Plasmid‐based, D‐aminopeptidase‐catalysed synthesis of (R)‐amino acids

The gene for D-aminopeptidase from Ochrobactrum anthropi SCRC Cl-38 was cloned in Escherichia coli JM109. An expression plasmid pCl38DP (4.5 kb) was constructed. The amount of the enzyme in a cell-free extract of E. coli JM109/pCl38DP was elevated up to 288000 units/liter culture, which is about 3600-fold over that of O. anthropi SCRC Cl-38. It was calculated that the enzyme comprised about 30% of the total extractable cellular protein. The intact cells of the E. coli transformant were used as a catalyst for (R)-stereospecific hydrolysis of several racemic amino amides HCl. Complete hydrolysis of (R)-alanine amide was achieved in a short time (4½h) from 5.0M racemic alanine amide HCl using cells of the E. coli transformant. The concentration of (R)-alanine reached 220 g/l. The cells or the cell-free extract catalyzed the synthesis of (R)-2-aminobutyric acid, (R)-methionine, (R)-norvaline and (R)-norleucine from their amides in a similar manner.