l-Asparaginase from Proteus vulgaris was purified by the following steps: cell lysis by lysozyme and toluene, pH treatment, ammonium sulfate fractionation, Sephadex G-100 gel filtration, DEAE-Sephadex chromatography, and crystallization by the addition of ammonium sulfate. This procedure yields the crystalline enzyme with a 30% recovery of the activity in crude extracts. The crystalline enzyme appears to be homogeneous, as judged by ultracentrifugation, disc electrophoresis, and isoelectric focusing with carrier ampholytes. Isoelectric point is 5.08. Specific activity of this crystalline enzyme is 300 IU/mg. The crystali.-Xi. sparaginase (L-asparagine amidohydrolase, EC 3.5.
To produce an immunologically and enzymologically new type of L-asparaginase, 108 strains of bacteria were screened for enzyme production. As a result, 13 bacteria belonging to the genera Alcaligenes, Bacterium, and Proteus were found to produce L-asparaginases in high levels. Among these L-asparaginases, partially purified L-asparaginases from B. cadaveris and P. vulgaris showed antitumor activity. A partially purified L-asparaginase preparation of P. vulgaris did not react with the antibody of Escherichia coli L-asparaginase on the Ouchterlony agar plate. Culture conditions for the production of L-asparaginase by P. vulgaris were investigated in detail. The enzyme was produced in high yields when cells were grown aerobically in a medium containing sodium fumarate and corn steep liquor. The addition of glucose or ammonium ion to the medium, however, resulted in depressed production of L-asparaginase. Under the optimum conditions, 3,700 international units of L-asparaginase was obtained from 1 liter of culture medium.
Immobilization of n-amino acid oxidase was investigated by covalently binding the enzyme to cyanogen bromide activated polysaccharides. Among polysaccharides tested, Sepharose 6B was found to be the best carrier. Some enzymatic properties of the immobilized enzyme were investigated and compared with those of the native enzyme. The optimum pH of the immobilized enzyme was shifted by 0.5pH units to the acid side in comparison with that of the native enzyme. With regard to substrate specificity, heat stability and effect of temperature, no significant differences were observed between the immobilized and native enzymes. The immobilized enzyme was conveniently used for a determination of D-amino acids and an analysis of optical purity of L-amino acids.
To establish an advantageous method for the production of l -amino acids, microbial isomerization of d - and dl -amino acids to l -amino acids was studied. Screening experiments on a number of microorganisms showed that cell suspensions of Pseudomonas fluorescens and P. miyamizu were capable of isomerizing d - and dl -phenylalanines to l -phenylalanine. Various conditions suitable for isomerization by these organisms were investigated. Cells grown in a medium containing d -phenylalanine showed highest isomerization activity, and almost completely converted d - or dl -phenylalanine into l -phenylalanine within 24 to 48 hr of incubation. Enzymatic studies on this isomerizing system suggested that the isomerization of d - or dl -phenylalanine is not catalyzed by a single enzyme, “amino acid isomerase,” but the conversion proceeds by a two step system as follows: d -pheylalanine is oxidized to phenylpyruvic acid by d -amino acid oxidase, and the acid is converted to l -phenylalanine by transamination or reductive amination.
To produce an immunologically and enzymologically new type of l -asparaginase, 108 strains of bacteria were screened for enzyme production. As a result, 13 bacteria belonging to the genera Alcaligenes, Bacterium , and Proteus were found to produce l -asparaginases in high levels. Among these l -asparaginases, partially purified l -asparaginases from B. cadaveris and P. vulgaris showed antitumor activity. A partially purified l -asparaginase preparation of P. vulgaris did not react with the antibody of Escherichia coli l -asparaginase on the Ouchterlony agar plate. Culture conditions for the production of l -asparaginase by P. vulgaris were investigated in detail. The enzyme was produced in high yields when cells were grown aerobically in a medium containing sodium fumarate and corn steep liquor. The addition of glucose or ammonium ion to the medium, however, resulted in depressed production of l -asparaginase. Under the optimum conditions, 3,700 international units of l -asparaginase was obtained from 1 liter of culture medium.
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