Immobilization of aminoacylase by adsorption to tannin immobilized on aminohexyl cellulose.

The immobilization of aminoacylase (N-acylamino acid amidohydrolase, EC 3.5.1.14) was investigated by using tannin immobilized on aminohexyl cellulose. The most active immobilized aminoacylase was obtained when aminoacylase was adsorbed to the immobilized tannin in a weak alkaline medium containing sodium chloride and n-butanol at 37 degrees C. The activity of the immobilized tannin-aminoacylase complex per unit volume was five times higher than that of the DEAE-Sephadex-aminoacylase complex used for industrial production of L-amino acids in our plants. The half-life of the immobilized tannin-aminoacylase complex was 20 days under continuous operation at a high concentration of substrate; on the contrary, that of the DEAE-Sephadex-aminoacylase complex was 0.5 days.     

A Facile Method of Preparing l-α-Methyl DOPA (l-3-(3,4-Dihydroxyphenyl)-2-methylalanine)

To develop a practical method for production of l-α-methyl DOPA, the optical resolution of its precursor, dl-3-(3,4-methylenedioxyphenyl)-2-methylalanine, was studied. The monohydrochloride of dl-3-(3,4-methylenedioxyphenyl)-2-methylalanine was resolved by a preferential crystallization procedure. Optically pure l-α-methyl DOPA was obtained in good yield. Industrial production of l-α-methyl DOPA by the present simple method is considered to be very promising.     

Studies on Amino Acids

The enzymatic resolution of acetyl-dl-methonine previously reported was studied further, in detail. As a result, it was found that metal ion plays an important role on the asymmetric hydrolysis of acyl-dl-methonines by the enzyme preparations of Aspergillus oryzae.     

Studies on Amino Acids. II

The enzymatic procedures for the resolution of dl-lysine such as asymmetric synthesis of acyl l-lysinc anilide and acyl dl-lysines have been studied. As a result, the procedure consisting in the enzymatic asymmetric hydrolysis of ε-benzoyl-α-acctyl-dl-lysine was found to be the most advantageous for the resolution of dl-lysine.     

Studies on Amino Acids. XI.

As a part of the studies intending to clarify biological significance of the presence of acylase, enzymatic activity which hydrolyzes acyl amino acids, its activity in yeast was investigated. As a results, the occurrence in yeast of acylase activity was confirmed for the first time.

In order to study the enzymatic properties of this acylase activity, experiments were carried out with the enzyme preparation from brewer’s yeast. As a result of the investigation, yeast acylase was found to be able to hydrolyze a number of acyl amino acids, of these chloroacetyl derivatives especially readily, as in the case of previously studied acylase activity in other sources such as mold acylase. Several observations on the influence of metal ions and inhibitors, optical specificity and others were also presented.     

Continuous production of NADP by immobilized Achromobacter aceris cells.

Several microorganisms having higher nicotinamide adenine dinucleotide kinase (NAD kinase, EC 2.7.1.23) activity were immobilized into polyacrylamide gel lattices. The enzyme activity field by immobilization was highest in Achromobacter aceris AKU 0120. By the incubation of the immobilized A. aceris cells at pH 4.0, the NAD kinase activity increased and the adenosine triphosphate (ATP)-degradation activity disappeared completely. Enzymatic properties of the immobilized A. aceris cells were investigated and compared with those of intact cells. The optimal pH and the optimal temperature of immobilized cells were the same as those of intact cells. Immobilized cell NAD kinase was more stable than that of intact cells. The operational half-life of immobilized cells was 20 days when the substrate solution was passed through a column packed with immobilized cells at a flow rate which gives a space velocity (SV) of 0.1 hr-1 at 37 degrees C. On the other hand, the half-life of the intact cells was only 6 hr.     

L-Glutamine formation by Flavobacterium rigense.

A penicillin-resistant mutant of Flavobacterium rigense designated as strain 703, FERM-P no. 3628, was obtained after ultraviolet treatment of F. rigense FERM-P no. 3556. The parent strain produces 0-2-hydroxypropylhomoserine from 1,2-propanediol. The mutant was found to be a good producer of L-glutamine. The physiological characteristics of strain 703 were different from the general group of L-glutamic acid-producing bacteria. Strain 703 required L-tryptophan and thiamine but not biotin for its growth. L-Glutamine formation on a specific basis, however, was independent of L-tryptophan and thiamine. Biotin and penicillin were also not effective. Only ammonium fumarate acted as an effective factor on L-glutamine formation. Accumulation of L-glutamine by strain 703 was 10 mg/ml at 30 degrees C for 48 h in a chemically defined medium containing 3% diammonium fumarate.     

Production of L-serine by Sarcina albida.

Conditions for the production of microbial L-serine hydroxymethyltransferase and for the conversion of glycine to L-serine were studied. A number of microorganisms were screened for their abilities to form and accululate L-serine from glycine, and Sarcina albida was selected as the best organism. Enzyme activity in this organism as high as 0.12 U/ml could be produced in shaken cultures at 30 degrees C in a medium containing glucose, ammonium sulfate, glycine, yeast extract, and inorganic salts. L-Serine was produced most efficiently by shaking cells at 30 degrees C in a reaction mixture containing 20% glycine, 5 X 10(-3) M formaldehyde, and 3 X 10(-4) M pyridoxal phosphate in yields of 22 mg of broth in 5 days. L-Serine was easily isolated in 84% yields by ion-exchange resin.     

Preparation and characteristics of microcapsules containing asparaginase

Conditions for the preparation of microcapsules containing asparaginase by interfacial polymerization were investigated. The activity of microcapsules prepared under the optimal conditions was about 37% compared with that of native asparaginase. Particle size of microcapsules could be controlled by determining the stirring rate and concentration of Span 85. The membranes of microcapsules were resistant to mechanical shock or attack of chymotrypsin, and no leakage of asparaginase from microcapsules was observed.