Modeling solid-to-solid biocatalysis: integration of six consecutive steps

A quantitative model for the conversion of a solid-substrate salt to a solid-product salt in a batch bioreactor seeded with product crystals is presented. The overall process consists of six serial steps (with dissolution and crystallization each in themselves complex multistep processes): solid-salt dissolution, salt dissociation into an ionic substrate and a counter-ion, bioconversion accompanied by biocatalyst inactivation, complexation of the ionic product with the counter-ion, and salt crystal growth. In the model, the consecutive steps are integrated, including biocatalyst inactivation and assuming that salt dissociation and complexation of ions are at equilibrium. Model parameters were determined previously in separate independent experiments. To validate the model, either dissolved or solid Ca-maleate was converted to solid Ca-D-malate by permeabilized Pseudomonas pseudoalcaligenes in a batch bioreactor seeded with Ca-D-malate crystals. The model very well predicted the concentrations of all components in the liquid phase (Ca-maleate, Ca(2+), maleate(2-), D-malate(2-), and Ca-D-malate) and the amounts of the solid phases (Ca-maleate. H(2)O and Ca-D-malate. 3H(2)O), especially when high initial amounts of Ca-maleate. H(2)O and Ca-D-malate. 3H(2)O were present.     

Preparation of L-aspartic acid by means of immobilized Alcaligenes metalcaligenes cells

Two types of biocatalysts based on immobilized cells of Alcaligenes metalcaligenes exhibiting aspartate ammonia-lyase activity (EC 4.3.1.1) were developed for the enzymic preparation of L-aspartic acid from ammonium fumarate. The first type of the biocatalyst consists in individual covalently crosslinked and permeabilized cells(I), while the second type is represented by cell aggregates (II). For the above preparation, biocatalyst I can be used only discontinuously in a mixed reactor. After termination of the reaction between individual cycles of its use, the biocatalyst is returned to the reactor in the form of a highly concentrated cell suspension or paste. Biocatalyst II can be used discontinuously or continuously in a fixed-bed column of the catalyst. The effects of pH, substrate concentration and temperature on the reaction velocity and effectivity of enzymic conversion was investigated. Optimal parameters of the reaction are as follows: pH 8.5, initial substrate concentration, 1.35 mol/L, temperature for discontinuous process, 37 degrees C, and temperature for continuous process, 25 degrees C. Under these conditions the enzymic conversion of substrate to product is quantitative. Under optimal toring conditions, the specific activity of both catalysts does not change within a period of one year. The operational half-life of the biocatalyst II during continuous use in a fixed-bed column of the catalyst under standard reaction conditions depends on the quality of the substrate. The discontinuous preparation of L-asparatic acid with the aid of biocatalyst I and continuous preparation of this product with the aid of biocatalyst II have been verified under pilot-plant conditions.     

Kinetic studies of alpha-galactosidase-containing mold pellets on PNPG hydrolysis.

Little is known about techniques for applying untreated microbial cells containing enzymes directly to industrial processes as a biocatalyst. The kinetic behavior of alpha-galactosidase-containing spherical pellets which are formed naturally under given conditions in a submerged culture of Mortierella vinacea was studied on the hydrolysis of PNPG (p-nitrophenyl-alpha-D-galactopyranoside). The effect on intraparticle diffusion on the overall reaction rate was assessed by the use of an effectiveness factor, which was calculated by the approximate solution to the equation derived from the mass balance within a pellet. The experimental effectiveness factors were found to be represented as a single function of the modified Thiele modulus, including such parameters as pellet size, enzyme concentration in the pellet, and substrate concentration. As the diffusional effect became more significant, the marked substrate inhibition as seen for a free enzyme disappeared gradually. The effect of product inhibition on the pellets was much weaker than that for a free enzyme at a given substrate concentration. In the region of diffusion controlled reaction, it was found that the rate is proportional to the square root of the enzyme concentration in the pellet. In addition, similarly to what was reported previously for a free enzyme,the reaction in a batch system was found to be approximately representable as simple first-order kinetics in which the rate constant was dependent on the initial substrate concentration.     

Kinetics of acetophenone reduction to (R)-1-phenylethanol by a whole-cell Pichia capsulata biocatalyst

(R)-1-phenylethanol is an important substance in fragrance and flavor industry. In this work, the reduction of acetophenone to (R)-1-phenylethanol in an aqueous medium was examined using Pichia capsulata as a whole-cell biocatalyst. Progress curve and initial rate measurements were used to obtain kinetic data. The experiments were carried out at pH 5, temperature of 25?°C, and in the presence of glucose to maintain in vivo regeneration of NADH. A model of the reversible reaction kinetics considering the substrate inhibition of the forward reaction was developed. Five kinetic parameters of this model were determined by a simultaneous fit of a reaction rate dependence on substrate concentration and 18 substrate and product concentration progress curves with very good accuracy. Equilibrium constant of the reaction and equilibrium conversion of acetophenone to (R)-1-phenylethanol were 13.7 and 93%, respectively.     

The use of microscale processing technologies for quantification of biocatalytic Baeyer-Villiger oxidation kinetics

Microscale processing techniques would be a useful tool for the rapid and efficient collection of biotransformation kinetic data as a basis for bioprocess design. Automated liquid handling systems can reduce labor intensity while the small scale reduces the demand for scarce materials such as substrate, product, and biocatalyst. Here we illustrate this concept by establishing the use of several microwell formats (96-round, 96-deep square and 24-round well microtiter plates) for quantification of the kinetics of the E. coli TOP10 [pQR239] resting cell catalyzed Baeyer-Villiger oxidation of bicyclo[3.2.0]hept-2en-6-one using glycerol as a source of reducing power. By increasing the biocatalyst concentration until the biotransformation rate was oxygen mass-transfer limited we can ensure that kinetic data collected are in the region away from oxygen limitation. Using a 96-round well plate the effect of substrate (bicyclo[3.2.0]hept-2en-6-one) concentration on the volumetric CHMO activity was examined and compared to data collected from 1.5-L stirred-tank experiments. The phenomenon and magnitude of substrate inhibition, observed at the larger scale, was accurately reproduced in the microwell format. We have used this as an illustrative example to demonstrate that under adequately defined conditions, automated microscale processing technologies can be used for the collection of quantitative kinetic data. Additionally, by using the experimentally determined stoichiometry for product formation and glycerol oxidation, we have estimated the maximum oxygen transfer rates as a function of well geometry and agitation rate. Oxygen-transfer rates with an upper limit of between 33 mmol. L(-1). h(-1) (based solely on product formation) and 390 mmol. L(-1). h(-1) (based on product formation and glycerol oxidation) were achieved using a 96-square well format plate shaken at 1300 rpm operated with a static surface area to volume ratio of 320 m(2). m(-3).     

Kinetic modelling of the synthesis of 2-hydroxy-5-hexenyl 2-chlorobutyrate ester by an immobilised lipase

An esterification process was developed for the direct synthesis of 2-hydroxy-5-hexenyl 2-chlorobutyrate ester from 2-chlorobutyric acid by using the epoxide 1,2-epoxy-5-hexene and Mucor miehei immobilised lipase as the biocatalyst in a batch reactor. The effect of temperature, catalyst concentration, and substrate and product concentration has been studied. An ordered Bi Uni enzymatic mechanism with competitive inhibition by the epoxide and acid has been proposed. The corresponding kinetic parameters were calculated by non-linear regression. Activation energy shows a value of 8.04kcal/mol. The thermodynamic parameters of the process, enthalpy and entropy, were 15.4kcal/mol and 45cal/mol, respectively.     

Kinetic effects of simultaneous inhibition by substrate and product

The starting point for the present investigations was the finding that increasing influent concentrations from 10 to 380 mmol/L glucose decreased the attainable growth rate of an acidogenic population in continuous culture from 0.52 to 0.05 h(-1) To account for this phenomenon, a new kinetic model is developed that combines substrate and product inhibition. Both effects are connected through the product yield, giving rise to a complex dependency of the growth rate on the substrate concentration. As a main feature, the maximum attainable growth rate decreases almost hyperbolically above some optimal substrate concentration in the influent. Furthermore, under certain conditions the kinetic model predicts the existence of three steady states: a high-conversion and a low-conversion state that are both stable and a metastable intermediate state. The latter states from the multiple-steady-state region are to be avoided, and eventual transitions to these states may have important consequences for the stability and the operation of such reaction systems. Substrate as well as product inhibition is reported for Propionibacterium freundenreichii and recently could be demonstrated for the above-mentioned acidogenic population. The proposed model allows optimization of anaerobic wastewater treatment processes and is applicable also to other fermentations.     

Kinetic modeling of enzymatic hydrolysis of cellulose in differently pretreated fibers from dairy manure

A kinetic model incorporating dynamic adsorption, enzymatic hydrolysis, and product inhibition was developed for enzymatic hydrolysis of differently pretreated fibers from a nitrogen-rich lignocellulosic material-dairy manure. The effects of manure proteins on the enzyme adsorption profile during hydrolysis have been discussed. Enzyme activity, instead of protein concentration, was used to describe the enzymatic hydrolysis in order to avoid the effect of manure protein on enzyme protein analysis. Dynamic enzyme adsorption was modeled based on a Langmiur-type isotherm. A first-order reaction was applied to model the hydrolysis with consideration being given for the product inhibition. The model satisfactorily predicted the behaviors of enzyme adsorption, hydrolysis, and product inhibition for all five sample manure fibers. The reaction conditions were the substrate concentrations of 10-50 g/L, enzyme loadings of 7-150 FPU/g total substrate, and the reaction temperature of 50 degrees C.     

Chemo-enzymatic epoxidation-process options for improving biocatalytic productivity

The reactor choice is crucial when designing a process where inactivation of the biocatalyst is a problem. The main bottleneck for the chemo-enzymatic epoxidation has been found to be enzyme inactivation by the hydrogen peroxide, H(2) O(2) , substrate. In the work reported here, the effect of reaction parameters on the reaction performance have been investigated and used to establish suitable operating strategies to minimize the inactivation of the enzyme, using rapeseed methyl ester (RME) as a substrate in a solvent-free system. The use of a controlled fed-batch reactor for maintaining H(2) O(2) concentration at 1.5 M resulted in increased productivity, up to 76 grams of product per gram of biocatalyst with higher retention of enzyme activity. Further investigation included a multistage design that separated the enzymatic reaction and the saturation of the RME substrate with H(2) O(2) into different vessels. This setup showed that the reaction rate as well as enzyme inactivation is strongly dependent on the H(2) O(2) concentration. A 20-fold improvement in enzymatic efficiency is required for reaching an economically feasible process. This will require a combination of enzyme modification and careful process design.     

Dynamic kinetic resolution: alternative approach in optimizing S-ibuprofen production

A lipase catalysed enantioselective hydrolysis process under in situ racemization of the remaining (R)-ibuprofen ester substrate with sodium hydroxide as the catalyst was developed for the production of S-ibuprofen from (R,S)-ibuprofen ester in isooctane. Detailed investigations on parameters study indicated that 0.5 M NaOH, addition of 20% (v/v) co-solvent (dimethyl sulphoxide), operating temperature of 45 °C, and 40 mmol/L substrate gave 86% conversion and 99.4% optical purity of S-ibuprofen in dynamic kinetic resolution. Meanwhile, in common enzymatic kinetic resolution process, only 42% conversion of the racemate and 93% enantiomeric excess of the product was obtained which are of lower values as compared to dynamic kinetic resolution. The S-ibuprofen produced during each process was evaluated and approximately 50% increment in concentration of S-acid product was produced when dynamic kinetic resolution was applied into the process.     

Substrate utilization kinetic model for biological treatment process

The applicability of Contois' kinetic equation to aerobic and anaerobic treatments of organic wastes is investigated. A refractory coefficient to account for the nonbiodegradable portion of the organic substrates in the digester is incorporated into the kinetic equation. The kinetic equation is applied to the data for aerobic digestions of organic substrates and for anaerobic treatment of dairy wastes. They all show a very good fit of the kinetic equation to the data. Furthermore, the kinetic parameters and the refractory coefficients are shown to be independent of influent organic substrate concentration. This study confirms previous reports that the effluent quality of biological treatment systems for organic wastes depends on influent organic waste concentration. The effect of temperature on the kinetic parameters and the refractory coefficient for anaerobic treatment of sewage sludge are studied. It shows that the kinetic parameters vary with temperature, while the refractory coefficient remains fairly constant. Equations to predict biodegradable treatment efficiency and volumetric substrate utilization rate are also briefly discussed.     

Design of immobilized enzyme reactors for the continuous production of fructose syrup from whey permeate

Biocatalyst inactivation is inherent to continuous operation of immobilized enzyme reactors, meaning that a strategy must exist to ensure a production of uniform quality and constant throughput. Flow rate can be profiled to compensate for enzyme inactivation maintaining substrate conversion constant. Throughput can be maintained within specified margins of variation by using several reactors operating in parallel but displaced in time. Enzyme inactivation has been usually modeled under non-reactive conditions, leaving aside the effect of substrate and products on enzyme stability. Results are presented for the design of enzyme reactors under the above operational strategy, considering first-order biocatalyst inactivation kinetics modulated by substrate and products. The continuous production of hydrolyzed-isomerized whey permeate with immobilized lactase and glucose isomerase in sequential packed-bed reactors is used as a case study. Kinetic and inactivation parameters for immobilized lactase have been determined by the authors; those for glucose isomerase were taken from the literature. Except for lactose, all other substrates and products were positive modulators of enzyme stability. Reactor design was done by iteration since it depends on enzyme inactivation kinetics. Reactor performance was determined based on a preliminary design considering non-modulated first-order inactivation kinetics and confronted to such pattern. The new pattern of inactivation was then used to redesign the reactor and the process repeated until reactor performance (considering modulation) matched the assumed pattern of inactivation. Convergence was very fast and only two iterations were needed.     

Kinetics of glucose isomerization to fructose by immobilized glucose isomerase in the presence of substrate protection

The activity of immobilized glucose isomerase of Streptomyces murinus has been tested batchwise under different conditions in order to gather the related kinetic parameters necessary to optimize an immobilized enzyme column for the continuous production of high fructose corn syrup (HFCS). To this purpose, the Briggs-Haldane model incorporating an apparent first-order inactivation constant has been used with success. A comparison of the equilibrium constants and of the maximum theoretical conversion yields calculated at different temperatures with those estimated for the native enzyme demonstrates that the immobilization favours the transformation of glucose to fructose only at T?>?70?°C, as a possible consequence of a combined effect of catalysis and equilibrium thermodynamics enhancement. Enzyme inactivation has also been tested at different temperatures and sugar concentrations to evaluate the related kinetic parameters under different conditions of substrate protection.     

n-carbamoyl-d-p-hydroxyphenylglycine production using immobilized d-hydantoinase from recombinant E. coli

An immobilized d-hydantoinase was characterized and employed to produce n-carbamoyl-d-p-hydroxyphenylglycine (CpHPG) in a repeated batch process. The V_max and K_m of the immobilized d-hydantoinase at 50°C were 6.28 mm min⁻¹ g⁻¹ biocatalyst and 71.6 mm, respectively. The product CpHPG did not inhibit the activity of d-hydantoinase. Optimal reaction temperature was 60°C. A decrease in activity of immobilized d-hydantoinase due to thermal inactivation could be described as first-order decay; the deactivation energy was 23.97Kcal mol⁻¹. Under process conditions (50°C, 10% w/v substrate, and pH 8.5), the half-life of the immobilized d-hydantoinase was eight batches. The attrition of immobilized d-hydantoinase particles with a large amount of insoluble substrate particles during stirring resulted in fine biocatalyst particles. In addition to the thermal inactivation, the loss of fine biocatalyst particles during the recovery step contributed to the low operational stability.     

Characterization of an industrial biocatalyst: immobilized glutaryl-7-ACA acylase

A batch of the immobilized industrial biocatalyst glutaryl-7-ACA acylase (GA), one of the two enzymes involved in the biotransformation of cephalosporin C (CefC) into 7-aminocephalosporanic acid (7-ACA), was characterized. K(m) value for glutaryl-7-ACA was 5 mM. Enzyme activity was found to be optimal at pH between 7 and 9.5 and to increase with temperature and in buffered solutions. To avoid product degradation, optimal reaction conditions were obtained working at 25 degrees C using a 50-mM phosphate buffer, pH 8.0. Immobilized GA showed good stability at pH value below 9 and at temperature up to 30 degrees C. The inactivation of immobilized GA in the presence of different amounts of H(2)O(2), a side product that might be present in the plant-scale industrial solutions of glutaryl-7-ACA, was also investigated, but the deactivation rates were negligible at H(2)O(2) concentration that might be reached under operative conditions. Finally, biocatalyst performance in the complete two-step enzymatic conversion process from CefC to 7-ACA was determined on a laboratory scale. Following the complete conversion of a 75 mM solution of CefC into glutaryl-7-ACA catalyzed by an immobilized D-amino acid oxidase (DAAO), immobilized GA was used for the transformation of this intermediate into the final product 7-ACA. This reaction was repeated for 42 cycles. An estimation of the residual activity of the biocatalyst showed that 50% inactivation of immobilized GA was reached after approximately 300 cycles, corresponding to an enzyme consumption of 0.4 kU per kg of isolated 7-ACA.     

Electrostatic effects on the kinetics of bound enzymes.

1. The effect of the interaction between the charged matrix and substrate on the kinetic behaviour of bound enzymes was investigated theoretically. 2. Simple expression is derived for the apparent Km. 3. The apparent Km can only be used for the characterization of the electrostatic effect of the ionic strength does not vary with the substrate concentration. 4. The deviations from Michaelis-Menton kinetics are graphically illustrated for cases when the ionic strength varies with the substrate concentration. 5. The inhibition of the bound enzyme by a charged inhibitor at constant ionic strength is characterized by an apparent Ki. 6. When both the inhibitor concentration and the ionic strength change there is no apparent Ki, and the inhibition profile is graphically illustrated for this case. 7. Under certain conditions the electrostatic effects manifest thenselves in a sigmoidal dependence of the enzyme activity on the concentration of the substrate or inhibitor.     

Comparison of two mathematical models for correlating the organic matter removal efficiency with hydraulic retention time in a hybrid anaerobic baffled reactor treating molasses

A modelling of the anaerobic digestion process of molasses was conducted in a 70-L multistage anaerobic biofilm reactor or hybrid anaerobic baffled reactor with six compartments at an operating temperature of 26 °C. Five hydraulic retention times (6, 16, 24, 72 and 120 h) were studied at a constant influent COD concentration of 10,000 mg/L. Two different kinetic models (one was based on a dispersion model with first-order kinetics for substrate consumption and the other based on a modification of the Young equation) were evaluated and compared to predict the organic matter removal efficiency or fractional conversion. The first-order kinetic constant obtained with the dispersion model was 0.28 h⁻¹, the Peclet dispersion number being 45, with a mean relative error of 2%. The model based on the Young equation predicted the behaviour of the reactor more accurately showing deviations lower than 10% between the theoretical and experimental values of the fractional conversion, the mean relative error being 0.9% in this case.     

Catalytic properties of glucoamylase immobilized on the synthetic carbon material Sibunit

Glucoamylase (commercial preparation Glucavamorin) was immobilized by sorption on a carbon support Sibunit. Starch saccharification by the resulting biocatalyst (dextrin hydrolysis) was studied. Investigation of the effect of adsorptional immobilization on kinetic parameters of glucoamylase, including the rate constant of thermal inactivation, showed that immobilization of Glucavamorin on Sibunit resulted in a thousandfold increase in glucoamylase stability in comparison with the dissolved enzyme. Presence of the substrate (dextrins) in the reaction mixture had a considerable stabilizing effect. Increase in dextrin concentration increases the thermostability of the immobilized enzyme. The overall factor of glucoamylase stabilization adsorbed on Sibunit with the presence of 53% dextrin solutions in comparison with the dissolved enzyme approximated 10(5). The biocatalyst for starch saccharification made on the base of Subunit-adsorbed Glucavamorin had a high operational stability. Its half-inactivation time at 60 degrees C exceeded 30 days.     

Catalytic properties of glucoamylase immobilized on synthetic carbon material Sibunit

Glucoamylase (commercial preparation Glucavamorin) was immobilized by sorption on a carbon support Sibunit. Starch saccharification by the resulting biocatalyst (dextrin hydrolysis) was studied. Investigation of the effect of adsorptional immobilization on kinetic parameters of glucoamylase, including the rate constant of thermal inactivation, showed that immobilization of Glucavamorin on Sibunit resulted in a thousand-fold increase in glucoamylase stability in comparison with the dissolved enzyme. Presence of the substrate (dextrins) in the reaction mixture had a considerable stabilizing effect. Increase in dextrin concentration increases the thermostability of the immobilized enzyme. The overall factor of glucoamylase stabilization adsorbed on Sibunit with the presence of 53% dextrin solutions in comparison with the dissolved enzyme approximated 10⁵. The biocatalyst for starch saccharification made on the base of Subunit-adsorbed Glucavamorin had a high operational stability. Its half-inactivation time at 60°C exceeded 30 days.