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

Abstract

Abstract.The gene for D-aminopeptidase from Ochrobactrum anthropi SCRC C1-38 was cloned in Escherichia coli JM109. An expression plasmid pC138DP (4.5 kb) was constructed. The amount of the enzyme in a cell-free extract of E. coli JM109/pC138DP was elevated up to 288000 units/liter culture, which is about 3600-fold over that of 0. anthropi SCRC C1-38. 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 HC1. Complete hydrolysis of (R)-alanine amide was achieved in a short time (44 h) from 5.OM racemic alanine amide HCI 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.

Recent Developments in Aminopeptidases, Racemases, and Oxidases

“…In the kinetic resolution of amino acid amides with the use of amidases, such as DAP and DaaA, it is possible to synthesize d-amino acids by kinetic resolution, selectively from racemic acid amides [16]. An Escherichia coli transformant highly expressing DAP catalyzed the synthesis of 2.5 M (about 220 g/l) d-alanine from 5 M racemic alanine amide in a 4.5-h reaction.…”

Hydrolysis of Amides

“…Intact cells as well as cell-free extracts of E. coli expressing this enzyme were used for the resolution of several racemic amino acid amides [269]. Starting from 5 M DL-alanine amide HCl, 2.5 M (220 g l À1 ) D-alanine was obtained in 4.5 h on applying whole cells.…”