Product Inhibition Constant Research Articles

Binding interactions in a kinase active site modulate background ATP hydrolysis

Kinases play central roles in many cellular processes, transferring the terminal phosphate groups of nucleoside triphosphates (NTPs) onto substrates. In the absence of substrates, kinases can also hydrolyse NTPs producing NDPs and inorganic phosphate. Hydrolysis is usually much less efficient than the native phosphoryl transfer reaction. This may be related to the fact that NTP hydrolysis is metabolically unfavorable as it unproductively consumes the cell's energy stores. It has been suggested that substrate interactions could drive changes in NTP binding pocket, activating catalysis only when substrates are present. Structural data show substrate-induced conformational rearrangements, however there is a lack of corresponding functional information. To better understand this phenomenon, we developed a suite of isothermal titration calorimetry (ITC) kinetics methods to characterize ATP hydrolysis by the antibiotic resistance enzyme aminoglycoside-3′-phosphotransferase-IIIa (APH(3′)-IIIa). We measured Km, kcat, and product inhibition constants and single-turnover kinetics in the presence and absence of non-substrate aminoglycosides (nsAmgs) that are structurally similar to the native substrates. We found that the presence of an nsAmg increased the chemical step of cleaving the ATP γ-phosphate by at least 10- to 20-fold under single-turnover conditions, supporting the existence of interactions that link substrate binding to substantially enhanced catalytic rates. Our detailed kinetic data on the association and dissociation rates of nsAmgs and ADP shed light on the biophysical processes underlying the enzyme's Theorell-Chance reaction mechanism. Furthermore, they provide clues on how to design small-molecule effectors that could trigger efficient ATP hydrolysis and generate selective pressure against bacteria harboring the APH(3′)-IIIa.

Diffusion, adsorption and reaction of glucose in glucose oxidase enzyme immobilized mesoporous silica (SBA-15) particles: Experiments and modeling

Glucose oxidase (GOD) enzyme is widely used as a sensing molecule for glucose and is thus immobilized in different porous hosts for sensing applications. We have synthesized different morphologies of mesoporous silica (SBA-15) host particles and impregnated GOD in it. Rod and cuboid shaped SBA-15 particles having internal pore diameters of 6.8 and 11.4nm respectively, were synthesized, in order to measure enzyme activity. The latter primarily determines the performance of a glucose sensor. In this quest, we therefore developed a mathematical model to predict variation of glucose concentration with time, by considering diffusion, adsorption and reaction rates of glucose by immobilized GOD. The model was validated with our own experiments, by fitting diffusion coefficient of glucose inside mesopores and the competitive product inhibition constant of GOD. The validated model indicates that, specific activity of GOD immobilized in SBA-15 is lower at higher GOD loading. Thus, this provides insight into the effect of pore-confinement on diffusion and reaction kinetics of the substrate (glucose) by an immobilized enzyme (GOD). Understanding the interplay of chemical transport and reaction kinetics issues in a porous solid nanoparticle host (SBA-15) can lead to superior detection and measurement of glucose concentration.

A New Nitrilase-Producing Strain Named Rhodobacter sphaeroides LHS-305: Biocatalytic Characterization and Substrate Specificity

The characteristics of the new nitrilase-producing strain Rhodobacter sphaeroides LHS-305 were investigated. By investigating several parameters influencing nitrilase production, the specific cell activity was ultimately increased from 24.5 to 75.0 μmol g(-1) min(-1), and hereinto, the choice of inducer proved the most important factor. The aromatic nitriles (such as 3-cyanopyridine and benzonitrile) were found to be the most favorable substrates of the nitrilase by analyzing the substrate spectrum. It was speculated that the unsaturated carbon atom attached to the cyano group was crucial for this type of nitrilase. The value of apparent K (m), substrate inhibition constant, and product inhibition constant of the nitrilase against 3-cyanopyridine were 4.5 × 10(-2), 29.2, and 8.6 × 10(-3) mol L(-1), respectively. When applied in nicotinic acid preparation, the nitrilase is able to hydrolyze 200 mmol L(-1) 3-cyanopyridine with 93% conversion rate in 13 h by 6.1 g L(-1) cells (dry cell weight).

A comparative study of activity and apparent inhibition of fungal β‐glucosidases

β-Glucosidases (BGs) from Aspergillus fumigates, Aspergillus niger, Aspergillus oryzae, Chaetomium globosum, Emericella nidulans, Magnaporthe grisea, Neurospora crassa, and Penicillium brasilianum were purified to homogeneity, and analyzed by isothermal titration calorimetry with respect to their hydrolytic activity and its sensitivity to glucose (product) using cellobiose as substrate. Global non-linear regression of several reactions, with or without added glucose, to a product inhibition equation enabled the concurrent derivation of the kinetic parameters k(cat), K(m), and the apparent product inhibition constant (app)K(i) for each of the enzymes. A more simple fit is not advisable to use as the determined (app)K(i) are in the same range as their K(m) for some of the tested BGs and produced glucose would in these cases interfere. The highest value for k(cat) was determined for A. fumigatus (768 s(-1)) and the lowest was a factor 9 less. K(m) varied by a factor of 3 with the lowest value determined for C. globosum (0.95 mM). The measured (app)K(i) varied a factor of 15; the hydrolytic activity of N. crassa being the most resistant to glucose with an apparent product inhibition constant of 10.1 mM. Determination of (app)K(i) using cellobiose as substrate is important as it reflects to what extent the different BGs are hydrolytically active under industrial conditions where natural substrates are hydrolyzed and the final glucose concentrations are high.

Precise, Facile Initial Rate Measurements

Progress curve analysis has been used sparingly in studies of enzyme-catalyzed reactions due largely to the complexity of the integrated rate expressions used in data analysis. Using an experimental design that simplifies the analysis, the advantages and limitations of progress curve experiments are explored in a study of four different enzyme-catalyzed reactions. The approach involves relatively simple protocols, requires 20-25% of the materials, and provides 10- to 20-fold signal enhancements compared to analogous initial rate studies. Product inhibition, which complicates integrated rate analysis, was circumvented using cloned, purified enzymes that remove the products and draw the reaction forward. The resulting progress curves can be transformed into the equivalent of thousands of initial rate and [S] measurements and, due to the absence of product inhibition, are plotted in the familiar, linear double-reciprocal format. Allowing product to accumulate during a reaction produces a continuously changing substrate/product ratio that can be used as the basis for obtaining product inhibition constants and to distinguish among the three classical inhibition mechanisms. Algebraic models describing the double-reciprocal patterns obtained from such inhibition studies are presented. The virtual continuum of substrate concentrations that occurs during a progress curve experiment provides a nearly errorless set of relative concentrations that results in remarkably precise data; kinetic constant standard deviations are on the order of 0.5%.

The Effects of Activated Protein C, Heparin, and Antithrombin On Thresholding Behaviour in Prothrombin Activation by Prothrombinase.

Abstract 2138Poster Board II-115The coagulation cascade results in considerable thrombin generation through a series of zymogen activations to enzymes initiated by factor VIIa-tissue factor complex. Thrombin generation consists of an initiation phase, where a small amount of thrombin (∼25 nM) sufficient to form a clot is made, and a propagation phase, which is characterized by a rapid increase (to ∼850 nM) and subsequent decay in the thrombin concentration. Thrombin generation is possibly described as under threshold limited control. Thresholding is observed in systems where a precursor in the presence of a stimulus gives rise to a response, which can both positively feedback and be inhibited. The threshold level of stimulus depends on the kinetics of feedback and inhibition. The coagulation cascade is intensely regulated by several mechanisms and inhibitors. In this study, prothrombin (the precursor) activation initiated by factor Xa (the stimulus) in the presence of factor V (the positive feedback loop) and antithrombin (the inhibitor) was investigated for threshold type behaviour. Reactions were started by adding factor Xa to the purified components prothrombin (140 nM), factor V (2 nM), phosphatidylcholine and phosphatidylserine vesicles (2 μM), CaCl2 (5 mM), antithrombin (280 nM), and Z-Gly-Gly-Arg-7-amido-4-methylcoumarin (Z-GGR-AMC, 400 μM), a fluorogenic thrombin substrate. Thrombin generation was observed as an increase in fluorescence over time due to Z-GGR-AMC hydrolysis. When thrombin generation ceased, the fluorescence plateaued. Under these conditions, the plateau in fluorescence was not a result of complete Z-GGR-AMC hydrolysis, but due to cessation of prothrombin activation. TableCurve 2D (Systat Software Inc., San Jose CA) was used to smooth the raw fluorescence data by drawing a best fit line, which then was used for further data manipulation. Three point running slopes over each time course then were calculated to produce rates of Z-GGR-AMC hydrolysis. The kinetic parameters, kcat (2.77 s-1), Km (92.2 μM), and Ki (product inhibition constant, 233 μM), for thrombin mediated Z-GGR-AMC hydrolysis were used to convert each slope to a free thrombin concentration. In each experimental condition tested, there was no detectable thrombin generated at low factor Xa concentrations. At higher factor Xa concentrations, thrombin generation was transient: the thrombin concentration rapidly rose to a peak followed by its decay. Thrombin potential, defined as the area under the thrombin versus time curve, was calculated by approximating the thrombin peak as a series of trapezoids. The dose-response curve (thrombin potential versus factor Xa concentration) resembled a sigmoid: at low factor Xa concentrations, the thrombin potential was almost zero; as the factor Xa concentration increased, the thrombin potential increased rapidly to a plateau. Under the above conditions, no thrombin generation was detected from 0 pM to approximately 0.5 pM factor Xa. The thrombin potential increased from approximately 0.5 pM to 3 pM factor Xa and past 3 pM thrombin potential was relatively constant. This type of dose-response curve is expected if thresholding exists. Adding activated protein C (aPC) at concentrations ranging from 0 nM to 5 nM increased the threshold concentration of factor Xa from approximately 0.5 pM to 10 pM. In addition, increasing the antithrombin concentration from 280 nM to 560 nM increased the threshold concentration of factor Xa from approximately 0.5 pM to 3 pM. Lastly, adding heparin at concentrations ranging from 0 μg/mL to 2 μg/mL increased the threshold concentration of factor Xa from approximately 0.5 pM to 50 pM. Because the dose-response curve displays a marked increase in thrombin potential over a small range of factor Xa concentrations and this pattern shifts to higher factor Xa concentrations with added aPC, heparin or increased antithrombin, prothrombin activation by prothrombinase is likely a threshold limited occurrence with respect to the initiating factor Xa concentration and the threshold level changes with the kinetics of inhibition. Disclosures:No relevant conflicts of interest to declare.

Immobilization of l-lactate dehydrogenase on magnetic nanoclusters for chiral synthesis of pharmaceutical compounds

In the present work, we study the covalent immobilization of l-lactate dehydrogenase (LDH) on silica-coated magnetic nanoclusters (SiMag). Immobilization of LDH on magnetic nanoclusters provides several advantages. First, it can ease the enzyme recovery through magnetic separation, rendering the immobilized LDH applicable for batch reactor which configuration can minimize the product inhibition. Second, the immobilization resulted in the increase of product inhibition constants of LDH which can further minimize the product inhibition. A 5-fold and a 1.6-fold increase of apparent competitive product inhibition constant of NAD + ( K iq ) and of lactate ( K iq K p / K q ) were observed, respectively. Third, the immobilized LDH showed improvement on thermal stability, with 5–7-fold reduced deactivation rate constant in second phase (A) of biphasic deactivation behavior, but with no significant difference in the first phase (B). Present study also develops a method to improve the immobilization process by understanding the surface interaction involved during the covalent immobilization of enzyme on magnetic nanoclusters. We found that the addition of sodium chloride during the immobilization could improve the relative activity of immobilized LDH from 50% to about 75%. This was due to the possibility of hydrophobic interaction in controlling a better LDH orientation on particle surface, and thus avoiding any severe deactivation. Protein representation using Jmol software also confirmed this possibility.

Stability and response of bioreactor: An analysis with reference to microbial reduction of SO2

Abstract A continuous B. Braun fermenter undergoing the first stage of bioreduction of gaseous pollutant, SO 2 —abiotically transformed to sulfite solution, using Desulfotomaculum nigrificans , purchased from NCIM, Pune (India), has been analysed both under steady and dynamic states. The kinetics of the microbial growth have been observed to face product inhibition in the entire range of inlet sulfite concentration of 0.904–1.696 g/dm 3 . The kinetic parameters like maximum specific growth rate ( μ m ), saturation constant ( K S ) and product inhibition constant ( K P ) have been evaluated by non-linear regression analysis and their values have been found to be 0.185 ks −1 , 6.512 × 10 −3 g/dm 3 and 0.045 g/dm 3 , respectively. The yield coefficient of generation of biomass with respect to the uptake of sulfite, Y X/S , has been observed to vary non-linearly with the fractional conversion of sulfite and the functional relationship has been determined as 1.918 exp(−1.984 X A ). Under the present range of values of dilution rate (2.778 × 10 −3 to 0.056 ks −1 ), the steady state conversion of sulfite has been observed to lie in the range of 99.6–6.12%. Non-ideality, i.e., the extent of deviation from the ideal hydrodynamic behavior of the bioreactor has been assessed using a two-parameter model. The parameters of this model have been evaluated conducting step-type tracer experiments for residence time distribution (RTD) of inlet tracer molecules in the fermenter. The values of the non-ideal parameters indicate that the reactor behaves almost ideally under the present investigation. For a priori determination of the stable regime of operation of this biosystem, in general, global and local stability analyses have been done by the techniques of separatrices and linear analysis, respectively. The process dynamics and the control behavior of the system have been assessed by the analysis of its response with respect to step changes in the forcing functions, namely flow rate and the sulfite concentration in the inlet stream of the bioreactor.

Use of Enrichment Culture for Directed Evolution of the Vibrio fluvialis JS17 ω-Transaminase, Which Is Resistant to Product Inhibition by Aliphatic Ketones

A novel high-throughput screening method that overcame product inhibition was used to isolate a mutant omega-transaminase from Vibrio fluvialis JS17. An enzyme library was generated using error-prone PCR mutagenesis and then enriched on minimal medium containing 2-aminoheptane as the sole nitrogen source and 2-butanone as an inhibitory ketone. An identified mutant enzyme, omega-TAmla, showed significantly reduced product inhibition by aliphatic ketone. The product inhibition constants of the mutant with 2-butanone and 2-heptanone were 6- and 4.5-fold higher than those of the wild type, respectively. Using omega-TAmla (50 U/ml) overexpressed in Escherichia coli BL21, 150 mM 2-aminoheptane was successfully resolved to (R)-2-aminoheptane (enantiomeric excess, >99%) with 53% conversion with an enantioselectivity of >100.

Batch kinetics and modelling of ethanolic fermentation of whey

SummaryThe fermentation of whey by Kluyveromyces marxianus strain MTCC 1288 was studied using varying lactose concentrations at constant temperature and pH. The increase in substrate concentration up to a certain limit was accompanied by an increase in ethanol formation, for example, at a substrate concentration of 10 g L−1, the production of ethanol was 0.618 g L−1 whereas at 50 g L−1 it was 3.98 g L−1. However, an increase in lactose concentration to 100 g L−1 led to a drastic decrease in product formation and substrate utilization. The maximum ethanol yield was obtained with an initial lactose concentration of 50 g L−1. A method of batch kinetics was utilized to formulate a mathematical model using substrate and product inhibition constants. The model successfully simulated the batch kinetics observed at S0 = 10 and 50 g L−1 but failed in case of S0 = 100 g L−1 because of strong substrate inhibition.

Effects of ultrasonic intensity and reactor scale on kinetics of enzymatic saccharification of various waste papers in continuously irradiated stirred tanks

Based on the enzymatic saccharification of the various pulps in the previous 0.8 l ultrasonic stirred tank reactor, the ultrasound-enhanced saccharification of waste papers such as newspaper, carton paper, office paper etc. was carried out in the same reactor as well as larger scale stirred tank reactors of size 3.2 and 6.4 l. The saccharification of each waste paper was less enhanced in the larger reactor at a given ultrasonic intensity. This could be attributed to the decrease in the ultrasonic intensity per reaction volume, i.e., the specific ultrasonic intensity. Most waste papers were more efficiently hydrolyzed with increasing specific ultrasonic intensities, although newspaper was less efficiently done for a too high specific intensity. Such an adverse effect might be due to the fact that some impurities in the newspaper such as lignin were activated by an intensive ultrasonic irradiation to form a rigid and closed network, which inhibited the access and adsorption of cellulase on to the substrate surface. The previous kinetic model was found to be applicable to analyze and simulate the saccharification of each waste paper in the different ultrasonic reactors. The ultimate conversion of a substrate based on the total sugar concentration estimated for an infinite reaction time could be correlated as a function of the ratio of initial substrate to enzyme concentrations at a fixed specific ultrasonic intensity. Either the apparent rate constant or the ultimate conversion increased and tended to approach a constant with an increase in the specific ultrasonic intensity except for the case of newspaper, while neither the apparent Michaelis constant, product inhibition constant nor glucose formation equilibrium constant was influenced by the specific ultrasonic intensity.

A kinetic study on enzymatic hydrolysis of a variety of pulps for its enhancement with continuous ultrasonic irradiation

The enzymatic hydrolyses of various pulps as a type of cellulosic materials were examined in a stirred flask reactor to establish a simplified kinetic model for the complicated process. To enhance the saccharification of a pulp with cellulase, the reaction was also carried out in a stirred tank reactor with continuous ultrasonic irradiation. The kinetic model was proposed by regarding the reaction system as a pseudo-homogeneous one, in which sugars would be produced from a hypothetical soluble substrate whose initial concentration would correspond to the concentration of total sugar produced ultimately. The validity of the model developed was confirmed by its successful simulation of the time courses of the total sugar and glucose during the saccharification either with or without ultrasonic irradiation. It was found that the variation in property of pulp as well as the intensity of ultrasonic irradiation exerted an effect on the apparent rate constant and ultimate conversion of substrate, but no effect on the apparent Michaelis and competitive product inhibition constants. The saccharification of the pulp called NUKP under the ultrasonic irradiation proceeded as a two-stage reaction, suggesting an almost complete conversion of the pulp to sugar. A mechanism was proposed for such an enhancement effect of the ultrasonic irradiation on enzymatic hydrolysis of cellulose.

Biosynthesis of ethyl butyrate using immobilized lipase: a statistical approach

Response surface methodology was used to determine optimum conditions for the esterification of ethanol and butyric acid to produce a flavor ester using immobilized lipase by Candida antarctica. Various reaction parameters including acid and/or alcohol concentration, enzyme concentration, temperature and reaction time affecting the synthesis of ethyl butyrate were investigated. The principal parameters influencing the esterification yield were incubation temperature and substrate concentration. The optimal conditions for the enzymic reaction were obtained at 0.04 M substrate concentration using 7% enzyme concentration at 34 °C for 96 h. Under these conditions the esterification percentage was 72.9%. The kinetics of commercial lipase was also studied and competitive inhibition by ethyl butyrate recorded. The product inhibition constant was 0.16 M.

Microcalorimetric Studies on the Product Inhibition of Hydrolysis of L-arginine Catalyzed by Bovine Liver Arginase

A new thermokinetic reduced extent method for the product inhibition of single substrate enzyme-catalyzed reactions is proposed and compared with the traditional initial rate method in this paper. The arginase-catalyzed hydrolysis of L-arginine to L-ornithine and urea was studied at 37°C in 40 mM sodium barbiturate-HCl buffer solution (pH=9.4). Michaelis constant (K m ) for arginine and maximum velocity (V m ) of the reaction were determined by initial method and thermokinetic method. The activation of exogenous manganese to this reaction was also studied. The product inhibition constant (K p ), which cannot be obtained directly from the initial rate method, was determined by thermokinetic without adding L-ornithine to the reaction system. When the concentration of Mn 2+ in cell is 0.1 mM, the enzyme gets its full activity. Incubation arginase with appropriate concentration of Mn 2+ resulted in increased V max and a higher sensitivity of the enzyme to product with no change in the K m for arginine. We suggest that the exogenous manganese ions in solution have just recovered the activity of arginase, which was lost in dissolving and dilution, but no effect on the mechanism of the reaction.

Temperature dependence of the kinetics of the acylase hydrolysis reaction by differential stopped flow microcalorimetry

The rates of the hydrolysis of N-acetylglycine, N-acetyl- l-methionine, and N-acetyl- l-phenylalanine by porcine acylase I in 0.1 M phosphate buffer, which is inhibited by acetate product formation, were monitored calorimetrically at temperatures between 15.2 and 45.3 °C by a differential stopped-flow microcalorimeter. Since the acylase is thermally stable and the pH of the phosphate buffer changes by less than 0.01 pH units over this temperature range, any temperature effect on the hydrolysis reaction can be attributed to the effect of temperature on the kinetics of the reaction. Analysis of the integrated heat released by the reaction as a function of time with regard to the integrated Michaelis–Menten equation yields apparent values for k cat and apparent values for K m that depend on the product inhibition constant. The apparent values for K m also exhibited a dependence on the initial substrate concentration because of the acetate product inhibition at each temperature. By assuming that the inhibition constant is independent of temperature over this temperature range and from extrapolation of K m to its value at zero substrate concentration, intrinsic values of K m and k cat were determined over the temperature range from 15.2 to 45.3 °C. The intrinsic values of K m exhibited very little variation over this temperature range while the intrinsic values of k cat exhibited an increase over the same temperature range. The heats of reaction also exhibited an increase with temperature over this range with an average heat capacity change of −94 J mol −1 K −1 for the three substrates.

Importance of product inhibition in the kinetics of the acylase hydrolysis reaction by differential stopped flow microcalorimetry

The hydrolysis of N-acetyl- l-methionine, N-acetylglycine, N-acetyl- l-phenylalanine, and N-acetyl- l-alanine at 298.35 K by porcine kidney acylase I (EC 3.5.1.14) was monitored by the heat released upon mixing of the substrate and enzyme in a differential stopped flow microcalorimeter. Values for the Michaelis constant ( K m) and the catalytic constant ( k cat) were determined from the progress of the reaction curve employing the integrated form of the Michaelis–Menten equation for each reaction mixture. When neglecting acetate product inhibition of the acylase, values for k cat were up to a factor of 2.3 larger than those values determined from reciprocal initial velocity–initial substrate concentration plots for at least four different reaction mixtures. In addition, values for K m were observed to increase linearly with an increase in the initial substrate concentration. When an acetate product inhibition constant of 600±31 M −1 , determined by isothermal titration calorimetry, was used in the progress curve analysis, values for K m and k cat were in closer agreement with their values determined from the reciprocal initial velocity versus initial substrate concentration plots. The reaction enthalpies, Δ r H(cal), which were determined from the integrated heat pulse per amount of substrate in the reaction mixture, ranged from −4.69±0.09 kJ mol −1 for N-acetyl- l-phenylalanine to −1.87±0.23 kJ mol −1 for N-acetyl- l-methionine.

Investigation of the kinetic mechanism of cytidine 5'-monophosphate N-acetylneuraminic acid synthetase from Haemophilus ducreyi with new insights on rate-limiting steps from product inhibition analysis.

The presence of sialic acid as a component of cell surface lipooligosaccharides or capsular polysaccharides has been shown to be correlated with the virulence of a number of Gram-negative mucosal pathogens, including several Haemophilus and Neisseria spp. As part of our efforts to evaluate the role of sialic acid in the pathobiology of these organisms, we have initiated studies of the enzymes from Haemophilus ducreyi (the infectious agent of chancroid) responsible for the activation and attachment of sialic acid to the lipooligosaccharide. In this report, we describe results of an investigation of the steady-state kinetic mechanism of the activating enzyme, cytidine 5'-monophosphate N-acetylneuraminic acid (CMP-NeuAc) synthetase. Using a combination of initial velocity, product inhibition, and dead-end inhibition studies, the reaction is shown to be freely reversible and to proceed through an ordered bi-bi kinetic mechanism in which CTP binds first and CMP-NeuAc dissociates last. In addition, a detailed analysis of the kinetic expressions for the observable constants is presented showing how the variation in apparent product inhibition constants (Kii) can be used to predict the rate-limiting step in kcat, which appears to be dissociation of CMP-NeuAc in this enzyme. To our knowledge, this relationship has not been previously recognized.

Kinetic modeling of omega-transamination for enzymatic kinetic resolution of alpha-methylbenzylamine.

A kinetic model for omega-transaminase from Bacillus thuringiensis JS64 was developed by using the King-Altman method to simulate the kinetic resolution of alpha-methylbenzylamine (alpha-MBA). Starting from a ping-pong bi-bi mechanism, a complete kinetic model including substrate inhibition only in the reverse reaction (i.e., transamination between acetophenone and L-alanine) was developed. The asymmetric synthesis of (S)-alpha-MBA proved to be difficult due to a much lower maximum reverse reaction rate than the maximum forward reaction rate, thermodynamically exergonic forward reaction (i.e., transamination between (S)-alpha-MBA and pyruvate), and the severe product and substrate inhibition of the reverse reaction. Experimental values for kinetic parameters show that the product inhibition constant of (S)-alpha-MBA is the most important parameter on determining the resolution reaction rate, suggesting that the resolution reaction rate will be very low unless (S)-alpha-MBA strongly inhibits the reverse reaction. Using the kinetic model, the kinetic resolution of alpha-MBA in aqueous buffer was simulated, and the simulation results showed a high degree of consistency with experimental data over a range of reaction conditions. Various simulation results suggest that the crucial bottleneck in the kinetic resolution of alpha-MBA lies mainly in the accumulation of acetophenone in reaction media as the reaction proceeds, whereas L-alanine exerts a little inhibitory effect on the reaction. The model predicts that removing acetophenone produced during the reaction can enhance the reaction rate dramatically. Indeed, the biphasic reaction system is capable of extracting acetophenone from the aqueous phase, showing a much higher reaction rate compared to a monophasic reaction system. The kinetic model was also useful in predicting the properties of other, better enzymes as well as the optimal concentrations of amino acceptor and enzyme in the resolution reaction.

Production of L(+)-lactic acid using immobilized rhizopus oryzae reactor performance based on kinetic model and simulation

The production of L(+)-lactic acid using alginate immobilizedRhizopus oryzae in tapered-column fluidized-bed batch reactor was tested and simulated using the kinetic data taken independently in shake-flask cultures. The data show saturation kinetics with substrate and product inhibitions in linear form. Analysis of the kinetic data gave kinetic constants:V m, 11.04 g lactic acid/(L-bead. h);K m, 20.9 g glucose/L; andK i, 365 g glucose/L for lactic acid production. The product inhibition constant,K p, was found to be 316 g lactic acid/L. The simulation results showed a good agreement with the experimental results when the initial lag phase was taken into account in the simulation model. Without the adjustment for the initial lag period, the kinetic model showed higher conversion. Starting with a glucose concentration of 150 g/L, it was possible to produce 73 g/L of L(+)-lactic acid in 44.5 h. The lactic acid yield was 64.8% by weight based on the amount of glucose consumed.

Characterization of alpha-glucosyltransferase from Pseudomonas mesoacidophila MX-45.

alpha-Glucosyltransferase was purified from Pseudomonas mesoacidophila MX-45. The molecular weight was estimated to be 63,000 by SDS-PAGE, and the isoelectric point was pI 5.4. For enzyme activity based on sucrose decomposition, the optimum pH and the optimum temperature were pH 5.8 and 40 degrees C, respectively. The ranges of stable pH and temperature were pH 5.1-6.7 and below 40 degrees C, respectively. The purified enzyme of MX-45 converted sucrose into trehalulose (1-O-alpha-D-glucopyranosyl- D-fructose) and isomaltulose (palatinose, 6-O-alpha-D-glucopyranosyl-D-fructose) simultaneously, and the ratio of trehalulose to isomaltulose increased at lower reaction temperatures. Therefore, optimum conditions for trehalulose production were pH 5.5-6.5 at 20 degrees C. The yield of trehalulose from sucrose (20-40% solution) was 91%. The Km for sucrose was 19.2 +/- 3.3 mM estimated by the Hanes-Woolf plot. Product inhibition was observed, and the product inhibition constant was 0.17 M. Hg2+, Fe3+, Cu2+, Mg2+, Ag+, Pb2+, glucono-1,5-lactone, and Tris(hydroxymethyl)aminomethane inhibited the reaction.