Showing papers on "Michaelis–Menten kinetics published in 2003"
TL;DR: The initial rate studies showed that the Michaelis constant for n-butanol was very low indicating lower affinity between the enzyme and the reactant, and the kinetics was found to obey the Ping-Pong bi-bi mechanism with n- butanol substrate inhibition.
136 citations
Journal Article•
TL;DR: The enzymatic hydrolysis of urea by jack bean urease was investigated at 25 °C over the pH range 4-9 and indicated that the inhibition mechanism was of the fully non-competitive type, with K p = 12.2 mmol l -1 .
Abstract: The enzymatic hydrolysis of urea by jack bean urease was investigated at 25 °C over the pH range 4-9. Reaction rate data were found to be well described by a modified Michaelis-Menten equation with a pH-dependent rate coefficient and a product inhibition term. The influence of pH on activity was interpreted in terms of perturbation of the enzyme distribution among three differently protonated forms. Kinetic analysis yielded a Michaelis constant of 3.21 mmol I -1 and indicated that the inhibition mechanism was of the fully non-competitive type, with K p = 12.2 mmol l -1 . The estimated activation energy was 35.3 kJ mol -1 . The resulting kinetic expression was tested by comparing model predictions with the experimental behaviour observed in unbuffered media and over a long-term period.
119 citations
TL;DR: Biochemical characteristics of a purified recombinant inulosucrase enzyme from Lactobacillus reuteri displayed Michaelis–Menten type of kinetics with substrate inhibition for the hydrolysis reaction.
70 citations
TL;DR: An efficient enzyme model exhibiting enantioselective esterase activity was prepared by using molecular imprinting techniques and a strong substrate selectivity of higher than three was found despite small differences in the structure of the substrate and template.
Abstract: An efficient enzyme model exhibiting enantioselective esterase activity was prepared by using molecular imprinting techniques. The enantiomerically pure phosphonic monoesters 4 L and 5 L were synthesized as stable transition-state analogues. They were used as templates connected by stoichiometric noncovalent interactions to two equivalents of the amidinium binding site monomer 1. After polymerization and removal of the template, the polymers were efficient catalysts for the hydrolysis of certain nonactivated amino acid phenylesters (2 L, 2 D, 3 L, 3 D) depending on the template used. Imprinted catalyst IP4 (imprinted with 4 L) enhanced the hydrolysis of the corresponding substrate 2 L by a factor of 325 relative to that of a buffered solution. Relative to a control polymer containing the same functionalities, prepared without template 4 L, the enhancement was still about 80-fold, showing the highest imprinting effect up to now. In cross-selectivity experiments a strong substrate selectivity of higher than three was found despite small differences in the structure of the substrate and template. Plots of initial velocities of the hydrolysis versus substrate concentration showed typical Michaelis-Menten kinetics with saturation behavior. From these curves, the Michaelis constant K(M) and the catalytic constant k(cat) can be calculated. The enantioselectivity shown in these values is most interesting. The ratio of the catalytic efficiency k(cat)/K(M), between the hydrolysis of 2 L- and 2 D-substrate with IP4, is 1.65. This enantioselectivity derives from both selective binding of the substrate (K(M)L/K(M)D=0.82), and from selective formation of the transition state (k(cat)L/k(cat)D=1.36). Thus, these catalysts give good catalysis as well as high imprinting and substrate selectivity. Strong competitive inhibition is caused by the template used in imprinting. This behavior is also quite similar to the behavior of natural enzymes, for which these catalysts are good models.
62 citations
TL;DR: The conservation of Trp-241 in all eukaryotic transglutaminases and the finding that W241Q-TG2 had a much lower kcat than wild-type enzyme suggest evolutionary specialization in the use of the indole group.
Abstract: Covalent posttranslational protein modifications by eukaryotic transglutaminases proceed by a kinetic pathway of acylation and deacylation. Ammonia is released as the acylenzyme is formed, whereas the cross-linked product is released later in the deacylation step. Superposition of the active sites of transglutaminase type 2 (TG2) and the structurally related cysteine protease, papain, indicates that in the formation of tetrahedral intermediates, the backbone nitrogen of the catalytic Cys-277 and the Ne1 nitrogen of Trp-241 of TG2 could contribute to transition-state stabilization. The importance of this Trp-241 side chain was demonstrated by examining the kinetics of dansylcadaverine incorporation into a model peptide. Although substitution of the Trp-241 side chain with Ala or Gly had only a small effect on the Michaelis constant Km (1.5-fold increase), it caused a >300-fold lowering of the catalytic rate constant kcat. The wild-type and mutant TG2-catalyzed release of ammonia showed kinetics similar to the kinetics for the formation of cross-linked product, indicating that transition-state stabilization in the acylation step was rate-limiting. In papain, a Gln residue is at the position of TG2-Trp-241. The conservation of Trp-241 in all eukaryotic transglutaminases and the finding that W241Q-TG2 had a much lower kcat than wild-type enzyme suggest evolutionary specialization in the use of the indole group. This notion is further supported by the observation that transition-state-stabilizing side chains of Tyr and His that operate in some serine and metalloproteases only partially substituted for Trp.
58 citations
TL;DR: It is shown that the resulting stochastic process for a large Michaelis-Menten constant behaves like the deterministic sequence x(n) arising by iterations of the function f(x)=x+x/(1+x).
54 citations
TL;DR: This paper presents the first description of an enzyme fraction exhibiting carotenoid cleavage activity isolated from fruit skin of Averrhoa carambola and proves that the isolated enzymes are closely related to aroma metabolism and release of star fruit.
50 citations
TL;DR: The combination of kinetic modelling tools and biological data has permitted the discrimination of at least three CYP enzymes involved in AMI N-demethylation in vitro by human liver microsomes.
Abstract: 1. To determine kinetic parameters (Vmax, K(m)) for cytochrome P450 (CYP) mediated metabolic pathways, nonlinear least squares regression is commonly used to fit a model equation (e.g., Michaelis Menten [MM]) to sets of data points (reaction velocity vs substrate concentration). This method can also be utilized to determine the parameters for more complex mechanisms involving allosteric or multi-enzyme systems. Akaike's Information Criterion (AIC), or an estimation of improvement of fit as successive parameters are introduced in the model (F-test), can be used to determine whether application of more complex models is helpful. To evaluate these approaches, we have examined the complex enzyme kinetics of amitriptyline (AMI) N-demethylation in vitro by human liver microsomes. 2. For a 15-point nortriptyline (NT) formation rate vs substrate (AMI) concentration curve, a two enzyme model, consisting of one enzyme with MM kinetics (Vmax = 1.2 nmol min-1 mg-1, K(m) = 24 microM) together with a sigmoidal component (described by an equation equivalent to the Hill equation for cooperative substrate binding; Vmax = 2.1 nmol min-1 mg-1, K' = 70 microM; Hill exponent n = 2.34), was favoured according to AIC and the F-test. 3. Data generated by incubating AMI under the same conditions but in the presence of 10 microM ketoconazole (KET), a CYP3A3/4 inhibitor, were consistent with a single enzyme model with substrate inhibition (Vmax = 0.74 nmol min-1 mg-1, K(m) = 186 microM, K1 = 0.0028 microM-1). 4. Sulphaphenazole (SPA), a CYP2C9 inhibitor, decreased the rate of NT formation in a concentration dependent manner, whereas a polyclonal rat liver CYP2C11 antibody, inhibitory for S-mephenytoin 4'-hydroxylation in humans, had no important effect on this reaction. 5. Incubation of AMI with 50 microM SPA resulted in a curve consistent with a two enzyme model, one with MM kinetics (Vmax = 0.72 nmol min-1 mg-1, K(m) = 54 microM) the other with 'Hill-kinetics' (Vmax = 2.1 nmol min-1 mg-1, K' = 195 microM; n = 2.38). 6. A fourth data-set was generated by incubating AMI with 10 microM KET and 50 microM SPA. The proposed model of best fit describes two activities, one obeying MM-kinetics (Vmax = 0.048 nmol min-1 mg-1, K(m) = 7 microM) and the other obeying MM kinetics but with substrate inhibition (Vmax = 0.8 nmol min-1 mg-1, K(m) = 443 microM, K1 = 0.0041 microM-1). 7. The combination of kinetic modelling tools and biological data has permitted the discrimination of at least three CYP enzymes involved in AMI N-demethylation. Two are identified as CYP3A3/4 and CYP2C9, although further work in several more livers is required to confirm the participation of the latter.
49 citations
TL;DR: The linearization of the Michaelis-Menten reaction by pseudo-first-order kinetics is revised and a phase-plane analysis allows the derivation of a new condition for its validity that is directly linked to the reaction efficiency, and contrary to widely established knowledge, is independent of the initial enzyme concentration.
47 citations
TL;DR: A phytase from Pseudomonas syringae MOK1 showed a high substrate specificity for sodium phytate with little or no activity on other phosphate conjugates and efficiently released orthophosphate from wheat bran and soybean meal.
Abstract: A phytase (EC 3.1.3.8) from Pseudomonas syringae MOK1 was purified to apparent homogeneity in two steps employing cation and an anion exchange chromatography. The molecular weight of the purified enzyme was estimated to be 45 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. The optimal activity occurred at pH 5.5 and 40°C. The Michaelis constant (K m ) and maximum reaction rate (Vmax) for sodium phytate were 0.38 mM and 769 U/mg of protein, respectively. The enzyme was strongly inhibited by Cu2+, Cd2+, Mn2+, and ethylenediaminetetraacetic acid (EDTA). It showed a high substrate specificity for sodium phytate with little or no activity on other phosphate conjugates. The enzyme efficiently released orthophosphate from wheat bran and soybean meal.
45 citations
TL;DR: The in-capillary reaction of angiotensin converting enzyme (ACE) with the tripeptide substrate hippuryl-L-histidyl- L-leucine was studied and an average Michaelis constant for ACE from rabbit lung was calculated to be 1.16 +/- 0.12 mM, a value consistent with previously reported data.
TL;DR: In this paper, the rate of hydrolysis of β-lactoglobulin (genetic variant B) by trypsin in aqueous solution at pH of 7.6 and ionic strength 0.08 M (NaC1) at 25.0°C increased with increasing hydrostatic pressure.
TL;DR: The reliability of kinetic substrate quantification by nonlinear fitting of the enzyme reaction curve to the integrated Michaelis–Menten equation was investigated by both simulation and preliminary experimentation and there were unique Am for best fitting of both the simulated and experimental reaction curves.
Abstract: The reliability of kinetic substrate quantification by nonlinear fitting of the enzyme reaction curve to the integrated Michaelis–Menten equation was investigated by both simulation and preliminary experimentation. For simulation, product absorptivity e was 3.00 mmol−1 L cm−1 and Km was 0.10 mmol L−1, and uniform absorbance error σ was randomly inserted into the error-free reaction curve of product absorbance Ai versus reaction time ti calculated according to the integrated Michaelis–Menten equation % MathType!MTEF!2!1!+- % feaafaart1ev1aaatCvAUfeBSjuyZL2yd9gzLbvyNv2CaerbuLwBLn % hiov2DGi1BTfMBaeXatLxBI9gBaerbd9wDYLwzYbItLDharqqtubsr % 4rNCHbGeaGqiVu0Je9sqqrpepC0xbbL8F4rqqrFfpeea0xe9Lq-Jc9 % vqaqpepm0xbba9pwe9Q8fs0-yqaqpepae9pg0FirpepeKkFr0xfr-x % fr-xb9adbaqaaeGaciGaaiaabeqaamaabaabaaGcbaGaciiBaiaac6 % gadaWadaqaaiaadgeacaWGTbGaai4lamaabmaabaGaamyqaiaad2ga % cqGHsislcaWGbbGaamyAaaGaayjkaiaawMcaaaGaay5waiaaw2faai % abgUcaRiaadgeacaWGPbGaai4lamaabmaabaGaeqyTduMaey41aqRa % am4saiaad2gaaiaawIcacaGLPaaacqGH9aqpdaqadaqaaiaadAfaca % WGTbGaai4laiaadUeacaWGTbaacaGLOaGaayzkaaGaey41aqRaamiD % aiaadMgaaaa!572F! $$
\ln \left[ {Am/\left( {Am - Ai} \right)} \right] + Ai/\left( {\varepsilon \times Km} \right) = \left( {Vm/Km} \right) \times ti
$$
. The experimental reaction curve of arylesterase acting on phenyl acetate was monitored by phenol absorbance at 270 nm. Maximal product absorbance Am was predicted by nonlinear fitting of the reaction curve to Eq. (1) with Km as constant. There were unique Am for best fitting of both the simulated and experimental reaction curves. Neither the error in reaction origin nor the variation of enzyme activity changed the background-corrected value of Am. But the range of data under analysis, the background absorbance, and absorbance error σ had an effect. By simulation, Am from 0.150 to 3.600 was predicted with reliability and linear response to substrate concentration when there was 80% consumption of substrate at σ of 0.001. Restriction of absorbance to 0.700 enabled Am up to 1.800 to be predicted at σ of 0.001. Detection limit reached Am of 0.090 at σ of 0.001. By experimentation, the reproducibility was 4.6% at substrate concentration twice the Km, and Am linearly responded to phenyl acetate with consistent absorptivity for phenol, and upper limit about twice the maximum of experimental absorbance. These results supported the reliability of this new kinetic method for enzymatic analysis with enhanced upper limit and precision.
TL;DR: In this paper, the enzymatic activities of wheat germ lipase (WGL) in water-in-oil (w/o) microemulsions of sodium bis-(2-ethylhexyl) sulfosuccinate (AOT)/cyclohexane/water have been investigated by using triolein as the substrate through measurements of absorbency of the product of hydrolysis at the wavelength of 707-nm in the course of reactions.
Abstract: The enzymatic activities of wheat germ lipase (WGL) (EC.3.1.1.3; triacylglycerol hydrolase) in water-in-oil (w/o) microemulsions of sodium bis-(2-ethylhexyl) sulfosuccinate (AOT)/cyclohexane/water have been investigated by using triolein as the substrate through measurements of absorbency of the product of hydrolysis at the wavelength of 707 nm in the course of reactions. Maximum enzyme activity was obtained at ω 0 ( molar ratio of water to surfactant )=9 , pH=7.4 and T =40 °C. The activation energy of the reaction was calculated from the Arrhenius plot. It was found that the hydrolysis reaction obeyed Michaelis–Menten kinetics in the investigated concentration range (0.01–0.15 mol/l) of the substrate, and the apparent Michaelis constant K m,a and the apparent maximal reaction rate V m were determined. The stability of wheat germ lipase in w/o microemulsions was also examined and discussed.
TL;DR: The rate of hydrolysis of 2-naphthyl acetate catalyzed by α-chymotrypsin has been measured in aqueous solutions of dodecyltrimethylammonium bromide at concentrations below and above the critical micelle concentration, as well as in the absence of surfactant, indicating that the interaction between the enzyme and micellelike aggregates alters the formation of the enzyme−substrate complexes.
Abstract: The rate of hydrolysis of 2-naphthyl acetate catalyzed by α-chymotrypsin has been measured in aqueous solutions of dodecyltrimethylammonium bromide at concentrations below and above the critical micelle concentration, as well as in the absence of surfactant. Under all the conditions employed, the reaction takes place following a Michaelis−Menten mechanism. The presence of the surfactant, at concentrations above its critical micellar concentration, increases the value of the Michaelis constant, Km, without significant changes in the catalytic rate constant, kcat. The increase in Km is larger than that expected from the incorporation of the substrate to the micellar pseudophase, indicating that the interaction between the enzyme and micellelike aggregates alters the formation of the enzyme−substrate complexes. This can be related to a partial unfolding of the enzyme, as is suggested by changes in its intrinsic fluorescence.
TL;DR: To obtain purer acid phosphatases than produced by prior art by operating under conditions that improve the final product, the use of a mild nonionic detergent, 40–80% saturation with (NH4)2SO4, maintained at low temperature to remove impurity, and theUse of chromatografic columns to concentrate the acid phosph atase and remove non-acid phosphatase proteins with lower or higher molecular weights is used.
Abstract: The objective of this study was to obtain purer acid phosphatases than produced by prior art by operating under conditions that improve the final product. The study features are the use of a mild nonionic detergent, 40–80% saturation with (NH4)2SO4, maintained at low temperature to remove impurity, and the use of chromatografic columns to concentrate the acid phosphatase and remove non-acid phosphatase proteins with lower or higher molecular weights. Acid phosphatase was isolated and purified from garlic seedlings by a streamline method without the use of proteolytic and lipolytic enzymes, butanol, or other organic solvents. Grown garlic seedlings of 10–15 cm height were homogenized with 0.1 M acetate buffer containing 0.1 M NaCl and 0.1% Triton X-100. After homogenization, the supernatant was filtered with paper filters. Filtrated supernatant was cooled to 4°C, followed by a threestep fractionation of the proteins with ammonium sulfate. The crude enzyme was isolated as a green precipitate that was dissolved in a small amount of 0.1 M acetate buffer containing 0.1 M NaCl and 0.1% Triton X-100. Garlic seedling acid phosphatase was purified with ion-exchange chromatography (DEAE cellulose). The column was equilibrated with 0.1 M acetate buffer. Acid phosphatase was purified 40-fold from the starting material. The specific activity of the pure enzyme was 168 U/mg. A variety of stability and activity profiles were determined for the purified garlic seedling acid phosphatase: optimum pH, optimum temperature, pH stability, temperature stability, thermal inactivation, substrate specificity, effect of enzyme concentration, effect of substrate concentration, activation energy, and effect of inhibitor and activator. The molecular mass of acid phosphatase was estimated to be 58 kDa by sodium dodecyl sulfate Polyacrylamide gel electrophoresis. The optimum pH was 5.7 and the optimum temperature was 50°C. The enzyme was stable at pH 4.0–10.0 and 40–60°C. Activation energy was between 10 and 20 kcal, and as Michaelis Menten coefficients, V m values were 100 and 20 mM/s and K m values were 21.27 and 8.33 mM for paranitrophenylphosphate and paranitrophenyl, respectively. Studies of the effect of metal ions on enzyme activity showed both an activating and a deactivating effect. While Cu, Mo, and Mn showed strong inhibitory effects, Na, Ca, and K were the significant activators of acid phosphatase.
TL;DR: Although the derivation of diagnostic criteria and closed form expressions of the cyclic voltammetric responses is privileged, numerical simulation procedures are described that are applicable to the systems under discussion, but also, with little adaptation to any other immobilized redox enzyme systems.
TL;DR: In this article, the authors used ammonium sulphate fractionation and DEAE-cellulose column chromatography to partially purify soluble acid invertases from storage tissue of an early maturing and high sucrose accumulating genotype Co89003.
Abstract: Soluble invertases were partially purified from storage tissue of an early maturing and high sucrose accumulating genotype Co89003 using ammonium sulphate fractionation and DEAE-cellulose column chromatography. The three different acid invertase isoforms named as A-I, A-II and A-III were identified at late grand growth stage of cane development, whereas only one neutral invertase isoform was detected at maturity stage of cane growth. The final purification achieved for A-I, A-II, A-III and neutral invertase isoform was 37, 45, 7 and 17-fold, respectively. The optimum pH values for A-I, A-II, A-III and neutral isoform were observed to be 5.0, 4.5, 4.5 and 7.0, respectively. Apparent Michaelis constant i.e. Km of different acidic isoforms at its optimum pH and temperature varied from 50.0 to 83.3 mM. For neutral invertase isoform, Km value was 44.0mM at its optimum pH and temperature. The highest Vmax/Km value of A-I isoform indicates the maximum kinetic perfection of this isoform and it may be the major vacuolar enzyme involved in sucrose hydrolysis. From the study of effect of temperature on Km and Vmax values of neutral invertase isoform, the enthalpy change, entropy change and energy of activation were observed to be -21.1 kJ mol-1,-40.5 JK-1 mol-1, and 42.5 kJmol-1, respectively. Among the different metal ions tested, manganese chloride strongly inhibited the activity of all soluble acid invertase isoforms and thus, may be utilized to induce early maturity and controlling sucrose inversion in sugarcane thereby increasing sugar recovery.
TL;DR: The EMMA was based on the plug‐plug interaction of enzyme and substrate plugs, which is essential to reduce quantities of materials and addressed the problem of incompatibility of the enzymatic reaction and separation of the reactants.
Abstract: We have investigated electrophoretically mediated microanalysis (EMMA) for the assay of a native glyco-enzyme. As a representative of this class of enzyme, beta-glucosidase was selected, and the reaction was analyzed. Our EMMA was based on the plug-plug interaction of enzyme and substrate plugs, which is essential to reduce quantities of materials. Furthermore, we have addressed the problem of incompatibility of the enzymatic reaction and separation of the reactants. As a result, EMMA of native glycosidase was achieved with a reaction volume of approximately 20 nL and the Michaelis constant was estimated according to the Lineweaver-Burk plot. The current method may have advantages over traditional assay methods, especially in terms of the amount of enzyme (ng order) and substrate (pmol order) required for a reaction.
TL;DR: In this article, a chiral symmetry breaking reaction model was constructed and its symmetry breaking transition resulting in the dominance of one enantiomer and the disappearance of the other was studied based on a chemical oscillation model which includes both an autocatalytic and a Michaelis-Menten type catalytic reaction.
Abstract: Based on a chemical oscillation model which includes both an autocatalytic and a Michaelis–Menten type catalytic reaction, a chiral symmetry breaking reaction model was constructed and its symmetry breaking transition resulting in the dominance of one enantiomer and the disappearance of the other was studied. Symmetry breaking behavior of the system depends not only on the stereoselectivity of the autocatalytic reaction but also on the stereospecificity of the Michaelis–Menten type catalytic reaction, and it was shown that a chiral symmetry breaking transition can take place even though the selectivities of these reactions are not so high. For example, when the Michaelis–Menten type catalytic reaction is perfectly stereospecific, a chirally asymmetric steady state appears even though the enantiomeric excess of the stereoselective autocatalytic reaction is less than 40%. That is, a stereoselective autocatalytic reaction is always needed for any chiral symmetry breaking transition, but its stereoselectivity need not to be high. Until now, many chemical oscillation systems, which always include an autocatalytic process, have been reported already. Accordingly, if the autocatalyst possesses optical isomers and each isomer catalyzes its own production, spontaneous appearance of a chiral symmetry breaking state can be observed in the chemical oscillation system under an appropriate experimental condition, even though the stereoselectivity in the autocatalytic reaction is not so high.
TL;DR: A conclusion could be made that modification of the enzymatic protein by the surfactant monomer should be responsible for the above-mentioned results.
Journal Article•
TL;DR: The oxidation of beta-D-glucose by the enzyme glucose oxidase was studied in aqueous medium, in solutions of surfactants AOT, TX-100 and in w/o microemulsion medium at different water/AOT mole ratio (omega), pH, temperature and in presence of additives.
Abstract: The oxidation of β-D-glucose by the enzyme glucose oxidase was studied in aqueous medium, in solutions of surfactants AOT (2-ethylhexylsulfosuccinate, sodium salt) TX-100 (polyethylene glycol p-tert octyl phenyl ether) and in w/o microemulsion medium (water/AOT/decane) at different water/AOT mole ratio ( ω), pH, temperature and in presence of additives. The time-dependent activities of the enzyme in aqueous and microemulsion media were determined. The catalytic process was retarded in the presence of TX-100 and AOT. In microemulsion medium, kcat values exhibited a deformed Wshaped profile with ω. At pH 7, a maximum value of kcat was observed at ω = 10.6. The kcat values were found to be higher in microemulsion medium than in aqueous medium at both pH’s 7 and 8. Activation parameters for the kinetic process were evaluated together with the thermodynamics of the enzyme-substrate Michaelis complex. The ΔG* was lower, whereas ΔH* and ΔS* were higher in microemulsion than in water. The Michaelis constant, KM was also lower in microemulsion. The inhibition effects of the additives, NaNO 3 and NaC were studied in both aqueous and microemulsion media by examining their influences on catalytic constant, kcat and Michaelis constant KM. In microemulsion, both the additives NaNO 3 and NaC produced non-competitive inhibition.
TL;DR: A two-parameter mathematical model was developed to calculate the effectiveness factor for immobilized enzymes in porous spherical particles, including simple Michaelis–Menten and product competitive inhibition kinetics.
TL;DR: In vitro experiments illustrated that the enzymatic activity of the derivatives of α-Chymotrypsin was adversely affected and the hydrolysis of selected food proteins becomes slower and the affinity of the enzyme to these substrates declined.
Abstract: α-Chymotrypsin was allowed to react with selected phenolic and related compounds (chlorogenic acid, m-, o-, p-dihydroxybenzene, p-benzoquinone). The derivatized enzymes obtained were characterized in terms of their activity. In vitro experiments illustrated that the enzymatic activity of the derivatives was adversely affected. The kinetics of the enzymatic reactions showed that the hydrolysis of selected food proteins becomes slower and the affinity of the enzyme to these substrates declined as measured by Michaelis-Menten constant and maximum velocity of the enzymatic reaction. This enzyme inhibition depended on the reactivity of the phenolic and related substances tested as well as on the degree of the derivatization. Further, influence of the enzyme-substrate ratio was also demonstrated. The effects of the derivatization are more pronounced with increasing concentration of the substrates.
TL;DR: The kinetics of enzymatic oxidative polymerization of 4-chloroguaiacol by laccase will be useful to understand those of the enzymatics oxidation of other phenolic compounds.
Abstract: The kinetics of enzymatic oxidative polymerization of 4-chloroguaiacol by laccase was studied. 4-Chloroguaiacol, a model compound of the chloroguaiacols formed in the bleaching process in the pulp industry, was oxidized by laccase from Trametes sp. Ha1 to eliminate chloroguaiacols from pulp wastewater by polymerization and precipitation. On the basis of the reaction rate obtained from the decrease in the 4-chloroguaiacol concentration, the optimum pH of the reaction was 4–5 and the apparent Michaelis constant Km for the reaction was more than 5 mmol dm–3. However, the value of Km obtained from the dependence of oxygen consumption rate on the concentration of 4-chloroguaiacol was 0.092 mmol dm–3. The reaction rate was expressed as the sum of the enzymatic oxidation rate of 4-chloroguaiacol and the polymerization rate of the radical formed in the enzymatic oxidation. The kinetics of the enzymatic oxidative polymerization shown in this study will be useful to understand those of the enzymatic oxidation of other phenolic compounds.
TL;DR: Electrical parameters of hydrolysis of acetylcholine and its 13 derivatives with different structures of ammonium group by cholinesterase, and N-acetoxyethylene-N-ethylpiperidinium iodide under action of horse blood serum ChE are determined.
Abstract: Taking into account the phenomenon of non-productive binding of substrate, kinetic parameters of hydrolysis of acetylcholine (ACh) and its 13 derivatives with different structures of ammonium group by cholinesterase (ChE) of human erythrocytes, ChE of horse blood serum, and ChE of optic ganglia of the Pacific squid Todarodes pacificus are determined. A dependence is revealed of values of parameters of their enzymatic hydrolysis and parameters of the non-productive binding on the substrate structure and ChE nature. Effects of salts, LiCl, NaCl, KCl, MgCl2, CaCl2 and BaCl2, on various kinetic parameters, including parameters of the non-productive binding of substrate, of enzymatic hydrolysis of iodides of ACh and N-acetoxyethylene-N-ethylpiperidinium under action of horse blood serum ChE are studied. Addition of the salts to the reaction mixture produced different effects on values of the catalytic center activity (ac) and the Michaelis constant (KM), depending on the cation nature and the substrate structure. At the same time, values of the ac/KM ratio that characterize to a degree the substrate affinity to the enzyme are equal to each other for two substrates differing in structure, regardless of the presence and nature of the studied cations. Parameters of the non-productive binding of N-acetoxyethylene-N-ethylpiperidinium iodide also depended on the salt nature; however, in that case, a question arises as to the correctness of the comparative analysis, when at determinations of the parameters the non-productive binding of ACh is ignored.
30 Jan 2003
TL;DR: To understand enzyme activity in the presence of PEG 400 or MPEG 550 molecules, a kinetic model was proposed and explained enzymatic reactions more exactly than that by the previously published model.
Abstract: The activity of Chromobacterium viscosum lipase (glycerol-ester hydrolase, EC 3.1.1.3) entrapped in AOT/isooctane reverse micelles was significantly increased by the addition of short chain polyethylene glycols (PEGs) or methoxypolyethylene glycols (MPEGs) for the hydrolysis of olive oil. To understand enzyme activity in the presence of PEG 400 or MPEG 550 molecules, a kinetic model was proposed. The validity of this model was verified by experimental data on the lipase-catalyzed hydrolysis of olive oil in AOT/isooctane reverse micellar systems, in which PEG 400 or MPEG 550 had been added. The large value of the equilibrium constant (kD) for enzyme activation indicated that the affinity between C viscosum lipase and PEG 400 or MPEG 550 molecules was very strong. The Michaelis constant (Km) predicted by the proposed model explained enzymatic reactions more exactly than that by the previously published model. Copyright © 2003 Society of Chemical Industry
01 Jan 2003
TL;DR: It is deduced that the reactor should be operated using low flow rates whereas for the latter high flow rates are optimal, and it is also shown that periodic behaviour is impossible.
Abstract: Enzyme immobilization is becoming a popular topic in recent years in food and biotechnology industry. Here an immobilised enzyme reactor (IER) is treated theoretically as a well-stirred flow reactor, with the restriction that bounded and unbounded (or freely suspended) enzyme species are constrained to remain within the reactor. Two cases in which the immobilized enzyme reactor is used to either reduce unwanted products or to synthesize a product have been considered. It has been deduced that for the former the reactor should be operated at low flow rates whereas for the latter high flow rates are desirable.
Journal Article•
TL;DR: The enzyme activity was stable under 45 degrees C and in the pH range of 3.0 - 9.5, and was enhanced by Cu2+, and was strongly inhibited by Fe2+.
Abstract: Laccase produced by Basidiomycete was purified to electrophoretic homogeneity by the steps of ammonium sulfate precipitation, DEAE-cellulose and hydrophobic interaction column chromatography. Purification of about 318.4 fold was achieved with an overall yield of 18.6%. Its molecular weight was estimated to be about 60.3 kD by SDS-PAGE, and that of it was 55.94 kD by mass spectrum. The optimum temperature and pH of the enzyme activity were 65 degrees C and 2.2 - 2.8 respectively. The isoelectric point was 4.02 (room temperature). Its N-terminal sequence was AIGPVTDL. The carbohydrate content was 49.2% by the phenol-sulfuric acid method. Michaelis constant of the enzyme for ABTS was 17.5 micromol/L. The enzyme activity was stable under 45 degrees C and in the pH range of 3.0 - 9.5. The activity was enhanced by Cu2+, and was strongly inhibited by Fe2+. While Mn2+ and Ag+ had no effect on laccase activity. Dithiothreitol and sodium azide inhibited completely the activity. Trp was possible essential residue for enzyme activity.