Repeated Batch Reactions Research Articles

Immobilization of thermotolerant N-carbamyl- d-amino acid amidohydrolase

N-Carbamyl- d-amino acid amidohydrolase (DCase), in which amino acid residues were substituted by mutation, followed by the selection based on thermotolerance, showed improved thermostability, by 5° or 10°C, compared to the native DCase. These DCases were immobilized on a macroporous phenol formaldehyde resin, Duolite A-568, and the immobilized thermotolerant enzymes showed higher activity than the immobilized native DCase. From the results of repeated batch reactions, the half-lives of the activities of immobilized thermotolerant DCase, in which Leu was substituted for Pro 203, and immobilized native DCase were 104 and 58 times, respectively. It was revealed that the higher thermotolerance enabled the immobilized enzymes to be more stable in reactions. A reductant, dithiothreitol, also stabilized the enzymes in reactions. Compared with soluble DCase, immobilized DCase was somewhat stable, and its activity was optimum at a lower pH.

Bioconversion using immobilized recombinant flocculent yeast cells carrying a fused enzyme gene in an `intelligent' bioreactor

Immobilized recombinant cells of the flocculent yeast Saccharomyces diastaticus carrying an expression plasmid for a fused enzyme between rat cytochrome P4501A1 and yeast NADPH-cytochrome P450 reductase were used in the bioconversion reaction from acetanilide (AA) to p-acetaminophene ( p-AAP). Immobilization of the strain within reticulated polyurethane foam biomass support particles (BSPs) was effected passively in situ in a fluidized-bed bioreactor using `draw and fill' operation. In repeated batch reactions both the final product concentration and the production rate were notably improved compared with the results obtained using freely suspended cells without BSPs. Cells immobilized within BSPs exhibited a significantly high level of expression of the fused enzyme. In addition, a high proportion of plasmid-carrying cells was maintained among the immobilized cells, in contrast to a much lower proportion among freely suspended cells released from the BSPs. Since the bioreactor became packed with highly expressing cells immobilized within BSPs as a consequence of spontaneous screening, it was termed an `intelligent' bioreactor, and is believed to offer significant potential for the further development of efficient production processes.

Stabilization of Nocardia EH1 epoxide hydrolase by immobilization

A partially purified epoxide hydrolase from Nocardia EH1 was stabilized by immobilization through ionic binding onto DEAE-cellulose. This biocatalyst showed more than twice the activity (225 %) of that of the free enzyme albeit at a marginal reduction in enantioselectivity. The addition of the nonionic detergent Triton X-100 during the immobilization further enhanced the stability as indicated by a dramatic shift in the temperature optimum from 35 to 45°C. The stabilized immobilized biocatalyst could be successfully employed in repeated batch reactions (residual activity of 55% after five cycles), which was not the case for whole cell reactions (residual activity ≤10 %). © Rapid Science Ltd. 1998

Immobilization of N-carbamyl-D-amino acid amidohydrolase.

N-Carbamyl-D-amino acid amidohydrolase (DCase), produced with recombinant Escherichia coli cells using a cloned gene from Agrobacterium sp. strain KNK712, has been immobilized for use in the production of D-amino acids. The porous polymers, Duolite A-568 and Chitopearl 3003, were much better than other resins for the activity and stability of the adsorbed enzyme. The activity of DCase expressed on Duolite A-568 and Chitopearl 3003 amounted to 96 units/g-wet-resin and 91 units/g-wet-resin, respectively. DCase immobilized on Duolite A-568 was found to be most stable at about pH 7, and it was further stabilized by reductants such as dithiothreitol, L-cysteine, cysteamine, and sodium hydrosulfite. The stability during the repeated batch reactions was greatly improved when dithiothreitol was in the reaction mixture, and the higher crosslinking degree with glutaraldehyde also stabilized the immobilized enzyme. After 14 times repeated reactions, the remaining activity of the immobilized enzyme cross-linked with 0.1% and 0.2% of glutaraldehyde, and 0.2% of glutaraldehyde with dithiothreitol in the reaction mixture was 12%, 18%, and 63%, respectively. DCase produced with Pseudomonas sp. strain KNK003A and Pseudomonas sp. strain KNK505, which are thermotolerant soil bacteria, and that with Agrobacterium sp. strain KNK712 were also immobilized on Duolite A-568. The stability of the enzymes of thermotolerant bacteria during reactions was superior to that of Agrobacterium sp. strain KNK712, though the activity was lower than that of strain KNK712.

Enzymatic synthesis of estolides by a bioreactor

AbstractAn attempt was made to synthesize ricinoleic acid estolides effectively with a bioreactor system containing immobilized lipase. The optimal water content for reaction changed during synthesis of estolide from ricinoleic acid. The water content needs to be controlled to optimize conditions for each reaction stage and to prepare estolide with a high degree of condensation. The estolide synthesis was attempted to confirm if the reaction would have proceeded to the targeted degree of condensation by controlling the reaction mixture under the optimal water content. The result showed that, with the bioreactor, the reaction could proceed rapidly. A repeated batch reaction was possible in the bioreactor. The amount of lipase used can thus be highly reduced compared with discarding it each time. The loading density of enzyme onto the carrier greatly affected enzymatic activity, with a loading level of 60 mg lipase/g carrier producing 60% more estolide per gram of enzyme than a loading level of 120 mg/g. The estolide product synthesized in the bioreactor showed no coloration during the reaction process. This fact confirmed the validity of the proposed method.

Immobilization of HLADH on polymer materials for reduction of cyclohexanone with NADH regeneration under two-phase conditions

Horse liver alcohol dehydrogenase (HLADH) was effectively immobilized by adsorption to poly(vinyl alcohol) (PVA), cross-linked polyacrylamide (PAA), or cross-linked chitosan beads (CP). The activity of the immobilized HLADH was estimated from the initial rate of the reduction of cyclohexanone coupled with NADH regeneration via oxidation of alcohols under aqueous/ n-heptane two-phase conditions. Both the activity and the specificity for alcohols of PAA-immobilized HLADH were almost the same as those of free HLADH. The activity of HLADH decreased upon immobilization on PVA or CP, but the specificity for alcohols was not altered markedly. For all the immobilized HLADH's, the apparent activity was dependent on water content and the amount of support material. More than 2.5% of water content in reaction solution was required for activation of the enzyme, and large amounts of support material led to a decrease in the reaction rate. The activity of immobilized HLADH decreased during repeated batch reactions, and the residual activity after 15 12-h reactions was 25–55% depending on the type of support material.

Enzymatic hydrolysis of the horn and hoof of cow and buffalo

With the aim of efficiently utilizing livestock waste as a protein resource, we studied their enzymatic hydrolysis. Particles of a mixture of horn and hoof from cow and buffalo with a diameter of less than 250 μm were selected for use as substrate, and a proteinase from Bacillus subtilis, selected from among various commercial proteolytic enzymes was used. For enzymatic hydrolysis of the substrate, heat treatment was absolutely necessary prior to digestion and the conditions for the enzymatic reaction were determined to be as follows: reaction time, 30 to 60 min; pH, 8.3; temperature, 50°C; weight ratio of substrate to enzyme, 1 : 0.05; and concentration of substrate, 62.5 g/ l. Even when a membrane reactor was used, the amount of enzyme required could not be reduced. However, repeated-batch reaction allowed us to reduce the added amount of enzyme. The enzyme was added only for the first round of the reaction in four rounds of repeated-batch reaction. As a result of this process, the weight ratio of substrate to the enzyme was reduced to 1 : 0.023 from 1 : 0.05 at a final substrate concentration of 61 g/ l.

Transphosphatidylation reaction of phosphatidylcholine to 4-methoxyphenol in water-immiscible organic solvents with immobilized phospholipase D

Immobilized phospholipase D (PLD) from Streptomyces sp. catalyzed the transfer reaction of the dipalmitoylphosphatidyl residue from 1,2-dipalmitoyl-3- sn-phosphatidylcholine (DPPC) to an aromatic hydroxy group on 4-methoxyphenol in water-immiscible organic solvents, to afford 1,2-dipalmitoyl-3- sn-phosphatidyl-4-methoxyphenol (DPP-PMP) with a 45% yield, accompanied by a trace amount of 1,2-dipalmitoyl-3- sn-phosphatidic acid sodium salt (DPPA-Na). The formation of DPP-PMP was affected by organic solvents used in the reaction. Benzene, toluene, and methylene chloride gave DPP-PMP with moderate yields but use of diethyl ether resulted in a low yield of DPP-PMP. In both ethyl acetate and water-miscible organic solvents, the transfer reaction did not take place. Immobilization of PLD was carried out by adding a 1 % volume of PLD solution to a suspension of a cation-exchange resin (Amberlite IRC-50, 5% w/v) in benzene with stirring and sonication. In a repeated batch reaction for DPP-PMP synthesis with immobilized PLD, after ten batch cycles the enzyme retained 74% of its initial activity.

Lipase-catalyzed alcoholysis of triglycerides for the preparation of 2-monoglycerides

Rhizopus arrhizus lipase immobilized on celite was used to prepare isomerically pure 2-monoglycerides by alcoholysis of triglycerides in organic media. Reaction parameters such as choice of solvent, choice of alcohol, and alcohol concentration were studied. When 12.5 mM tripalmitin was used as substrate, methyl- tert-butyl ether was the best solvent for alcoholysis at water activity 0.11. Ethanol gave the highest yield (97%) at an optimal ethanol concentration of 200–300 mM. At higher alcohol concentrations, the enzyme activity was substantially lowered. The enzyme preparation showed high stability in repeated-batch reactions.

Sorbitol production in charged membrane bioreactor with coenzyme regeneration system: II. Theoretical analysis of a continuous reaction with retained and regenerated NADPH

A theoretical model was constructed in order to study charged membrane bioreactors (CMBRs). In this model, it was postulated that a native nicotinamide coenzyme NADP(H) can be partially retained by a charged membrane in continuous operation. A multienzyme system composed of NADPH-dependent aldose reductase (AR) and glucose dehydrogenase (GDH) was used for the production of sorbitol and gluconic acid from glucose and for the conjugated enzymatic regeneration of NADP(H). Both enzymes were studied with respect to their reaction kinetics. AR was determined to obey the Theorell-Chance mechanism. GDH reaction was approximated by the initial velocity equation of the sequential Bi-Bi mechanism since the reverse reaction could be neglected. Significant inhibitions of both enzymes by sorbitol, gluconic acid, and glucose were observed, and the mode of inhibition was estimated to modify the velocity equations. The differential equation system for each component was derived and numerically analyzed according to the model. The theoretical model elucidated several features of the CMBR. (1) When compared at the same productivity, higher retainment was found to bring about a higher coenzyme turnover number, indicating that the feed coenzyme concentration can be reduced. (2) Under constant conversion, a contradictory relationship between turnover number and residence time arises if the feed concentration of a coenzyme varies. The theoretical model predicts that there is a practically optimal concentration for using NADP(H) efficiently. This concentration was consistent with that yielding the estimated minimum total cost. (3) In this system, excess-GDH-to-AR activity was required because of differences in their kinetic constants. The amount of regeneration enzyme required can be reduced by the accumulation of excels NADPH due to coenzyme retainment. (4) Comparison with an ideal repeated batch reaction revealed that the continuously operated CMBR was vastly superior with respect to productivity as well as operation ability.

Biological preparation of an optically activ alcohol. Part III. Enantioselective hydrolysis of .ALPHA.-cyano-3-phenoxybenzyl acetate with Arthrobacter lipase.

Lipase-catalyzed enantioselective hydrolysis of the acetic ester of racemic α-cyano-3-phenoxybenzyl alcohol (CPBA) was examined to prepare (S)-CPBA. Most of the lipases tested hydrolyzed (S)-CPBA acetate preferentially, while Candida cylindracea lipase favored (R)-CPBA acetate. Enantioselective hydrolysis by Arthrobacter lipase gave the optically pure (S)-CPBA in the reaction mixture of pH 4.0. The kinetic studies showed that (R)-CPBA acetate reacted as a competitive inhibitor. The Arthrobacter lipase solution in the water/oil biphasic reaction system could be used repeatedly. The lipase immobilized to resins had insufficient activity or low operational stability for the repeated batch reaction. The unhydrolyzed (R)-CPBA acetate was racemized by heating with triethylamine and could be reused as the substrate of the enzymatic hydrolysis. A chemico-enzymatic process for the preparation of (S)-CPBA was developed based on these studies.

Utilization of powdered pig bone as a support for immobilization of lipase

Pig bone was examined for its suitability as a support material for lipase immobilization. It was observed that pig bone (PB) particles dispersed readily in both polar and nonpolar solvents, and lipase was easily adsorbed. In particular lipase adsorbed on olive oil-soaked pig bone (OPB) particles exhibited a higher hydrolytic activity than that in lipase adsorbed on a selection of other representative supports, regardless of removing the presoaked olive oil from the particles after immobilization of lipase. The optimum pH and temperature for hydrolytic activity of OPB-adsorbed lipase were the same as those for free lipase, although thermal resistance was increased by immobilization. When OPB-adsorbed lipase was used for repeated batch reactions of olive oil hydrolysis, an activity of more than 80% of the initial activity of each run could he retained after 46 h reaction. The results suggest that PB is an excellent support material.

Isolation of various hyperactive mutants of phenylalanine ammonia-lyase containing yeasts

Various strains of phenylalanine ammonia-lyase (PAL) containing yeasts were mutagenised using ultraviolet (UV) irradiation. Two of the yeast, SPA 1 and SPA 17, produced UV kill curves with a distinct shoulder reflecting their diploid nature. Of the analogues used, p-fluoro-D,L-phenylalanine and β-2-thienyl-D,L-alanine selected the greatest frequency of mutants with the highest PAL activities: one such mutant, FP10M6, exhibited five times the PAL activity of the parent SPA 10. Mutants constitutive for PAL synthesis could not be isolated using any selection regime, including resistance to relatively high concentrations of 2-deoxyglucose. Three of the hyperactive PAL mutants examined were not impaired for growth and their PAL-induction profiles were not different from the respective parents. Inactivation of PAL shortly after peak synthesis was found with all mutants examined, although this was not extensively investigated. Of the 21 strains mutagenised, mutants from three of the wild-type yeasts were found to exhibit moderate growth at 37 °C, while growth of the parent strains was greatly impaired. The latter temperature-resistant mutants exhibited a twofold increase in PAL activity as compared with the parent strains at either 30 or 37 °C. Under biotransformation reaction conditions, several of the mutants were capable of producing more than 15 g L-phenylalanine∙L−1. Although the stability of the PAL catalyst varied markedly among different mutants, some of the most productive mutants did exhibit good reuseability under sequentially repeated batch reaction conditions.

Immobilization of enzymes masks their active site.

We studied the effect of immobilizing cellulase to carboxycellulose sodium by radiation polymerization on the masking of the active site of the enzyme. Masking of the enzyme during the preparation of immobilized enzyme was assayed at low temperature. The activity of immobilized enzyme was retained during repeated batch reactions, indicating that the enzyme was firmly trapped in the polymer matrix. Various compounds (designated monomers) were used to dissolve the carboxymethylcellulose; enzyme activity was affected by the nature of the monomer, by the monomer concentration, and by the solubility of the substrate in monomer.

Effect of growth phase on stability of fumarase activity of Brevibacterium flavum cells immobilized with .KAPPA.-carrageenan.

Brevibacterium flavum cells obtained from different growth phases were immobilized with κ-carrageenan and the stability of the fumarase activity was investigated. The stability of fumarase activity of the immobilized preparation of cells of the stationary growth phase was highest. The highest stability of the immobilized cells seemed to be correlated to the high stability of fumarase activity in free cells of the stationary phase. High rigidity of the cell wall and membrane of B. flavum cells of the stationary phase and firm binding of fumarase protein to the cell membrane were suggested from several lines of evidence obtained on treatment of the cells with lysozyme and detergents or sonication of the cells. Electronmicrographs showed that the cells of the stationary phase retained the original shape after repeated batch reactions. Solubilized fumarase prepared from cells of the stationary phase showed the highest stability. Experiments using the partially purified enzyme strongly suggested the existence ...

9?-Hydroxylation of 4-androstene-3,17-dione by gel-entrapped Corynebacterium sp. cells

Whole cells of Corynebacterium sp. having steroid 9α-hydroxylation system were immobilized by entrapment with photo-crosslinkable resin prepolymers, urethane prepolymers or several kinds of polysaccharides. Of various entrapment methods tested, cells entrapped in photo-crosslinked gels showed the highest activity to hydroxylate 4-androstene-3,17-dione at 9α-position. The properties of the photo-crosslinkable resin prepolymers, such as the hydrophobicity and the chain length of the prepolymers, affected markedly the activity of the entrapped cells. Addition of dimethyl sulfoxide to a buffer system at 15 vol. % was effective to solubilize the product, 9α-hydroxy-4-androstene-3,17-dione, and gave the highest yield. In an aqueous system, the activity of hydrophilic gel-entrapped cells was higher than that of hydrophobic gel-entrapped cells. In 15% of dimethyl sulfoxide, the hydrophobic gel-entrapped cells showed almost the same activity as the hydrophilic gel-entrapped cells probably due to extraction of the product from gels to the external solvent before its metabolic degradation. Entrapment significantly enhanced the conversion ratio at high substrate concentrations and the operational stability of the 9α-hydroxylation system in the cells. In the presence of nutrients in the reaction mixture, entrapped growing cells maintained fully the original activity at least during 10 times of repeated batch reactions for 5 days.