Mechanism of stereospecific production of L-amino acids from the corresponding 5-substituted hydantoins by Bacillus brevis.

Abstract

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.

N-Carbamyl-L-Amino Acid Amidohydrolase ofPseudomonassp. Strain NS671: Purification and Some Properties of the Enzyme Expressed inEscherichia coli

“…2 ) Among N-carbamyl-L-amino acid amidohydrolases purified partially, 1.9 -12) the enzymes from B. brevis AJ-12299 9 ) and Zvmobacterium oroticum 11 ) required Mn 2 + or Fe 2 + for thei; activities. The N-carbamyl-amino acid amidohydrolase of strain NS671 had a broad specificity for N-carbamyl-L-~-amino acids.…”

Microbial Conversion ofDL-5-Substituted Hydantoins to the CorrespondingL5-Amino Acids byBacillus stearothermophilusNS1122A

“…10) The molecular mass of a denatured hydantoinase was estimated by SOS-polyacrylamide gel electrophoresis by the method of Laemmli.…”

Cloning and sequencing of the genes involved in the conversion of 5-substituted hydantoins to the corresponding L-amino acids from the native plasmid of Pseudomonas sp. strain NS671

“…2, lane 4). To determine the configuration of the N-carbamylmethionine produced from D-5-(2-methylthioethyl)hydantoin, we converted it chemically to methionine by the sodium nitrite method (36), and it was found to be the D-isomer judging from the configuration of this methionine. Thus, E. coli carrying pHPB12 is able to convert D-and L-5-(2-methylthioethyl)hydantoin to N-carbamyl-D-and N-carbamyl-L-methionine, respectively, and to convert N-carbamyl-L-methionine to L-methionine.…”

“…It has been reported that some bacteria belonging to the genera Bacillus (33,35,37), Flavobacterium (22,27), and Arthrobacter (30), etc., can convert racemic 5-substituted hydantoins to the corresponding L-amino acids. The bacterial conversion consists of the following three successive steps (22,30,31,35,36,39). The first step is either spontaneous or the enzymatic racemization of 5-substituted hydantoins.…”