Deactivation of free and stabilized acid phosphatase by urea

Tests on acid phosphatase (E.G. 3.1.3.2) deactivation by urea have been performed at two pH values. Two conditions have been used: native enzyme operating batch-wise in dilute solution and stabilized enzyme in continuous flow ultrafiltration membrane reactor. Stabilization is achieved by confining the enzyme within a concentrated solution of a linear chain polymer that forms a polarization layer over the membrane. The results provide significant information on the kinetics and thermodynamics of the complex phenomena taking place during deactivation. Deactivation by urea is also compared with thermal deactivation.     

Enzyme inactivation and stabilization studies in an ultrafiltration reactor

Summary An ultrafiltration membrane enzymatic reactor is used in connection with different reacting systems.The experimental conditions are such that the enzyme, which operates at fairly high concentration levels because of the concentration polarization phenomena taking place in the reactor, is still in soluble form.The analysis of the system unsteady-state response enables the identification of the mechanism of enzyme deactivation and the extraction of the kinetic parameters of both the deactivation and the main reaction.The stabilizing effect observed in connection with enzyme entrapment within an inert gel deposited onto the U.F. membrane active surface is also discussed.List of Symbols A U.F. membrane cross sectional area cm² - C_E Enzyme concentration mg/ml - C_EI Enzyme concentration at the active membrane surface mg/ml - C_E Mean enzyme concentration mg/ml - c _s ^o Substrate concentration in the feed m moles/ml - c _s ^u Substrate concentration in the outlet m moles/ml - D_E Enzyme diffusivity cm²/s - Km michaelis constant mM - k₂ Kinetic constant of the enzymatic reaction m moles/mg s - k_d Kinetic constant of the enzyme deactivation reaction s^–1 - N^o Initial amount of active enzyme mg - N(t) Active enzyme amount at reaction time t mg - Q Flow rate ml/s - r Rate of the main reaction m moles/ml s - t Reaction time s - t^* Reaction time at which product concentration in the outlet is within ± 2% of the steady-state value s - v Fluid velocity cm/s - V Cell volume ml - V_B Volume within which 99% of the enzyme fed is contained at the steady-state ml - V_S Volume within which 99% of the total substrate concentration drop occurs at the steady-state ml - x Distance upstream the membrane measured from the membrane surface cm     

Optimal design for CSTR's in series performing enzymatic lactose hydrolysis

Analytical expressions are derived for the optimal design (based on minimum overall reactors volume) of a series of N CSTR's performing enzymatic lactose hydrolysis. It is assumed that lactose hydrolysis obeys Michaelis-Menten kinetics with competitive product (galactose) inhibition and no enzyme deactivation occurs. The optimum design of a cascade of ideally mixed reactors are compared with equal size reactors and with plug flow reactor required for a given overall degree of lactose conversion. The effect of operating parameters such as temperature, lactose initial (feed) concentration and conversion, enzyme and product initial concentration on the optimal overall holding time are also investigated. Optimization results for a series of N CSTR's up to five are obtained and compared with plug flow reactor.     

Active protein and substrate flow effects in a tubular immobilized invertase reactor

Investigations of invertase (EC 3.2.1.26) immobilized inside modified nylon tubes showed that between 4% and 20% (w/w) of the protein exposed to binding sites on the tube was immobilized. An enhanced activity consistent with enzyme purification during immobilization was also evident, suggesting that, in scaled-up commercial applications, nylon tube invertase would be a more economical converter of sucrose than the free enzyme. The quantity and specific activity of the immobilized protein were not stochiometrical with the amount used in the coupling solution and, in the system studied, a concentration of 2 mg ml^?1 was optimal. K_m and V_max values confirmed higher rates of immobilized invertase catalysis when the rates of substrate flow through the reactor were higher. Higher rates of substrate flow imply a shortened residence time in the reactor and would lower the fractional conversion per pass of the substrate, reducing the efficiency of the reactor in flow-through situations. Thus, these higher catalysis rates, attributable at the higher flow rates to a reduction of the diffusion barrier between enzyme and substrate, would not translate into improved economy in the commercial flow-through processes at which the reactor is aimed.     

Enzymatic hydrolysis of cellulose. Regulatory effect of the non-soluble substrate on the effectiveness of the enzymatic reaction

It was shown that one of the cellulase components, i.e. cellobiase, can be adsorbed on cellulose surface with the concomitant decrease of activity (by 10 times and more). The specific activity of the adsorbed cellobiase depends on the enzyme concentration in the adsorption layer and is increased with the increase in the surface concentration of cellobiase. It was found that variations in the amount of non-soluble cellulose and the corresponding changes in cellobiase activity in the system (as a result of the adsorption) can lead to a certain alteration in the shape of the kinetic curves for formation of intermediate cellobiose, which in its turn controls the rate of formation of the end product, i.e. glucose. Thus, the substrate surface causes a regulatory effect on the rate and kinetic mechanism of the enzymatic conversion of cellulose to glucose due to the adsorption effects.     

Continuous production of erythrulose using transketolase in a membrane reactor

Transketolase can be used for synthesis of chiral intermediates and carbohydrates. However the enzyme is strongly deactivated by the educts. This deactivation depends on the reactor employed. An enzyme membrane reactor allows the continuous production of L-erythrulose with high conversion and stable operational points. A productivity (space-time yield) of 45g L d was reached.     

Lipase-catalyzed kinetic resolution of (R,S)-1-phenylethyl propionate in an enzyme membrane reactor

Enzymatic stereoselective hydrolysis of (R,S)-1-phenylethyl propionate was performed in a stirred tank and in a biphasic enzyme membrane reactor. Lipase from Pseudomonas sp. was proved to be a good enantioselective catalyst for this reaction. The enzyme was covalently immobilized in a porous polyamide membrane (flat sheet as well as hollow-fibres) via glutaraldehyde. An influence of membrane hydrophobicity on reactor performance was observed. Initial lipase activity and productivity in the processes were equal to 1.05 × 10^?4, 1.3 × 10^?5 and 1.0 × 10^?5 mole/(h × mg of enzyme) in the case of native lipase, in the aromatic polyamide hydrophobic membrane reactor and in the hydrophilic polyamide-6 membrane reactor, respectively. The influence of some factors such as temperature, pH, buffer concentration, initial substrate concentration and addition of β-cyclodextrin derivatives on reaction rate and enantioselectivity was investigated and discussed. In the enzyme membrane reactor both organic and aqueous phases circulated countercurrently on both sides of the membrane. At a conversion degree of under 55–60%, pure enantiomer of the remaining ester (i.e. > 98%) was obtained.     

A CSTR-hollow fiber system for continuous hydrolysis of proteins. Performance and kinetics

The effect of four operating variables (enzyme concentration, substrate concentration, flow rate, and reaction volume) on the performance of CSTR-hollow fiber membrane reactor was studied for the continuous hydrolysis of a soy protein isolate using Pronase. Based on a residence time distribution study, the reactor system was modeled as an ideal CSTR in combination with the Michaelis-Menten equation of enzyme kinetics. This kinetic model correlated conversion with a space-time parameter modified to include all four independent variables. An empirical model based on curvilinear regression analysis was also developed. Both models predicted conversion fairly well, although the kinetic model slightly underpredicts at high conversion.     

Effect of immobilized enzyme deactivation in fixed- and fluid-bed reactors

A two-parameter theoretical model is developed to evaluate the effect of immobilized enzyme deactivation on substrate conversion in fixed- and fluid-bed reactors under diffusion-free conditions. The method describes a simple reaction in which three different immobilized enzyme deactivation forms are considered, and an expression is developed to evaluate the effect of immobilized enzyme deactivation on yield in a consecutive reaction. Comparison of reactor performances for the two reactor types reduces to a comparison of the appropriate dimensionless parameters. The practical implications of the development are illustrated through an example.     

Enzyme stabilization towards thermal,chemical and proteolytic deactivation

An enzyme stabilization technique which consists of entrapping protein within a polymeric network has been discussed. The high macromolecular concentration levels which lead to formation of the network are produced as a consequence of polarization phenomena which take place within an unstirred ultrafiltration membrane reactor. Increases in enzyme half-life were generally produced in connection with simple and complex deactivation phenomena of widely different natures (thermal, chemical and proteolytic). Experimental tests have been carried out on the following enzymes: β-d-glucosidase (β-d-glucoside glucohydrolase, EC 3.2.1.21), β-d-fructofuranosidase (β-d-fructofuranoside fructohydrolase, EC 3.2.1.26), acid phosphatase [orthophosphoric-monoester phosphohydrolase (acid optimum), EC 3.1.3.2] and β-d-galactosidase (β-d-galactoside galactohydrolase, EC 3.2.1.23).     

Modeling of a Catalyzed Reaction Using Lipase Immobilized in a Poly(vinyl alcohol) Membrane

A mathematical model for the hydrolysis reaction of p‐nitro phenol laurate catalyzed by a lipase immobilized in a membrane was developed. In an earlier study this model reaction was found to show very different reaction rates when it was performed in aqueous micellar solution with free enzyme and with membrane immobilized enzyme. It was assumed that a local accumulation of substrate in the membrane is responsible for the observed rate enhancement. The conversion of p‐nitro phenol ester within the membrane was modeled by considering a combination of the convective flow through poly(vinyl alcohol) membrane pores, concentration polarization of substrate containing micelles at the membrane surface and the kinetics of the reaction with free enzymes. It was demonstrated that the model offered a comprehensive understanding of the interaction of the involved phenomena. The modeling results are in good agreement with the experimental data from 10 runs with different enzyme and substrate concentrations. The substrate concentration at the membrane surface increased by up to a factor of 3 compared to the feed concentration. This effect explains the observed rate enhancement. Moreover, the model was used to determine the unknown parameters, i.e., the intrinsic retention and the mass transfer coefficient, by fitting the model to the experimental data. The model may also be used to calculate the optimum operating conditions and design parameters of such a reactor.     

Comparative study of reactor performance for the resolution of d,l-amino acids

The racemic resolution of l-valine and l-serine by fungal aminoacylase has been evaluated by comparing the performance of various reactor configurations including an anion exchange nylon tangential flow membrane reactor, a tubular reactor with aminoacylase adsorbed onto DEAE-Sephadex as support and a continuous stirred tank reactor with enzyme recycling using a flat ultrafiltration module (CSTR/UF). Among the substrates tested, the N-chloroacetyl-d,l-amino acids were the preferred substrates, showing the highest catalytic efficiency (V_m/K_m).Optimum reactor operational conditions obtained in discontinuous assays were selected to study the behaviour of the reactors in a continuous mode. DEAE-Sephadex loaded six-fold more enzyme than anion exchange nylon (60 and 10 gE/litre, respectively, related to reactor volume), whereas enzyme concentration within the CSTR/UF reactor was limited only by enzyme solubility.The tangential flow membrane reactor configuration with a 10 g/litre enzyme concentration produced higher productivity values (0·35 kg l-valine/litre per day, and 80% conversion degree) and operational stability (t = 161 days) than the CSTR/UF reactor (0·24 kg l-valine/litre per day, and 80% conversion degree) performing with the same enzyme concentration. The tubular reactor with the enzyme adsorbed onto DEAE-Sephadex (60 g/litre enzyme load) showed higher productivity values (1·9 kg l-valine/litre per day, and 80% conversion degree) and operational stability (t = 70 days) than the CSTR/UF reactor (1·05 kg l-valine/litre per day, and 80% conversion degree). However, the CSTR/UF reactor was the preferred configuration, as it had the highest enzyme load and productivity (1·95 kg l-valine/litre per day of reactor volume, and 80% conversion degree), a half-life of 55 days at 50°C, and the possibility of easy continuous enzyme addition.     

Coated-wall microreactor for continuous biocatalytic transformations using immobilized enzymes

Microstructured flow reactors are emerging tools for biocatalytic process development. A compelling design is that of the coated-wall reactor where enzyme is present as a surface layer attached to microchannel walls. However, preparation of a highly active wall biocatalyst remains a problem. Here, a stainless steel microreactor was developed where covalent immobilization of the enzyme in multiple linear flow channels of the reaction plate was supported by a macroporous wash-coat layer of gamma-aluminum oxide. Using surface functionalization with aminopropyl triethoxysilane followed by activation with glutardialdehyde, the thermophilic beta-glycosidase CelB from Pyrococcus furiosus was bound with retention of half of the specific activity of the free enzyme (800 U/mg), yielding a high catalyst loading of about 500 U/mL. This microreactor was employed for the continuous hydrolysis of lactose (100 mM) at 80 degrees C, providing a space-time yield of 500 mg glucose/(mL h) at a stable conversion of > or =70%. The immobilized enzyme displayed a half-life of 15 days under the operational conditions. Due to the absence of hydrophobic solute-material interactions, which limit the scope of microstructures fabricated from poly(dimethylsiloxane) for biocatalytic applications, the new microreactor was fully compatible with the alternate enzyme substrate 2-nitro-phenyl-beta-D-galactoside and the 2-nitro-phenol product resulting from its hydrolysis catalyzed by CelB.     

Kinetic Modeling of Acetophenone Reduction Catalyzed by Alcohol Dehydrogenase from <Emphasis Type="Italic">Thermoanaerobacter</Emphasis> sp.

NADPH-dependent alcohol dehydrogenase (ADH) from Thermoanaerobacter sp. was kinetically characterized using reduction of acetophenone as a model. To achieve 98% conversion of acetophenone, cofactor regeneration by oxidation of 2-propanol with the same enzyme was used. The enzyme was stable in the batch reactor. It was enantioselective towards (S)-1-phenylethanol (ee>99.5%). Due to its high deactivation in continuously operated stirred tank reactor (k_d=0.0141 min⁻¹) there was no way to keep high conversion of acetophenone at 98%. The deactivation occurred in the repetitive batch as well. A mathematical model for the acetophenone reduction with cofactor regeneration describing the behaviour in a batch, repetitive-batch and continuously stirred tank reactor was developed.     

固定化纤维二糖酶的研究

黑曲霉 (AspergillusnigerLORRE 0 12 )的孢子中富含纤维二糖酶 ,将这些孢子用海藻酸钙凝胶包埋后 ,可以方便有效地固定纤维二糖酶。固定化后的纤维二糖酶性能稳定 ,半衰期为 38d ,耐热性和适宜的pH范围均比固定化前有所增加 ,其Km 和Vmax值分别为 6 .0 1mmol L和 7.0 6mmol (min·L)。利用固定化纤维二糖酶重复分批酶解10g L的纤维二糖 ,连续 10批的酶解得率均可保持在 97%以上 ;采用连续酶解工艺 ,当稀释率为 0 .4h- 1 ,酶解得率可达 98.5 %。玉米芯经稀酸预处理后 ,其纤维残渣用里氏木霉 (Trichodermareesei)纤维素酶降解 ,酶解得率为6 9.5 % ;通过固定化纤维二糖酶的进一步作用 ,上述水解液中因纤维二糖积累所造成的反馈抑制作用得以消除 ,酶解得率提高到 84.2 % ,还原糖中葡萄糖的比例由 5 3 .6 %升至 89.5 % ,该研究结果在纤维原料酶水解工艺中具有良好的应用前景。     

Membrane reactor coupled with electrophoresis for enzymatic production of aspartic acid

Aspartic acid production by aspartase reaction on ammonium fumarate was carried out in a membrane reactor coupled with electrophoresis. A pressurized, stirred vessel attached with an ultrafiltration membrane was used as a membrane reactor. An electric field was applied across the membrane to preferentially remove the product aspartate from the reactor into the permeate stream. The charged molecule, aspartate, is much smaller than the molecular-weight cutoff of the membrane (10(4)) so that the ions would move freely through pores of the membrane. The concentration of aspartate in the permeate stream is determined by the electromigration velocity of the ions and the permeation rate of solvent (water) through the membrane. The permeation rate of solvent could be controlled by the applied pressure, and the migration velocity of the ions could be controlled by the electric field strength applied. The equilibrium conversion of ammonium fumarate to the aspartate was 70%. In the presence of electric field, the aspartase activity was not disturbed. Also, it is shown that the aspartate concentration in the permeate stream was 20% higher than that in the reaction solution with the permeate flow rate of 0.7 mL/min. The steady-state conversion was 60%. Instead of aspartate, aspartic acid can be recovered directly from the permeate stream by controlling the circulation of buffer electrolyte in the anode compartment.     

Effects of mass-transfer resistance on apparent stability and performance of fixed-bed immobilized enzyme reactors: theory and experiments with immobilized invertase

Taking the hydrolysis of sucrose by invertase immobilized on anion-exchange resin as an example, the effects of mass-transfer resistance on the apparent stability of immobilized enzyme (IME) and the optimal policy for an IME reaction in a fixed-bed reactor have been studied theoretically and experimentally. The following results were obtained: (1) The effect of mass-transfer resistance on the effective deactivation rate of IME is summarized in two parameters concerning the intraparticle diffusion alpha(p) and the interparticle alpha(f). (2) At a constant processed amount of raw materials, there exists an optimal flow rate of reaction fluid to enhance the reactor performance while the mass-transfer resistance shifts the optimal point. (3) The intrinsic deactivation rate of IME has been estimated from the relationship between the fractional conversion at the reactor outlet and the operation time.     

Stabilization of cellobiase by covalent coupling to soluble polysaccharide.

Cellobiase from Aspergillus niger was glycosylated by covalent coupling to cyanogen bromide activated dextran. The conjugated enzyme retained 62% of the original specific activity exhibited by the native cellobiase. The optimum pH as well as the pH stability of the conjugated form remain almost the same as for the native enzyme. Compared to the native enzyme, the conjugated form exhibited a higher optimal reaction temperature and energy of activation, a higher K(m) (Michaelis constant) and lower Vmax (maximal reaction rate), and improved thermal stability. The thermal deactivation of the native and conjugated cellobiase obeyed the first-order kinetics. The calculated half-life values of heat inactivation at 60, 70 and 80 degrees C was 10.7, 6.25, and 4.05 h, respectively, whereas at these temperatures the native enzyme was less stable (half-life of 3.5, 1.69, and 0.83 h, respectively). The deactivation rate constant at 80 degrees C for the conjugated cellobiase is about 7.9 x 10(-2) h-1, which is lower than that of the native enzyme (36.0 x 10(-2) h-1). The activation energy for denaturation of the native enzyme is about 10.58 kcal/mol, which is 7.25 kcal/mol lower than that of the conjugated enzyme. The effect of different surfactants and some metal ions on the activity of the conjugated cellobiase has been investigated.     

The possibility for improvement of ceramic membrane ultrafiltration of an enzyme solution

Ultrafiltration represents an attractive downstream processing technique for enzymes concentration and their primary purification. However, the process efficiency is often limited by protein fouling and shear-induced enzyme deactivation, resulting in permeate flux decline and loss of enzyme activity. The objective of this work was to investigate the possibility for improvement of ceramic membrane ultrafiltration of endo-pectinase solution. Experimental investigations were performed on a 5 nm ceramic membrane with the Kenics static mixer placed inside the membrane in order to improve the process performance. The use of the static mixer resulted in the flux improvement of about 45% at a volume concentration factor (VCF) of 3 leading to the reduction of operation time of 25% and the energy saving of about 40%. Although the rejection of endo-pectinase was higher than 96%, the extensive loss of the enzyme activity during operation indicated that the modification of the feed solution is essential for improved ultrafiltration performance. Addition of pectin to the original endo-pectinase solution led to a significant reduction of the enzyme deactivation: the enzyme activity yield was 90% at a VCF of 1.6 during operation with the static mixer.