Initial Enzyme Concentration Research Articles

The Hydrophobic Hinge Region of Rat DNA Polymerase β Is Critical for Substrate Binding Pocket Geometry

The hydrophobic hinge of DNA polymerase beta facilitates closing and stabilization of the enzyme once the nucleotide substrate has bound. Alteration of the hydrophobic nature of the hinge by the introduction of a hydrophilic glutamine residue in place of isoleucine 260 results in an inaccurate polymerase. The kinetic basis of infidelity is lack of discrimination during the binding of substrate. The I260Q polymerase beta variant has lower affinity than wild type enzyme for the correct substrate and much higher affinity for the incorrect substrate. Our results demonstrate that the hinge is important for formation of the substrate binding pocket. Our results are also consistent with the interpretation that DNA polymerase beta discriminates the correct from incorrect substrate during the binding step.

Mechanistic Studies on a Novel, Highly Potent Gold-Phosphole Inhibitor of Human Glutathione Reductase

The homodimeric flavoprotein glutathione reductase (GR) is a central player of cellular redox metabolism, connecting NADPH to the large pool of redox-active thiols. In this work, the inhibition of human GR by a novel gold-phosphole inhibitor (GoPI) has been studied in vitro. Two modes of inhibition are observed, reversible inhibition that is competitive with GSSG followed by irreversible inhibition. When approximately 1 nm GoPI is incubated with NADPH-reduced GR (1.4 nm) the enzyme becomes 50% inhibited. This appears to be the most potent stable inhibitor of human GR to date. Analyzing the monophasic oxidative half-reaction of reduced GR with GSSG at pH 6.9 revealed a K(d)((app)) for GSSG of 63 microm, and a k((obs)max) of 106 s(-1) at 4 degrees C. The reversible inhibition by the gold-phosphole complex [{1-phenyl-2,5-di(2-pyridyl)phosphole}AuCl] involves formation of a complex at the GSSG-binding site of GR (K(d) = 0.46 microm) followed by nucleophilic attack of an active site cysteine residue that leads to covalent modification and complete inactivation of the enzyme. Data from titration spectra, molecular modeling, stopped-flow, and steady-state kinetics support this theory. In addition, covalent binding of the inhibitor to human GR was demonstrated by mass spectrometry. The extraordinary properties of the compound and its derivatives might be exploited for cell biological studies or medical applications, e.g. as an anti-tumor or antiparasitic drug. Preliminary experiments with glioblastoma cells cultured in vitro indicate an anti-proliferative effect of the inhibitor in the lower micromolar range.

Determination of the Depolymerisation Kinetics of Amylose, Amylopectin and Maltodextrin by Aspergillus niger Glucoamylase Using a 2-p-toluidinylnaphthalene-6-sulfonate/Flow-injection Analysis System with Fluorimetric Detection

This work describes the determination of depolymerisation kinetics of amylose, amylopectin and maltodextrin by Aspergillus niger glucoamylase using a flow-injection analysis system with fluorimetric detection and 2- p-toluidinylnaphthalene-2-sulfonate as the fluorescent probe. Experimental data corresponding to the time evolution of the concentration of detectable substrate were fitted to a single exponential decay curve in the case of amylose (linear substrate) and to a double exponential decay curve in the case of amylopectin and maltodextrin (ramified substrates). For all the substrates assayed, the depolymerisation rates at time zero correlated well with the initial substrate and enzyme concentrations through the Michaelis-Menten hyperbola. Therefore, this methodology allowed the determination of glucoamylase activity using any of these substrates. The determined value of the enzymic constant K m was lower for amylose than for amylopectin and maltodextrin, thus reflecting the higher difficulty of glucoamylase to hydrolyse the (1,6) when compared to the (1,4) linkages. In contrast, the values obtained for the rate constant k3 were very similar for all the substrates assayed.

Méthodes de qualification des immobilisations chimiques d'une enzyme sur un support solide

Carbonic anhydrase was immobilized on a polymer thermoplastic to promote gaseous CO2 hydration into bicarbonate ions. Catalyst immobilization was realized through a series of chemical reactions enabling enzyme covalent binding to polyamide support. Different initial enzyme concentrations of 0.25, 0.50, 0.75, 1, and 2 mg/ml were verified. Different techniques were developed to assess enzyme attachment. The amount of bound protein was determined using Bradford dosage of proteins remaining in solution following CA II incubation with solid support. ELISA has given a qualitative evaluation of the protein, enabling a follow up of enzyme binding robustness as a function of time. p-nitrophenyl acetate hydrolysis and CO2 hydration were assessed by spectrophotometry and electrometry, respectively. The percentage of active enzyme following immobilization was measured using an esterase linear model. Catalytic transformation rates for gaseous CO2 hydration were calculated for each type of immobilization.

Hydrolysis of New Phthalimide-Derived Esters Catalyzed by Immobilized Lipase

The last step of the production of four phthalimide-derived acids, designed to act as antiasthma drugs, was performed by enzymatic hydrolysis of the respective methyl or ethyl esters. The esters 4-ethyl-[2-(1,3-dioxo-1,3-dihydro- 2-isoindoylyl)]-phenoxyacetic methyl ester (PHT-MET), 4-ethyl-[2- (1,3-dioxo-1,3-dihydro-2-isoindoylyl)]-phenoxyacetic ethyl ester, 4-(1, 3-dioxo-1,3-dihydro-2-isoindoylyl)-phenoxyacetic ethyl ester, and 2-(1,3-dioxo-1, 3-dihydro-2-isoindoylyl)-phenoxyacetic ethyl ester were hydrolyzed by immobilized lipase. The enzymatic reaction could be used only to produce the desired 4-substituted compounds. The best result that was found to hydrolysis of PHT-MET, and, therefore, that ester was selected for optimization experiments in a three-phase system. Reactions were performed with solid biocatalyst (Lipozyme RM IM), organic solvent phase (ethyl acetate), and aqueous phase (saturated Na2CO3 solution). To optimize the reaction conditions, an experimental design optimization procedure was used. The variables studied were the amount of enzyme, the temperature, and the volume of the aqueous solution. Time course experiments were then performed for different initial enzyme concentrations (0.5, 0.9, and 1.4 UH/mL of solvent). The optimized reaction conditions found were 20 mg of Lipozyme (0.9 UH/mL solvent) and 5.0 mL of Na2CO3(sat) at 40 degrees C for 6 h.

Enzymatic hydrolysis by immobilized lipase applied to a new prototype anti-asthma drug

The last step of the production of 4-ethyl-(2-(1,3-dioxo-1,3-dihydro-2-isoindoylyl))-phenoxy acetic acid (LASSBio 482), designed to act as an anti-asthma drug, was performed by enzymatic hydrolysis of the methyl ester. Reactions were performed in a three-phase system with a solid biocatalyst (Lipozyme ® RM IM), an organic solvent phase (ethyl acetate) and an aqueous phase (saturated Na 2CO 3 solution). In order to optimize the reaction conditions, experimental design techniques were used. The variables studied were the amount of enzyme, the reaction temperature and the total volume of saturated Na 2CO 3 solution. The experimental results were analysed statistically and the main effects of the variables were computed and analysed. Dynamic experiments were then performed under optimised reaction conditions for different initial enzyme concentrations (0.5, 0.9 and 1.4 U H Lipozyme/mL solvent). An empirical nonlinear model was then proposed to describe the process, and experimental data were used to estimate the model parameters. The optimised reaction conditions were 20 mg Lipozyme ® (0.9 U H/mL solvent), 5.0 mL Na 2CO 3 (sat), a temperature of 40 °C and batch reaction time of 6 h. The results showed that enzymatic hydrolysis is a promising technique for the synthesis of the desired product.

Kinetic study of the enzymatic cycling reaction conducted with 3α-hydroxysteroid dehydrogenase in the presence of excessive thio-NAD + and NADH

We have established a simple kinetic model applicable to the enzyme cycling reaction for the determination of 3α-hydroxysteroids. This reaction was conducted under the reversible catalytic function of a single 3α-hydroxysteroid dehydrogenase (3α-HSD) with nucleotide cofactors, thio-NAD + (one of the NAD + analogues) for the oxidation of 3α-hydroxysteroids and NADH for the reduction of 3-oxosteroids. This model was constructed based on the reaction mechanism of 3α-HSD, following an ordered bi–bi mechanism with cofactor binding first, under the assumption that the respective enzyme-cofactor complexes were distributed according to the initial ratio of thio-NAD + to NADH by the rapid equilibrium of both enzyme-cofactor complexes. The cycling rate in the new kinetic model could be expressed with the dissociation constants of enzyme-cofactor complexes and the initial concentrations of cofactors and enzyme. The cycling rate was verified by a comparison with the experimental data using 3α-HSD from Pseudomonas sp. B-0831. The results showed that the experimental data corresponded well with the results obtained from the kinetic model.

Immobilization of enzymes in mesoporous materials: controlling the entrance to nanospace

The immobilization behavior of lysozyme (LYZ) in mesoporous silicas (MPSs) with controlled morphologies has been investigated. The effects of entrance amount (EA) on the quantity and rate of immobilization of enzymes have been investigated due to the successful morphological control of MPSs. EA increases with the decrease of particle size of MPSs, leading to a significant improvement of immobilization ability. Rod-like SBA-15 (∼1–2 μm in length) shows a faster adsorption rate and larger immobilization amount than that for conventional SBA-15 (∼20 μm in length). It is observed that the adsorption rate of enzyme within MPSs is independent of the initial enzyme concentration. Increasing temperature favors a fast immobilization process of LYZ into MPSs. A maximum equilibrium adsorption amount of LYZ into rod-like SBA-15 is ∼500 mg/g at all temperatures under study (4–55 °C). However, for conventional SBA-15, this maximum equilibrium adsorption amount cannot be observed within 48 h even at high temperature. A strong interaction between LYZ and MPSs is observed, resulting in very small amount of LYZ released by MPSs. Such conclusions may be important to understand the mechanism of protein immobilization within MPSs and potentially useful for applications of MPSs in biocatalysts or biosensors.

The condition for pseudo-first-order kinetics in enzymatic reactions is independent of the initial enzyme concentration

The condition for pseudo-first-order kinetics in enzymatic reactions is independent of the initial enzyme concentration

The condition for pseudo-first-order kinetics in enzymatic reactions is independent of the initial enzyme concentration

The linearization of the Michaelis–Menten reaction by pseudo-first-order kinetics is revised. 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.

Immobilization and catalytic properties of lipase on chitosan for hydrolysis and esterification reactions

The objective of this study was to evaluate the immobilization of lipase on a chitosan support by physical adsorption, aiming at its application in hydrolytic and synthetic reactions. Two types of chitosan (flakes and porous) were used for immobilizing lipase from a microbial source (Candida rugosa) and animal cells (porcine pancreas). The best results for recovery of total activity after immobilization were obtained for microbial lipase and porous chitosan beads. This set was selected for further immobilization studies, including full characterization of the immobilized derivative in aqueous and organic media. In aqueous medium, the operational and thermal stabilities of this preparation were quantified. In organic medium, the direct synthesis of n-butyl butyrate in organic solvent was chosen as a model reaction. The influence of several parameters, such as temperature, initial butyric acid concentration and amount of enzyme in the reaction system, was analyzed. Production of n-butyl butyrate was optimized and an ester yield response equation was obtained, making it possible to predict ester yields from known values of the three main factors. Use of this immobilized preparation was extended to the direct esterification of a large range of carboxylic acids (from C2 to C12) with a variety of alcohols (from C2 to C10).

Modelling of potato starch saccharification by an Aspergillus niger glucoamylase

The kinetics of hydrolysis of a soluble potato starch catalysed by free glucoamylase was investigated in a batch reactor. Seventeen progress curves of product concentration, obtained at varying initial substrate and enzyme concentrations, were fitted simultaneously using six different models of saccharification process. A good description of the whole course of the progress curves was obtained using a six-parameter model based on a kinetic equation, which contained product and substrate inhibition terms. An essential feature of the model was that starch was structured into susceptible and resistant fractions that differed in the rate constants of hydrolysis. The model parameters were estimated with a good accuracy and were in a good agreement with the available literature information.

A Century of Enzyme Kinetics: Reliability of the K M and v max Estimates

The application of the quasi-steady-state approximation (QSSA) in biochemical kinetics allows the reduction of a complex biochemical system with an initial fast transient into a simpler system. The simplified system yields insights into the behavior of the biochemical reaction, and analytical approximations can be obtained to determine its kinetic parameters. However, this process can lead to inaccuracies due to the inappropriate application of the QSSA. Here we present a number of approximate solutions and determine in which regions of the initial enzyme and substrate concentration parameter space they are valid. In particular, this illustrates that experimentalists must be careful to use the correct approximation appropriate to the initial conditions within the parameter space.

Modelling of the kinetics of thermal inactivation of glucoamylase from Aspergillus niger

Thermal inactivation of glucoamylase (GA) from Aspergillus niger was investigated by incubating the enzyme in buffer solutions in a broad temperature range. Preliminary experiments, made at different initial enzyme concentrations, showed the absence of association/aggregation reaction in the inactivation pathway. A biphasic character of inactivation was identified owing to the frequent sampling, conduction of experiments until the almost complete activity loss, and good accuracy of activity measurement. A further kinetic analysis showed that the biphasic inactivation could not be explained be the behaviour of different isozyme forms. The method of multitemperature evaluation was then applied to fit simultaneously all data with a model based on the Lumry–Eyring mechanism that incorporated the influence of temperature on the rate constants through the Arrhenius equation. A very close fit of activity values and good accuracies of the model parameters, the rate constants at the reference temperature of 60°C and activation energies, validated the use of the model for the description of thermal inactivation of glucoamylase inactivation.

Empirical evaluation of cellulase on enzymatic hydrolysis of waste office paper

Enzymatic hydrolysis of waste office paper was evaluated using three commercial cellulases, Acremonium cellulase, Meicelase, and Cellulosin T2. Varying the enzyme loading from 1 to 10% (w/w) conversion of waste office paper to reducing sugar was investigated. The conversion increased with the increase in the enzyme loading: in the case of enzyme loading of 10% (w/w), Acremonium cellulase yielded 79% conversion of waste office paper, which was 17% higher compared to Meicelase, 13% higher than that of Cellulosin T2. Empirical model for the conversion (%) of waste office paper to reducing sugar (x) was derived from experimental results as follow,x=kE m t (aE+b) wherek, m, a, and d denote empirical constants.E indicates initial enzyme concentration.

Inhibition of enzymatic reactions. A rapid method to determine the index pI50.

The activity of every substance I inhibiting an enzymatic reaction can be approximately evaluated by the index PI50. This paper describes a simple and fast method of estimate and/ or determination of this index. The method is based on the linearity of the dependence of the ratio of reaction rates of uninhibited and inhibited reaction vs. concentration of the inhibitor at constant initial substrate and enzyme concentrations for fully competitive, noncompetitive, uncompetitive and mixed type of inhibition by the one inhibitor. The validity of the method is demonstrated by four inhibitors of hydrolysis of acetylthiocholine by butyrylcholine esterase.

Preparation of reversibly immobilized lysozyme onto Procion Green H-E4BD-attached poly(hydroxyethylmethacrylate) film for hydrolysis of bacterial cells

Abstract The flat sheet support for enzyme immobilization was prepared by UV-initiated photopolymerization of 2-hydroxyethyl-methacrylate (HEMA) in the presence of an initiator (αα′-azoisobutyronitrile; AIBN). An affinity dye, Procion Green H-E4BD, was attached covalently under alkaline conditions and the pHEMA-Procion Green H-E4BD-attached film was used for the immobilization of lysozyme via adsorption. The amount of attached dye on the pHEMA film was 160 μmol m 2 and the water content of the dye-attached pHEMA film was 69%. The lysozyme adsorption capacity of the dye-attached pHEMA film was determined under conditions of different pH and with different initial concentrations of enzyme in the medium. The maximum lysozyme adsorption capacity of the dye-attached pHEMA film, under the specified experimental conditions was 3.92 g m −2 . Non-specific adsorption of the lyzozyme on the pHEMA film was negligible. Optimum reaction pH was 6.0 for the free and 7.0 for adsorbed enzyme. The free enzyme had an optimum temperature of 35°C, whereas it shifted to 40°C for the immobilized enzyme system. The enzyme could be repeatedly adsorbed and desorbed from the dye-attached pHEMA film without any significant loss in adsorption capacity.

Kinetics of enzyme action of Cartazyme NS-10 prior to bleaching of kraft pulp

The kinetics of enzyme treatment of hardwood kraft pulp with commercial xylanase Cartazyme NS-10 was investigated. The enzyme treatment process was found to follow closely the topochemical modified equation of Prout–Tompkins. The influence of the initial enzyme concentration was studied and the applicability of the power kinetic equation was established for the initial rate of the process. An equation of practical use was obtained, which provides the temperature function of the amount of the reducing substances, depending on the enzyme initial concentration. This function made it possible to control the process of prior enzyme treatment of unbleached kraft pulp.

Lysozyme inactivation and aggregation in stirred-reactor

Mechanisms of enzyme inactivation and aggregation are still poorly understood. In this work, we are considering the characterisation of both inactivation and aggregation in stirred tank reactor, with lysozyme as the model enzyme.The inactivation kinetics are first order. For stirring speeds in the range of 0–700 rpm, the kinetic constant is found to be proportional to the power brought by the impeller. It suggests that inactivation depends on collisions between enzyme molecules. Efficient collisions between native and inactive molecules induce native molecules to turn into inactive molecules and lead to lysozyme aggregation.During inactivation, enzymes are found to aggregate as shown by light scattering measurements. The structure of aggregates was studied on samples treated for chemical denaturation and reduction. The aggregates are supramolecular edifices, mainly made up of inactivated enzymes linked by weak forces. But aggregates are also made up of dimers and trimers of lysozyme, linked by disulfide bridges. Dimers and trimers are 18% and 5%, respectively, of the total amount of lysozyme aggregates.Whatever the stage of aggregate formation and the initial enzyme concentration are, these aggregates are irreversibly inactivated. Enzyme activity is definitely lost even if stirring is stopped and/or temperature decreased.This study points out the importance of hydrodynamics in bioreactors and highlights the nature of the aggregates resulting from the interactions between native and inactive enzymes.

The use of commercial pectinase in fruit juice industry. Part 3: Immobilized pectinase for mash treatment

Enzymatic mash treatment is a well-known modern process for gaining more juice from fruits and vegetables. According to the technique, cell wall and middle-lamina pectin of the fruit are degraded by pectinase activities. Besides increasing press capacity and the yield of juice up to 20%, it has also a positive effect to achieve high carotene and dry matter content of the product. The aim of the research was to investigate the activity and reusability of immobilized commercial pectinase named as “Pectinex Ultra SP-L” on carrot puree. Immobilization process was carried out by using ion exchange resin particles washed with 0.05 M phosphate buffer at pH 4.5. Pectinase activity of immobilized enzyme was determined by the measurement of viscosity reduction of pectin solution model system at pH 4.5 and 35°C and found to be 1.252-pectin (w/v, %)/s ml. Activity loss was only 20% after nine batches run model system studies. The optimum initial enzyme concentration was detected by measuring the highest pectinase activity in pectin solution and found to be 6% (v/v). Enzyme immobilized particles were added to the carrot puree with an amount of 1.5 g particle/100 g puree at pH 4.5 and 35°C to degrade soluble and insoluble pectin and haze-provoking polysaccharides. The activity of immobilized enzyme was determined by measuring pH, dry matter content and viscosity of the puree. Immobilized enzyme preparation reduced the viscosity of the carrot puree from 90 to 6.5 Poise, after 60 min of incubation. While the viscosity and pH of the puree were decreased, dry matter content and total yield were found to be increased because of the polysaccharide degradation. An average yield increment was 30.23% with respect to the yield obtained from non-enzymic processed carrot juice. Immobilized enzyme was used 5 times in carrot puree medium at the above described conditions and the activity loss was found to be only 6.5%. Activity of the immobilized enzyme was quite stable.