Kazuhiro Nakanishi

On the specific resistance of cakes of microorganisms

The mean specific resistance of the cakes of various microorganisms was evaluated by measurement of either a change in the amount of permeate with time or of steady-state flux under constant pressure. The mean specific resistance was different with different shapes and sizes of microorganisms. The large differences arose from different packing structures of the cake. The effect of a filter aid on the filtration rate and cake structure was studied experimentally and theoretically. The effects of a filter aid were best explained by a series model, in which a cake layer composed of microbial cells and a layer of randomly distributed microbial cells and filter aid are packed on the membrane surface in series with respect to the directions of permeation.

Properties of immobilized β-d-galactosidase from Bacillus circulans

Partially purified β-d-galactosidase (β-d-galactoside galactohydrolase, EC 3.2.1.23) from Bacillus circulans showed high activity towards both pure lactose and lactose in skim milk, and a better thermal stability than the enzyme from yeast or Escherichia coli. During the course of hydrolysis of lactose catalysed by the enzyme, considerable amounts of oligosaccharides were produced. β-d-Galactosidase from B. circulans was immobilized onto Duolite ES-762, Dowex MWA-1 and sintered alumina by adsorption with glutaraldehyde treatment. The highest activity for hydrolysis of lactose was obtained with immobilization onto Duolite ES-762. During a continuous hydrolysis of lactose, the immobilized enzyme was reversibly inactivated, probably due to oligosaccharides accumulating in the gel. The inactivation was reduced when a continuous reaction was operated at a high percent conversion of lactose in a continuous stirred tank reactor (CSTR). The half-life of the immobilized enzyme was estimated to be 50 and 15 days at 50 and 55°C, respectively, when the reaction was carried out in a CSTR with a percent conversion of lactose >70%.

Continuous production of galacto-oligosaccharides from lactose using immobilized β-galactosidase from Bacillus circulans

Summaryβ-Galactosidase-2 (β-d-galactoside galactohydrolase, EC 3.2.1.23) from Bacillus circulans was purified using hydroxyapatite gel chromatography and immobilized onto Duolite ES-762 (phenolformaldehyde resin) and Merckogel (controlled pore silica gel) for continuous production of galacto-oligosaccharides using lactose as the substrate. The maximum amount of ologosaccharides produced by the immobilized enzyme was 35–40% of the total sugar during hydrolysis of 4.56% lactose. Partially purified β-galactosidase from B. circulans was also immobilized onto various supports for the same purpose. The stability of the immobilized β-galactosidase-2 or partially purified enzyme during a continuous reaction depended on their supports and specific activity. Of the supports tested, Merckogel was best for operational stability. With this support, the enzyme was quite stable with specific activity up to 15 units/g of wet gel; it was reversibly inactivated with more.

Long-term continuous synthesis of aspartame precursor in a column reactor with an immobilized thermolysin

SummaryN-(Benzyloxycarbonyl)-l-asparty-l-phenylalanine methyl ester, the precursor of the synthetic sweetener aspartame, was continuously synthesized in an immobilized thermolysin plug-flow type reactor at 25° C with the substrates (N-benzyloxycarbonyl-l-aspartic acid and l-phenylalanine methyl ester) dissolved in ethyl acetate. The immobilized enzyme was quite stable in ethyl acetate containing 2.5% 0.01 M 2-(N-morpholino)ethanesulphonic acid-NaOH buffer, pH 6.0, and 20 mM CaCl2 with or without the substrate at 25° C. By periodically washing the column, we could conduct a continuous reaction for over 500 h with an average yield of 95% and a space velocity of 1.85 h −1.

Enzymatic synthesis of the precursor of Leu-enkephalin in water-immiscible organic solvent systems

The precursor of Leu-enkephalin, Z-L-TyrGlyGly-L-Phe-L-LeuOEt, was synthesized from amino acid derivatives with three proteinases without the protection of the side chain of L-Tyr. First, Z-GlyGlyOBut and Z-L-TyrGlyGlyOBut were synthesized in quite a high yield, 83% and 99%, in an aqueous/organic biphasic system by papain and alpha-chymotrypsin, respectively. Then, Z-L-Phe-L-LeuOEt was synthesized by thermolysin from Z-L-Phe and L-LeuOEt either in buffer or in a biphasic system; the yields were 95% and 100%, respectively. The synthesis of Z-L-TyrGlyGly-L-Phe-L-LeuOEt from Z-L-TyrGlyGly and L-Phe-L-LeuOEt was performed effectively by thermolysin immobilized on Amberlite XAD-7 in a buffer and in an aqueous/organic biphasic system, as well as in saturated ethyl acetate, while the yield was low in reactions by free thermolysin. In the reaction with the immobilized enzyme (IME) in saturated ethyl acetate, the maximum yield of the precursor of Leu-enkephalin was 68%. The reasons for effective synthesis with IME are: (1) higher concentration of L-Phe-L-LeuOEt inside support, which resulted in rising the rate of the synthesis reaction and protecting the competitive hydrolysis of Z-L-TyrGlyGly by thermolysin, (2) entrapment of the product inside the support where thermolysin could not act in the case of reaction in buffer, and (3) extraction of the product with the organic solvent in the case of reaction in a biphasic system or in saturated organic solvent.

Repeated batch and continuous syntheses of N-(benzyloxycarbonyl)-l-phenylalanyl-l-phenylalanine methyl ester with immobilized thermolysin

SummaryN-(Benzyloxycarbonyl)-l-phenylalanyl-l-phenylalanine methyl ester was synthesized from N-(benzyloxycarbonyl)-l-phenylalanine and l-phenylalanine methyl ester in an aqueous solution (aqueous phasic reaction), in an aqueous/organic biphasic system (biphasic reaction), and in an organic solvent (organic phasic reaction) with immobilized thermolysin. In the aqueous phasic reaction with thermolysin immobilized on Amberlite XAD-7, the whole product was trapped inside the support; extraction with ethyl acetate was needed to recover the product, and the equilibrium yield was low (about 65%). With the biphasic and organic phasic reactions with ethyl acetate as an organic solvent, the yield was around 95%. Because of the high yield and feasibility of operation, repeated batch and continuous reactions were done in the biphasic and organic phasic systems, respectively. The half-lives of the activity for the immobilized enzyme used in the biphasic system at 40°C by repeated batch operation and in a plug flow reactor fed with substrate dissolved in ethyl acetate at 40°C and 30°C were estimated to be about 200 h (67 batches), 420 h, and 1100 h, respectively.

Effect of glutaraldehyde on oligosaccharide production by β-galactosidase from Bacillus circulans

SummaryThe oligosaccharide-producing activity of β-galactosidase-1, one of the isomers of β-galactosidase (β-d-galactoside galactohydrolase, EC 3.2.1.23) from Bacillus circulans was changed after immobilization onto porous silica gel (Merckogel) by crosslinkage with glutaraldehyde. The reason for this modification was studied by treating the free enzyme with glutaraldehyde. Glutaraldehyde of 0.025% to 3% modified 40% to 90% of the free amino groups with or without intermolecular crosslinking. The maximum yield of oligosaccharides increased from 12% to 40% depending upon degree of modification, while native enzyme gave only 6% trisaccharides during hydrolysis of 127 mM lactose. The Km value for the enzyme treated with glutaraldehyde was also increased.

Mechanism for reversible inactivation of immobilized β-galactosidase from Bacillus circulans during continuous production of galacto-oligosaccharides

The specific activity-dependent stability of the immobilized β-galactosidase-2 (β-d-galactoside galactohydrolase, EC 3.2.1.23) from Bacillus circulans during the continuous production of galactooligosaccharides from lactose was studied. This was done by measuring the elution pattern of saccharides from the various immobilized Merckogel (controlled pore silica gel) columns and the amount of saccharides remaining in the gel. It was suggested that oligosaccharides produced were trapped inside the three dimensional enzyme aggregate with the immobilized enzyme having a specific activity of 240 units/g of wet gel, causing gradual inactivation, while the immobilized enzyme with 15 units/g of wet gel was stable since the oligosaccharides were not accumulated.

Long term continuous ATP regeneration by enzymes of the alcohol fermentation pathway and kinases of yeast

SummaryContinuous ATP regeneration from adenosine using enzymes of the alcohol fermentation pathway, adenosine kinase and adenylate kinase of bakers yeast was investigated using a reactor equipped with a semipermeable membrane. The addition of DTT, which protected the thiol groups of some enzymes against oxidation, increased the duration of the period of ATP formation from 42 h (previously the longest period) to 100 h. With the addition of a yeast extract containing intermediates of alcohol fermentation, a stable steady state was attained in which a yield of more than 75% ATP continued for 2 weeks. These results suggest that the long term continuous ATP formation attained might be due to the protection and stabilization of enzymes by the yeast extract which was added to prevent inactivation.

Diffusion of Saccharides and Amino Acids in Cross-linked Polymers

Diffusion coefficients of saccharides and amino acids were measured in cross-linked polymers such as dextran gels, polyacrylamide gels and photo-crosslinkable resins of different gel concentrations or various degrees of crosslinkage. Comparison of diffusion coefficients in gels with those in polymer solutions indicated that the diffusional velocity in gels was restricted not only by the interaction between diffusing substances and gel components but also by the steric hindrance of gel matrix. The diffusion coefficients in dextran gels and polyacrylamide gels were appreciably lower than those in polymer solutions, and the degree of lowering depended on both the gel concentration and the size of diffusing substances. The photo-crosslinkable resins basically composed of polyethylene glycol showed a higher permeability than dextran and polyacrylamide gels on account of a weak interaction between diffusing substances and resin component. Furthermore, an attempt was made to correlate the decrease of diffusion c...