Optimization of a reactor assembly for the production of 6-APA from penicillin-V

The design and operation of an industrial penicillin-V deacylation reactor is simulated, using a kinetic expression and mass transport parameters for the immobilized enzyme particles which were determined experimentally in a previous study. It is desirable to use a series of equalsized plug flow reactors with pH control at the entrance to each reactor, and with a possibility of recycling reactant in each reactor. These measures are necessary to avoid a steep pH profile through the reactor; the deacylation reaction is accompanied by an increase of acidity of the reaction medium, and H(+) is a strong inhibitor and may deactivate the enzyme. The optimization study which is carried out at a fixed penicillin conversion of x = 0.99 shows that it is uneconomical to use penicillin feed concentrations above 150mM-175mM, and that the buffer concentration in the reaction medium should not be less than 50mM-75mM. Increasing the number of reactors from 4 to 8 or 10 leads to higher productivity of 6-APA, and a moderate recycle in the first couple of reactors diminishes the sharp decrease in pH which will be found in a straight plug flow reactor operation of the equipment. Higher pumping costs and lower productivity are unavoidable drawbacks of an operation mode where the separation costs for the product mixture are desired to be low.     

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.     

Effect of pH on the production of lipolyzed butter oil by a calf pregastric esterase immobilized in a hollow-fiber reactor: I. uniresponse kinetics

A calf pregastric esterase immobilized in a hollow-fiber reactor was employed to hydrolyze milkfat, thereby producing a lipolyzed butteroil. The reaction kinetics can be modeled by a two-parameter model of the general Michaelis-Menten form based on a ping-pong bi-bi mechanism; the rate of enzyme deactivation can be modeled as a first-order reaction. The initial concentration of accessible glyceride bonds, [G](O), was estimated by complete saponification of the substrate butteroil as 2400 mM. An extra sum of squares test indicated that not only the parameters of the kinetic generalized Michaelis-Menten model, but also the deactivation-rate constant varied significantly with pH. The optimum pH, for lypolysis is near 6.0 at a temperature of 40 degrees C because at this pH the rate of deactivation of the esterase is minimized.     

Hydrolysis of Butteroil by Immobilized Lipase Using a Hollow-Fiber Reactor: Part VI. Multiresponse Analyses of Temperature and pH Effects

A lipase from Aspergillus niger, immobilized by physical adsorption on hydrophobic hollow fibers made of microporous polypropylene, was used to effect the hydrolysis of the glycerides of melted butterfat at 40, 50, 55, and 60°C (pH 7.0), and at pH 3.0, 4.0, 5.0, 7.0, 8.0, and 9.0 (40°C). McIlvane buffer and melted butterfat were pumped cocurrently through the hollow fiber reactor. The concentrations of ten different free fatty acids in the effluent oil stream were measured by HPLC. Multiresponse nonlinear regression methods were employed to fit the data to multisubstrate rate expressions derived from a Ping Pong Bi Bi mechanism in which the rate controlling step is deacylation of the enzyme. Thermal deactivation of the immobilized lipase was also included in the mathematical model of reactor performance. A postulated normal distribution of v_max with respect to the number of carbon atoms of the fatty acid residue (with an additive correction for the number of double bonds) was found to provide the best statistical fit of the data. The models developed can be used to independently predict the effects of either the pH or the temperature, as well as the reactor space time and the time elapsed after immobilization, on the free fatty acid profile of the lipolyzed butteroil product.     

A process for bioconversion of cephalosporin C by Rhodotorula gracilis D-amino acid oxidase

Summary A process for the production (in a stirred tank reactor) of glutaryl-7-ACA from cephalosporin C using immobilized D-amino acid oxidase is described. Results so obtained under optimal conditions (1.2 mg coupled enzyme/L, pH 8.5, 2 mM cephalosporin C) point to a system which shows high conversion efficiency and a remarkable operational stability. No exogenous H₂O₂ is requested to shift the reaction equilibrium toward glutaryl-7-ACA production, nor any side product is detected. The immobilized system productivity was 54 g/day/mg of enzyme. This process represents the first reported case of a reactor successfully developed with a DAAO for bioconversion of cephalosporin C.     

Deacylation of benzylpenicillin by immobilized penicillin acylase

Immobilized penicillin acylase preparations have much higher activities per unit volume than immobilized cell preparations. Many parameters of the deacylation reaction are dependent on pH and both reactant and one of the products, 6-aminopenicillanic acid, are acid and alkali labile. Acid is produced as result of the deacylation reaction and must be neutralised. The influence of these pH effects on the design of the catalyst and the reactor is discussed.     

A comparative study of immobilized amyloglucosidase in a packed bed reactor and a continuous feed stirred tank reactor

The catalytic activity of amyloglucosidase covalently attached to DEAE-cellulose was studied in a packed bed reactor and a continuous feed stirred tank reactor (CSTR) for the reaction maltose → glucose. At low flow rates mass-transfer limitations in the bed reactor lead to lower conversions for this reactor compared to the CSTR. Simple theoretical expressions for these reactors were compared with the experimental results. There are significant differences between the kinetic parameters and pH profile of the immobilized and free enzyme. The immobilized enzyme also showed greater stability at 50°C than did free amyloglucosidase. The temperature dependence of the reaction rate was the same for immobilized and free enzyme.     

Hydrolysis of butteroil by immobilized lipase using a hollow-fiber reactor: IV. Effects of temperature

A lipase from Aspergillus niger, immobilized by adsorption on microporous polypropylene hollow fibers, was used to effect the hydrolysis of the glycerides of melted butterfat at pH. 7.0 at 40, 50, 55, and 60 degrees C. Mcllvane buffer was pumped upward through the lumen, and melted butterfat was pumped upward through the shell side of a hollow fiber reactor. Nonlinear regression methods were employed to determine the kinetic parameters of models based on combinations of three nested rate expressions for the hydrolysis reaction with three nested rate expressions for thermal deactivation of the enzyme. A rate expression containing four lumped parameters is sufficient to model the release of free fatty acids as a function of reactor space time and time elapsed after immobilization. Nonlinear regression methods were also employed in global fits of the data to rate expressions containing an explicit dependence on temperature. For the reaction conditions used in this research, a 14-parameter rate expression is necessary to accurately model the overall release of free fatty acids as a continuous function of the absolute temperature, initial substrate concentrations, reactor space time, and time elapsed after immobilization of the lipase.     

Efficient production of S-(+)-2-chlorophenylglycine by immobilized penicillin G acylase in a recirculating packed bed reactor

(S)-(+)-2-Chlorophenylglycine 1 is an important intermediate in the synthesis of Clopidogrel. A recirculating packed bed reactor (RPBR) was constructed for efficient production of (S)-1 by kinetic resolution of racemic N-phenylacetyl-2- chlorophenylglycine 2 using immobilized penicillin G acylase (PGA). The immobilized PGA exhibited maximum activity at 50 °C and pH 8.0 with (R,S)-2 as substrate. The kinetic constants (K_m and v_max) of immobilized PGA were calculated to be 20.61 mM and 83.2 mM/min/g, respectively. The substrate displayed inhibitory effect on immobilized PGA with inhibition constant of 221.23 mM. The immobilized PGA showed a strict enantiospecificity for substrate at different temperature, pH and substrate concentration examined. The performance and productivity of RPBR were evaluated by several critical parameters, including immobilized PGA load, substrate feeding rate, height to diameter ratio and so on. The kinetic resolution process shows higher initial reaction rate and conversion by recycling 100 mL of substrate solution (80 mM) through RPBRs packed with 6.0 g immobilized PGA with a feeding rate of 1.5 mL/min while the H/D ratio was 4.0. The immobilized PGA-catalyzed kinetic resolution of (R,S)-2 was successfully operated in the RPBR for 60 batches, with an average productivity of 1.2 g/L/h for (S)-1 in high optical purity (>97% enantiomeric excess) in semi-continuous operation. The residual (R)-2 can be easily racemized and then used as substrate.     

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.     

Activity and storage stability of immobilized glucose oxidase onto magnesium silicate

Glucose oxidase (GOD) was covalently immobilized onto florisil (magnesium silicate) carrier via glutaraldehyde. Immobilization conditions were optimized: the amount of initial GOD per grams of carrier as 5 mg, pH as 5.5, immobilization time as 120 min and temperature as 10 °C. Under the optimized reaction conditions activities of free and immobilized GOD were measured. Free and immobilized GOD samples were characterized with their kinetic parameters, and thermal and storage stabilities. K_M and V_max values were 68.2 mM and 435 U mg GOD⁻¹ for free and 259 mM and 217 U mg GOD⁻¹ for immobilized enzymes, respectively. Operational stability of the immobilized enzyme was also determined by using a stirred batch type column reactor. Immobilized GOD was retained 40% of its initial activity after 50 reuses. Storage stabilities of the immobilized GOD samples stored in the mediums with different relative humidity in the range of 0–100% were investigated during 2 months. The highest storage stability was determined for the samples stored in the medium of 60% relative humidity. Increased relative humidity from 0% to 60% caused increased storage stability of immobilized GODs, however, further increase in relative humidity from 80% to 100% caused a significant decrease in storage stability of samples.     

Use of a lipase immobilized in a membrane reactor to hydrolyze the glycerides of butteroil

A lipase from A spergillus niger, immobilized by adsorption on a microporous, polypropylene flat-sheet membrane, was used to effect the continous hydrolysis of the glycerides of melted butterfat at 35°C. For the reaction conditions used in this research, a pseudo-zero order rate expression can be used to model the kinetics of the overall hydrolysis of butterfat. Multiresponse nonlinear regression methods were employed to determine the kinetic parameters of a multisubstrate rate expression derived fro ma mechanism based on the general Michaëlis–Menten approach. For the multiresponse data taken at pH 7.0, the dependence of the maximum rate of release of each fatty acid residue of butterfat on its carbon chain length is accurately described by a skewed, bell-shaped (or Γ-type) distribution. Data taken at five different pH values were fit assuming a Dixon–Webb diprotic model for the pH dependence of the reaction rate. The thermal deactivation of the immobilized lipase obeyed first-order kinetics with a half-life of 19.9 days at 35°C. The multisubstrate model is useful for the prediction of the free fatty acid profile of lipolyzed butterfat, whereas the lumped-substrate model provides an estimate of the overall degree of hydrolysis as a function of the reactor space time.     

Hydrolysis of butteroil by immobilized lipase using a hollow-fiber reactor: part II. Uniresponse kinetic studies

A lipase from Aspergillus niger immobilized by adsorption on microporous, polypropylene hollow fibers was used to effect the hydrolysis of the glycerides of melted butterfat at 40 degrees C and pH 7.0. Mcllvane buffer was pumped through the lumen and melted butterfat was pumped courrently through the shell side of a shell-and-tube reactor. Nonlinear regression methods were employed to determine the kinetic parameters of three nested rate expressions derived from a Ping Pong Bi Bi enzymatic mechanism coupled with three nested rate expressions for the thermal deactivation of the enzyme. For the reaction conditions used in this research, a four-parameter rate expression (which includes a two-parameter deactivation rate expression and a two-parameter hydrolysis rate expression) is sufficient to model the overall release of free fatty acids from the triglycerides of butterfat as a function of space time and time elapsed after immobilization. At a space time of 3.7 h immediately after immobilization of lipase, 50% of the fatty acid residues esterified in the sn-1,3 positions of the triglycerides can be released in the hollow-fiber reactor.     

R-mandelic acid production with immobilized recombinant Escherichia coli cells in a recirculating packed bed reactor

A recirculating packed bed reactor (RPBR) was used for efficient production of R-mandelic acid (R-MA) by kinetic resolution of racemic R,S-mandelonitrile (R,S-MN) using the recombinant E. coli cells crosslinked with diatomite (DA)/glutaraldehyde (GA)/polyethyleneimine (PEI). The performance and productivity of RPBR were evaluated by several parameters, including cell load, substrate feeding rate, height diameter (H/D) ratio, reactor structures, and operation stability. The kinetic resolution process showed higher initial reaction rate (1.52?mM/min) and yield (100%) by recycling 100?mL of substrate solution (70?mM) through RPBR packed with 6.0?g immobilized cells at a substrate-feeding rate of 19?mL/min while the H/D ratio was 2.8. The immobilized cells were successfully applied into kinetic resolution of R,S-MN in the RPBR for 50 batches with an average productivity of 4.12?g/L/h for R-MA with >99% of enantiomeric excess.     

Kinetic behavior of immobilized Penicillin acylase

Penicillin acylase has been immobilized to carboxymethylcellulose and to the resin Amberlite XAD7. The reaction kinetics of the enzyme were affected by both intrinsic (molecular) and microenvironmental effects. The Michaelis constant for the enzyme increased after immobilization as a result of an intrinsic effect of the reagent, glutaraldehyde, used for enzyme immobilization. Microenvironmental effects were of two types: diffusional limitation of access of substrate and a reaction-generated pH depression in the support particles. This depression of internal pH was observed in all the preparations and could be reduced by addition of pH buffering salts to reactor. An adsorbed pH-indicating dyc was used to determine the surface and internal pH of particles of XAD7–penicillin acylase under various reaction conditions. The extent of diffusional rate limitation in XAD7–penicillin acylase was related to the penetration depth of protein into the porous support particles. The penetration depth of protein and thus the diffusional limitation of the reaction rate could be controlled by the conditions of preparation of the immobilized enzyme. A staining technique was used to observe the location of the protein.     

Kinetic evaluation of biotransformation of benzaldehyde to L-phenylacetylcarbinol by immobilized pyruvate decarboxylase from Candida utilis

Biotransformation of benzaldehyde to L-phenylacetylcarbinol (L-PAC) as a key intermediate for L-ephedrine has been evaluated using immobilized pyruvate decarboxylase (PDC) from Candida utilis. PDC immobilized in spherical polyacrylamide beads was found to have a longer half-life compared with free enzyme. In a batch process, the immobilized PDC generally produced lower L-PAC than free enzyme at the same concentrations of substrates due to increased by-products acetaldehyde and acetoin and reduced benzaldehyde uptake. With immobilized PDC, L-PAC formation occurred at higher benzaldehyde concentrations (up to 300 mM) with the highest L-PAC concentration being 181 mM (27.1 g/L). For a continuous process, when 50 mM benzaldehyde and 100 mM sodium pyruvate were fed into a packed-bed reactor at 4 degrees C and pH 6.5, a productivity of 3.7 mM/h (0.56 g/L . h) L-PAC was obtained at an average concentration of 30 mM (4.5 g/L). The half-life of immobilized PDC reactor was 32 days. (c) 1996 John Wiley & Sons, Inc.     

Bioconversion of D-galactose to D-tagatose: continuous packed bed reaction with an immobilized thermostable L-arabinose isomerase and efficient purification by selective microbial degradation

The continuous enzymatic conversion of D-galactose to D-tagatose with an immobilized thermostable L-arabinose isomerase in packed-bed reactor and a novel method for D-tagatose purification were studied. L-arabinose isomerase from Thermoanaerobacter mathranii (TMAI) was recombinantly overexpressed and immobilized in calcium alginate. The effects of pH and temperature on D-tagatose production reaction catalyzed by free and immobilized TMAI were investigated. The optimal condition for free enzyme was pH 8.0, 60°C, 5 mM MnCl(2). However, that for immobilized enzyme was pH 7.5, 75°C, 5 mM MnCl(2). In addition, the catalytic activity of immobilized enzyme at high temperature and low pH was significantly improved compared with free enzyme. The optimum reaction yield with immobilized TMAI increased by four percentage points to 43.9% compared with that of free TMAI. The highest productivity of 10 g/L h was achieved with the yield of 23.3%. Continuous production was performed at 70°C; after 168 h, the reaction yield was still above 30%. The resultant syrup was then incubated with Saccharomyces cerevisiae L1 cells. The selective degradation of D-galactose was achieved, obtaining D-tagatose with the purity above 95%. The established production and separation methods further potentiate the industrial production of D-tagatose via bioconversion and biopurification processes.     

Revisiting the effect of water content on enzymatic peptide synthesis in non-aqueous medium

Enzymatic acyl-transfer reaction in organic medium competes with the hydrolytic side reaction depending on the water content. The effect of water content on aminolysis activity of -chymotrypsin for the synthesis of Bz-Tyr-Val-NH₂ in acetonitrile was examined under the conditions which were devoid of the hydrolytic deacylation. Excess H-Val-NH₂ (880 mM) was employed to keep the hydrolysis negligible. The aminolysis rate increased abruptly between 4 and 5% (v/v) water but a further increase in the water content did not affect the reaction rate. This suggests that water added more than 5% (v/v) does not enhance intrinsic enzyme activity but acts only as a nucleophile for the hydrolytic deacylation.     

Immobilization of Escherichia coli Cells Containing Aspartase Activity with Polyurethane and Its Application for l-Aspartic Acid Production

Whole cells of Escherichia coli containing aspartase activity were immobilized by mixing a cell suspension with a liquid isocyanate-capped polyurethane prepolymer (Hypol). The immobilized cell preparation was used to convert ammonium fumarate to l-aspartic acid. Properties of the immobilized E. coli cells containing aspartase were investigated with a batch reactor. A 1.67-fold increase in the l-aspartic acid production rate was observed at 37 degrees C as compared to 25 degrees C operating temperature. The pH optimum was broad, ranging from 8.5 to 9.2. Increasing the concentration of ammonium fumarate to 1.5 M from 1.0 M negatively affected the reaction rate. l-Aspartic acid was produced at an average rate of 2.18 x 10 mol/min per g (wet weight) of immobilized E. coli cells with a 37 degrees C substrate solution consisting of 1.0 M ammonium fumarate with 1 mM Mg (pH 9.0).