Our stock cultures were screened for microorganisms that can produce galacto-oligosaccharide (Gal-OS) from lactose. Of the 574 strains of bacteria and yeasts tested, Sterigmatomyces elviae CBS8119, Rhodotorula minuta IFO879, and Sirobasidium magnum CBS6803 were found to be efficient producers of Gal-OS from lactose and S. elviae CBS8119 was selected as a representative, high-level producing strain. With toluenetreated resting S. elviae CBS8119 cells, 135 mg of Gal-OS per ml was produced from 360-mg/ml lactose. During this reaction, the by-product glucose was found to inhibit Gal-OS production. Therefore, in order to remove the glucose from the reaction mixture, a culture method in which cell growth followed the enzymatic reaction was devised, which increased the yield of Gal-OS considerably because of the consumption of glucose for cell growth. Under such conditions, 232 mg of Gal-OS per ml was produced from 360-mg/ml lactose after incubation at 30؇C for 60 h. The structure of the major product was identified as O--D-galactopyranosyl-(134)-O--D-galactopyranosyl-(134)-D-glucopyranose (4-galactosyl-lactose) by 13 C nuclear magnetic resonance spectroscopy.
The mechanism of stereospecific production of L-amino acids from the corresponding 5substituted hydantoins by Bacillus brevis AJ-12299 was studied. The enzymes involved in the reaction were partially purified by DEAE-Toyopearl 650M column chromatography and their properties were investigated. The conversion of DL-5-substituted hydantoins to the corresponding L-amino acids consisted of the following two successive reactions. The first step was the ring-opening hydrolysis to N-carbamoyl amino acids catalyzed by an ATP dependent L-5-substituted hydantoin hydrolase. This reaction was stereospecific and the A^-carbamoyl amino acid produced was exclusively the L-forrri. N-Carbamoyl-L-amino acid was also produced from the D-form of 5substituted hydantoin, which suggests that spontaneous racemization occurred in the reaction mixture. In the second step, N-carbamoyl-L-amino acid was hydrolyzed to L-amino acid by an Ncarbamoyl-L-amino acid hydrolase, which was also an L-specific enzyme. The ATPdependency of the L-5-substituted hydantoin hydrolase was supposed to be the limiting factor in the production of L-amino acids from the corresponding 5-substituted hydantoins by this bacterium.
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