Microbial Conversion of DL-5-Substituted Hydantoins to the Corresponding L-Amino Acids by Bacillus stearothermophilus NS1122A

Abstract

A moderate thermophilic bacterium that converts DL-5-(2-methylthioethyl)hydantoin stereospecifically to L-methionine was isolated from soil and designated NS1122A. The bacterial strain NS1122A was identified as Bacillus stearothermophilus. The optimal temperature and pH of the reaction with the resting cells were 60-70°C and around 8, respectively. The addition of Co²⁺ or Mn²⁺ stimulated the production of L-methionine, while Cu²⁺ and Zn²⁺ strongly inhibited it. Under adequate conditions, 7. 6 mg/ml of L-methionine was produced from 10 mg/ml of DL-5-(2-methylthioethyl)hydantoin with a molar yield of 89%. The enzymes responsible for the conversion were purified from the cells of B. stearothermophilus NS1122A. The purified hydantoinase was homogeneous by the criterion of SDS-polyacrylamide gel electrophoresis. The molecular mass of the native enzyme was approximately 200 kDa by gel filtration. The hydantoinase was capable of hydrolyzing L- and D-5-substituted hydantoins, and the D-form of 5-substituted hydantoins were more effectively hydrolyzed than the L-form. The partially purified N-carbamyl-amino acid amidohydrolase showed a broad substrate specificity with strict L-stereospecificities. The N-carbamyl-L-amino acid amidohydrolase required Co²⁺ (0. 5 mM), Mn²⁺ (0. 5 mM), or Ni²⁺ (5. 0 mM) ion for the activity.