D-and L-amino acids were produced from L-and D-amino acid amides by D-aminopeptidase from Ochrobactrum anthropi C1-38 and L-amino acid amidase from Pseudomonas azotoformans IAM 1603, respectively, in the presence of ␣-amino--caprolactam racemase from Achromobacter obae as the catalyst by dynamic kinetic resolution of amino acid amides.Several dynamic kinetic enzymatic resolutions of synthetic substrates have been developed for the industrial production of chiral amino acids. The use of L-␣-amino-ε-caprolactam (ACL)-hydrolase in the presence of ACL racemase is a typical example of L-lysine production (1, 2, 12). L-cysteine is produced by hydrolases acting on amino-⌬ 2 -thiazoline-4-carboxylic acid in the presence of a racemase (23). Used in combination with D-specific hydantoinase (22,26), either with spontaneous racemization of hydantoin or with hydantoin racemase (21,24), N-carbamoyl-D-amino acid amidohydrolase catalyzes the hydrolysis of N-carbamoyl-D-amino acids to form D-amino acids (3,(16)(17)(18)(19)(20). N-acyl-D-amino acid amidohydrolase (27) and N-acyl amino acid racemase (25) are utilized together to form D-amino acids. Fig. 1 compares several dynamic kinetic enzymatic resolutions of synthetic substrates. The optically active amino acids can be synthesized in three steps with amidases from aldehydes via aminonitrile and amino acid amide, while the D-amino acylase route requires four steps via aminonitrile, amino acid, and N-acyl amino acid.We isolated and characterized several D-stereospecific amino acid amidases, such as D-aminopeptidase (DAP), Damino acid amidase, alkaline D-peptidase, and R-amidase, and utilized them in the kinetic resolution of amino acid amides (4-9, 14). L-Amino acid amide hydrolases such as LaaA and LaaA Bd have been characterized previously (15).In this report, the effect of a high concentration of the substrate on the dynamic kinetic resolution of amino acid amide, together with the optimum pH, was investigated using DAP or LaaA in the presence of ACL racemase (10, 11).ACL racemase was purified from Escherichia coli JM109/ pACL60, and its activity was assayed as described previously (10). The expression level of ACL racemase was about 300 U/liter culture (5% of the total soluble protein). The amount of inclusion body was not measured. DAP expressed in E. coli JM109 was purified by a similar procedure, and its activity was assayed as described previously (8). About 8,000 U/liter of DAP could be produced maximally in the cell extract (about 6% of the total soluble protein). LaaA activity toward L-alanine amide was determined by measuring the amount of Lalanine formed. The reaction mixture (1.0 ml) contained 0.1 M potassium phosphate buffer (KPB; pH 7.0), 0.1 M L-alanine amide, and an appropriate amount of LaaA. The reaction was performed at 30°C for 10 min and stopped by adding 200 l 2 N HClO 4 . The amount of L-alanine formed was determined by the method used for D-ACL, and the enzyme activity was measured by the same method as described previously (10). Recombinant E. coli JM1...