaver-Burk plot to be 760 µM and 4.14s-1, respectively. The enzyme showed narrow substrate specificity and not phytate, but GTP was dephosphorylated with the highest relative rate of hydrolysis. However, according to the kcat/KM values, phytate was concluded to be the in vivo substrate of the enzyme. Optimal activity was determined at pH 4.5 and 45-55°C. The enzyme was strongly inhibited by Fe3+, Cu2+, Zn2+, molybdate, vanadate, fluoride and phosphate (1 mM). Key-words: Enterobacter sakazakii; phytate-degrading enzyme; phytate, purification

Influence of Inorganic Nutrients on the activity of Enzyme, Nitrate reductase in the leaves of Mulberry, Morus alba (L) (M-5 variety)

Abstract: The protein content of mulberry leaves is directly related with the potential of nitrate reductase enzyme. Effect of kinetic parameters and inorganic mineral nutrients (Mg; Zn and Mo) on the velocity of nitrate reductase catalyzed biochemical reaction was studied using the leaves of mulberry, Morus alba (L) (M-5 variety). Maximum velocity (Vmax) was found registered for PH=7.4; temperature=32 0 C; incubation period= 30 minutes with vaccum infiltration manually at 5 minutes interval. For the purpose to determine Michaelis Menten constant (Km), the substrate concentration at which, the velocity of enzyme catalyzed biochemical reaction attain half of its maximum, attempt has been made towards the transformation of data on [S] and v. The key quotient: [(2v Vmax +S) ÷ v] – [S(1+Vmax) ÷ Vmax] was calculated. Plotting the key quotient verses the substrate concentration [S] has illuminated into a straight line intersecting both, X and Y axes at a point which correspond to : [(2Vmax 2 + Km)÷Vmax]. The equation of the plot correspond to be derived as: Y = - [S] + [(2Vmax 2 +Km)÷ Vmax]. This plot is to be recognized as Punyamayee plot of enzyme kinetics. Accordingly the Michaelis Menten constant (Km) of nitrate reductase catalyzed biochemical reaction in assay sample of mulberry leaves was found elevated in assay sample of leaves of mulberry plant recipient of foliar spray of magnesium sulphate, Zinc sulphate and ammonium molybdate. The optimum dose for magnesium sulphate and Zinc sulphate was 2.5mM, while with the ammonium molybdate, it was 0.01mM. The enzyme nitrate reductase was found significantly influenced with the optimum dosage of inorganic nutrients like MgSO4; Zn and (NH4)2 Mo O4. The nitrate reductase activity may be considered as predictive test for protein rich yield of leaves in mulberry. Efficient use of inorganic nutrients for qualitative protein levels in mulberry leaves serve to orchestrate the moriculture practices and thereby the qualitative improvement in cocoon yield of silkworm, Bombyx mori(L).

Purification and Characterization of an Alkaliphilic Choline Oxidase of Fusarium oxysporum

Abstract: A novel choline oxidase found in a fungus, Fusarium oxysporum strain V2, was purified to homogeneity as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The enzyme has a molecular mass of 128 kDa and consists of two identical subunits. The purified enzyme showed adsorption peaks at 340 nm and 450 nm. It showed alkaliphilic pH characteristics: its optimum pH was 9.0–10.0, and it was stable at pH 8.0–10.2. The Michaelis constant (K m) values for choline and betaine aldehyde were 0.28 mM and 0.39 mM respectively. Trimethylamino-alcohols, dimethylamino-alcohols, and diethylaminoethanol were substrates for the enzyme, but the K m values for them increased with decreasing numbers of methyl groups on the ammonium headgroup. A marked decrease in the maximum velocity (V max) and V max/K m values was observed when N-replaced choline analogs were used as substrate instead of choline. The enzyme had a remarkably higher affinity for choline and betaine aldehyde than do previously reported enzym...

Kinetics and docking studies on the effect of chemical modification of NADH for redox reactions with dehydrogenases

Abstract: Cofactor analogs promise important applications in biosynthesis. The effect of chemical modification on the reactivity of NADH for redox reactions catalyzed by dehydrogenases was examined in this work. Compared with the native NADH, kinetics and molecular docking studies with 8-(6-aminohexyl)-amino-NADH showed that its binding with alcohol dehydrogenase (ADH) was not much affected or even enhanced by a factor of 4.9-fold with lactate dehydrogenase (LDH), but complicated the binding of substrates to the enzymes. For ADH, the Michaelis constant for acetaldehyde decreased from 0.47 to 0.048 mM, while that of sodium pyruvate with LDH increased to 0.81 from 0.18 mM. On the other hand, the modified coenzyme showed a 19.3-fold decrease in turnover number (k(cat)) with ADH, while a slight increase with LDH. Molecular docking analysis showed that the hexanediamine arm on the modified coenzyme generated an extra hydrogen bond at the active site of ADH, as well as additional hydrophobic interactions with both ADH and LDH. It appeared that the apparently decreased reactivity of modified cofactor with ADH was caused mainly by the enhanced stability of ternary coenzyme-enzyme-substrate complex, while in the case of LDH, the reduced substrate binding as a result of the chemical modification of NADH led to a slight increase in the overall reaction reactivity. (C) 2012 Elsevier B.V. All rights reserved.

Michaelis-Menten kinetics in shear flow: Similarity solutions for multi-step reactions.

Abstract: Models for chemical reaction kinetics typically assume well-mixed conditions, in which chemical compositions change in time but are uniform in space. In contrast, many biological and microfluidic systems of interest involve non-uniform flows where gradients in flow velocity dynamically alter the effective reaction volume. Here, we present a theoretical framework for characterizing multi-step reactions that occur when an enzyme or enzymatic substrate is released from a flat solid surface into a linear shear flow. Similarity solutions are developed for situations where the reactions are sufficiently slow compared to a convective time scale, allowing a regular perturbation approach to be employed. For the specific case of Michaelis-Menten reactions, we establish that the transversally averaged concentration of product scales with the distance x downstream as x5/3. We generalize the analysis to n-step reactions, and we discuss the implications for designing new microfluidic kinetic assays to probe the effect of flow on biochemical processes.