The production of 6-aminopenicillanic acid by a multistage tubular reactor packed with immobilized penicillin amidase

A theoretical model equation was derived to find the correlation between the conversion and the amount of immobilized penicillin amidase in column. The theoretical values of the conversion were predicted form this correlation and compared with experimental results. It was observed in a column reactor that the pH drop along the column path was linear versus the enzyme loading and that the enzyme activity was also linearly dependent on pH up to 8.0. In order to diminish the effect of pH drop, a continuous two-stage plug-flow reactor (PFR) with pH adjustment between the two columns was used was used in the experiments, and two- and three-stage PFRs were simulated by computer. In the case of the two-stage PFR, the maximum productivity was demonstrated experimentally and theoretically as well. when an equal amount of the immobilized enzyme was packed in both columns. It was also predicted in the tree-stage PFR system that the optimal distributions of enzyme loading in three columns were found to be 1:1:1. It was demonstrated that the increased number of reactors in series could enhance the level of the maximum productivity with a given amount of enzyme loading.     

Effect of mass transfer in a recirculation batch reactor system for immobilized penicillin amidase

The effect of external mass transfer resistance on the overall reaction rate of the immobilized whole cell penicillin amidase of E. coli in a recirculation batch reactor was investigated. The internal diffusional resistance was found negligible as indicated by the value of effectiveness factor, 0.95. The local environmental change in a column due to the pH drop was successfully overcome by employing buffer solution. The reaction rate was measured by pH-stat method and was found to follow the simple Michaelis-Menten law at the initial stage of the reaction. The values of the net reaction rate experimentally determined were used to calculate the substrate concentration at the external surface of the catalyst pellet and then to calculate the mass transfer coefficient, k(L), at various flow rates and substrate concentrations. The correlation proposed by Chilton and Colburn represented adequately the experimental data. The linear change of log j(D) at low log N(Re) with negative slope was ascribed to the fact that the external mass transfer approached the state of pure diffusion in the limit of zero superficial velocity.     

Kinetic and operational study of a cross-flow reactor with immobilized pectolytic enzymes.

The kinetics and operational behavior of a nylon membrane derivative with immobilized pectolytic enzymes in a cross-flow reactor were analyzed. A high dependence on the recycling flow rate was observed. A design equation of the system has been proposed by taking into account both the shear stress deactivation and the control of the external diffusional limitations. Integration of the resulting design equation allowed us to study the effect of different operational parameters on substrate conversion. The catalytic efficiency of the immobilized derivative in a cross-flow reactor showed the highest pectin hydrolysis capability when it was compared with two different configurations of packed-bed reactors.     

Deacylation of benzylpenicillin by immobilized penicillin acylase in a continuous four-stage stirred-tank reactor

Immobilized penicillin acylase has been used for the deacylation of benzylpenicillin at 37°C in a continuous reactor consisting of four 1 liter stirred tanks connected in series. There was good agreement between the predicted and actual conversions obtained in each tank under steady-state conditions. The operational stability of the immobilized enzyme in the tanks depended on the pH and the rate of addition and concentration of alkali needed to neutralize the acid produced during the reaction. At pH 7 with the addition of 2M NaOH, the half-life for enzyme stability was greater than 400 hr in all tanks. This was over half the value for the immobilized enzyme when stored at 37°C and pH 7.     

Ceramic membrane microfilter as an immobilized enzyme reactor.

This study investigated the use of a ceramic microfilter as an immobilized enzyme reactor. In this type of reactor, the substrate solution permeates the ceramic membrane and reacts with an enzyme that has been immobilized within its porous interior. The objective of this study was to examine the effect of permeation rate on the observed kinetic parameters for the immobilized enzyme in order to assess possible mass transfer influences or shear effects. Kinetic parameters were found to be independent of flow rate for immobilized penicillinase and lactate dehydrogenase. Therefore, neither mass transfer nor shear effects were observed for enzymes immobilized within the ceramic membrane. Both the residence time and the conversion in the microfilter reactor could be controlled simply by regulating the transmembrane pressure drop. This study suggests that a ceramic microfilter reactor can be a desirable alternative to a packed bed of porous particles, especially when an immobilized enzyme has high activity and a low Michaelis constant.     

Dynamic modeling of immobilized cell reactor: application to ethanol fermentation

A mathematical model has been developed for the unsteady-state operation of an immobilized cell reactor. The substrate solution flows through a mixed-flow reactor in which cells immobilized in gel beads are retained. The substrate diffuses from the external surface of the gel beads to some internal location where reaction occurs. The product diffuses from the gel beads into liquid medium which flows out of the reactor. The model combines simultaneous diffusion and reaction, as well as cell growth, and it can predict how the rates of substrate consumption, product formation, and cell growth vary with time and with initial conditions. Ethanol fermentation was chosen as a representative reaction in the immobilized cell reactor, and numerical calculations were carried out. Excellent agreement was observed between model predictions and experimental data available in the literature.     

Prediction of continuous reactors performance based on batch reactor deactivation kinetics data of immobilized lipase

Experiments on deactivation kinetics of immobilized lipase enzyme fromCandida cylindracea were performed in stirred batch reactor using rice bran oil as the substrate and temperature as the deactivation parameter. The data were fitted in first order deactivation model. The effect of temperature on deactivation rate was represented by Arrhenius equation. Theoretical equations were developed based on pseudo-steady state approximation and Michaelis-Menten rate expression to predict the time course of conversion due to enzyme deactivation and apparent half-life of the immobilized enzyme activity in PFR and CSTR under constant feed rate policy for no diffusion limitation and diffusion limitation of first order. Stability of enzyme in these continuous reactors was predicted and factors affecting the stability were analyzed.     

Production of 6-APA using a recirculated packed bed batch reactor

Summary A recirculated packed bed batch reactor has been designed for the production of 6-aminopenicillanic acid. It was observed that the flow rate of penicillin G solution is a rate limiting step for its hydrolysis. Under the conditions used, the maximum rate of hydrolysis of penicillin G was observed at a flow rate of 3.0 L/min.     

Studies on improved techniques for immobilizing and stabilizing penicillin amidase associated with E. coli cells.

A method for catalyst development has been suggested for immobilizing whole E. coli cells containing penicillin amidase. Conventional methods have limitations, such as permeation of substrate and product through cellular membranes, leaching of protein and other cellular components into the reaction phase, lower specific activity compared to immobilized enzyme system, etc. The whole cell immobilization technique has been optimized for different process parameters. The most suitable conditions for this process were pH, 4.25; cell concentration, 3.75%; concentration of glutaraldehyde, 1.5%; level of bovine serum albumin as additional support, 2 mg ml-1. The reaction was continued for 2 h. The granular catalyst has good mechanical strength, low protein leachability, and high retention of penicillin amidase activity.     

Ca2+ and calmodulin antagonists inhibit the proton gradients associated with non-cyclic and cyclic photophosphorylation in spinach chloroplasts

The synthesis of benzylpenicillin (BP) after mixing phenyl-acetyl-glycine(PAG), 6-aminopenicillanic acid (6-APA) and free or immobilized penicillin amidase (E.C.3.5.1.11.) was studied as a function of pH and ionic strength. Before the final equilibrium was reached a kinetically controlled synthesis of BP was observed. Then a transient maximum concentration in BP much larger than the final equilibrium content was synthesized in the acyl-transfer process. The factors influencing this maximum have been analyzed. Increasing ionic strength markedly decreased the maximum in BP and the rate of deacylation of phenyl-acetyl-penicillin amidase by 6-APA. The change was largest when the enzyme was immobilized in a positively charged support, where at low ionic strength the concentration of 6-APA around the enzyme is larger than the bulk concentration due to the partitioning of charged solutes.     

Properties of penicillin amidase immobilized by copolymerization with acrylamide.

An enzyme preparation in a spherical granule form was obtained by copolymerization of penicillin amidase (EC 3.5.1.11) (previously modified with maleic anhydride) and acrylamide via a crosslinking agent. As compared with the native enzyme, immobilized amidase is more resistant to heating, has a lower affinity to benzylpenicillin, and is less inhibited by phenylacetate. Its substrate specificity and optimum pH remain unchanged.     

Self‐renaturing enzymes: Design of an enzyme‐chaperone chimera as a new approach to enzyme stabilization

Molecular chaperones in aqueous‐organic mixtures can broaden the utility of biocatalysis by stabilizing enzymes in denaturing conditions. We have designed a self‐renaturing enzyme‐chaperone chimera consisting of penicillin amidase and a thermophilic chaperonin that functions in aqueous‐organic mixtures. The flexible linker separating the enzyme and chaperone domains was optimized and the design was extended to incorporate a chitin binding domain to facilitate immobilization of the chimera to a chitin support. The initial specific activity of penicillin amidase was not compromised by the enzyme‐chaperone fusion or by immobilization. The total turnover number of immobilized chimera for amoxicillin synthesis in aqueous‐methanol mixtures was 2.8 times higher after 95 h than the total turnover number of the immobilized penicillin amidase lacking a chaperone domain. Similarly, in 32% methanol the soluble chimera was active for over three times longer than the enzyme alone. This approach could easily be extended to other enzyme systems. Biotechnol. Bioeng. 2009;102: 1316–1322. © 2009 Wiley Periodicals, Inc.     

Enzymes in polyelectrolyte complexes. The effect of phase transition on thermal stability

Penicillin amidase, alpha-chymotrypsin and urease have been immobilized in water-soluble nonstoichiometric polyelectrolyte complexes (N-PEC). N-PEC are formed by modified poly(N-ethyl-4-vinyl-pyridinium bromide) (polycation) and excess poly(methylacrylic acid) (polyanion). N-PEC are a new class of polymers capable, characteristically, of phase transitions solution in equilibrium precipitate induced by slight change in pH or ionic strength. Neither the chemical structure of the carrier nor the number of cross-linkages between an enzyme and a carrier change on phase transition. That gives an unique opportunity to elucidate the difference between enzymes immobilized on water-soluble and water-insoluble supports. A detailed study of the phase transition effect on thermal stability of the enzymes and protein-protein interactions has been carried out. The following effects were found. Pronounced thermal stabilization of penicillin amidase and urease may be achieved on two conditions: the enzyme is in the precipitate; (b) the enzyme is linked to the N-PEC nucleus. Then the thermal stability of N-PEC-bound penicillin amidase increases 7-fold at pH 5.7, 60 degrees C, and 300-fold at pH 3.1, 25 degrees C, compared to the native enzyme. For urease, the thermal stabilization increases 20-fold at pH 5.0, 70 degrees C. The localization of enzyme on N-PEC has been established by titration of alpha-chymotrypsin bound to a polycation or polyanion with basic pancreatic trypsin inhibitor. Both in solution (pH 6.1) and in N-PEC precipitate (pH 5.7), an alpha-chymotrypsin molecule bound to a polyanion is fully exposed to the solution. If the enzyme is bound to a polycation, only 20% of alpha-chymotrypsin molecules in the precipitate and 40% in solution retain their ability for protein-protein interactions. This means that a polycation-bound enzyme is localized in the hydrophobic nucleus of the complex, whereas the polyanion-bound enzyme sits on the hydrophilic shell of the complex. On pH-induced phase transition (pH decreases from 6.1 to 5.7), there occurs a stepwise decrease in penicillin amidase activity which is due to a 9.8-fold increase in the Km for 2-nitro-4-phenylacetamidobenzoic acid. Change of the catalytic activity and thermal stability of N-PEC-bound penicillin amidase is fully reversible and reproducible. Such soluble-insoluble immobilized enzymes with controllable thermal stability and activity may be used for simulating events in vivo and in biotechnology.     

Hydrolysis of particulate tributyrin in a fluidized lipase reactor.

Pancreatic lipase has been immobilized onto stainless steel beads by adsorption followed by crosslinking, and onto polyacrylamide by covalent bonding. The activities of the two types of immobilized enzyme toward the particulate substrate, tributyrin emulsion droplets, were determined experimentally, and rate constants based on Michaelis-Menten kinetics were calculated. The activity of the stainless steel-lipase was determined for various flow conditions and for various support sizes by the use of a differential fluidized bed recycle reactor. The rate constants calculated indicate that the experimental reaction rate is free from mass transfer influences, since the observed Michaelis constant does not vary with the fluidization velocity or with the support particle size. In addition, the Michaelis constant of the stainless steel-lipase was found to be equal to that of the free enzyme, suggesting that adsorption and subsequent crosslinking does not alter the enzyme-substrate affinity. The emulsion substrate mass transfer rates, calculated from the filtration theory, indicate that each substrate particle which contact the immobilized enzyme is hydrolyzed to a significant extent. The experimentally determined kinetic rate constants may be used directly to predict the size of integral fluidized bed reactors.     

Immobilized heparinase: In vitro reactor model

Heparinase immobilized to agarose has previously been shown to be useful in degrading heparin and thereby preventing thromboembolytic complications when this anticoagulant has been used in extracorporeal perfusions. The current study examined the kinetics of this immobilized enzyme. When heparinase is covalently bound to 8% agarose, the partition coefficient of heparin in the catalytic particle is 0.36 +/- 0.048 (N = 10). The immobilized enzyme has a K(m) of 0.15 +/- 0.03 mg/mL and an activation energy of 10.3 +/- 0.57 kcal/gmol (N = 5). These values are statistically indistinguishable from the values for the free enzyme. The immobilized enzyme showed a pH activity optimum between 7.0 and 7.4, compared to the optimum pH of 6.5 for the soluble enzyme. The activity optimum of immobilized heparinase with respect to salt concentration was between 0 and 0.1M. A reactor containing immobilized heparinase recirculating internally at 1300 mL/min behaved as a continuously stirred tank reactor (CSTR) when solutions at a flow rate of 120 mL/min were passed through the device. The residence time distribution was determined using blue dextran (molecular weight 2 x 10(6) daltons), which is sterically excluded from the agarose catalyst. A model of the heparinase reactor based on ideal CSTR behavior and the immobilized enzyme kinetic parameters was developed. It accurately predicted experimental conversions over a range of catalyst volumes, enzyme loadings, and substrate concentrations to within 7% in most cases and with a maximum deviation of 13%.     

Continuous alcohol fermentation in an immobilized cell rotating disk reactor

The increasing interest in alcohol fermentation over these last years because of the energy crisis has been demonstrated by an increase in scientific research. After a brief analysis of the main results of the literature in the field of alcohol fermentation reactors, the use of a new type of immobilized cell reactor [the rotating biological surface (RBS) reactor] was studied. As is well known, the RBS reactor is a form of fixed-film reactor and can be described as a dynamic trickling filter. Our experimental apparatus employed a spongy material to trap the yeast cells on the disks. The results of fermentations carried out in the RBS reactor working in batch, in continuous with cell support, and in continuous without cell support have been presented in order to compare the different productivities and to assess the performance of the RBS immobilized cell reactor. An ethanol productivity of 7.1 g/L h was achieved in the RBS-ICR at a dilution rate of 0.3 h(-1), 2.5 times higher than the maximum productivity obtained in the RBS reactor without support at a lower dilution rate. The adoption of a spongy material as a cell immobilizer, combined with the use of the RBS reactor, enhances the particular advantages of both systems.     

Urea hydrolysis by immobilized urease in a fixed-bed reactor: analysis and kinetic parameter estimation

Urea hydrolysis by urease immobilized onto ion exchange resins in a fixed-bed reactor has been studied. A modified Michaelis-Menten rate expression is used to describe the pH-dependent, substrate- and product-inhibited kinetics. Ionic equilibria of product and buffer species are included to account for pH changes generated by reaction. An isothermal, heterogeneous plug-flow reactor model has been developed. An effectiveness factor is used to describe the reaction-diffusion process within the particle phase. The procedure for covalent immobilization of urease onto macroporous cation exchangers is described. Urea conversion data are used to estimate kinetic parameters by a simplex optimization method. The best-fitted parameters are then used to predict the outlet conversions and pH values for systems with various inlet pH values, inlet urea and ammonia concentrations, buffers, particle sizes, and spacetimes. Very good agreement is obtained between experimental data and model predictions. This immobilized urease system exhibits quite different kinetic behavior from soluble urease because the pH near the enzyme active sites is different from that of the pore fluid. This effect results in a shift of the optimal pH value of the V(max) (pH) curve from 6.6 (soluble urease) to ca. 7.6 in dialysate solution, and ca. pH 8.0 in 20mM phosphate buffer. The reactor model is especially useful for estimating intrinsic kinetic parameters of immobilized enzymes and for designing urea removal columns.     

Production of penicillins and cephalosporins in an immobilized enzyme reactor

Summary Four enzymes required for the biosynthesis of pencillins and cephalosporins by Streptomyces clavuligerus have been immobilized on an anion exchange resin. The capabilities of the system have been studied by circulation of reaction mixtures through the immobilized enzyme reactor. Within 30 min, all of the substrate -(l--aminoadipyl)-l-cysteinyl-d-valine is consumed and converted to a mixture of penicillins and cephalosporins. After 60 min the major antibiotic products are (iso)penicillin N and desacetylcephalosporin C. The activity of the immobilized enzyme reactor activity is stable to storage at temperatures below 4°C but activity is lost on repeated use.     

A novel enzyme reactor using gluten membrane entrapping cell-associated enzyme.

A membrane enzyme reactor consisting of variable pieces of replaceable cell-immobilized membranes was proposed for the continuous production of bioproducts. To demonstrate the characteristics of the reactor, cell-immobilized membranes were prepared by the entrapment of permeabilized recombinant Escherichia coli cells containing penicillin G acylase within the gluten matrices. A stainless-steel net that was created with a mesh frame was used to support each gluten membrane so that the membranes could be filled into the rectangular-shaped reactor. The reactor equipped with either six or 12 pieces of cell-immobilized gluten membranes containing a biomass concentration of 5%, w/w was effective in catalyzing the production of 6-aminopenicillanic acid from penicillin G. In comparison with intact cells, the cell-immobilized preparation was more stable and the half-life time of the immobilized cell-associate enzyme in gluten membrane was estimated to be 36 days by a long-term operation. As the substrate solution was forced to flow through the reactor equipped with six membranes and in the direction perpendicular to the membranes, the pressure drop was determined to be less than 50 cm H(2)O with a flow-rate up to 50 mL/min. This low pressure due to the porous structure of gluten membrane would lead to a lower operational cost. Increasing either the number of membranes or the area of each cell-immobilized membrane can easily do scaling-up of this membrane reactor.     

Biotransformation of acrylonitrile to acrylamide using immobilized whole cells of Brevibacterium CH1 in a recycle fed-batch reactor

Acrylamide was produced from acrylonitrile using immobilized Brevibacterium CH1 cells that were isolated from soil and found to possess nitrile hydratase activity. The reaction conditions and stability of the enzyme activity were studied. The conversion yield was nearly 100%, including a trace amount of acrylic acid. This strain showed strong activity of nitrile hydratase toward acrylonitrile and extremely low activity of amidase toward acrylamide. A packed bed reactor was operated in a fed-batch manner for acrylamide production of high concentration. The acrylonitrile concentration was maintained below 3% and the operating temperature at 4 degrees C to minimize enzyme deactivation.