Production of aromatic D-amino acids from alpha-keto acids and ammonia by coupling of four enzyme reactions

A multi-enzyme system composed of glutamate racemase, thermostable D-amino acid aminotransferase, glutamate dehydrogenase and formate dehydrogenase wa s employed for the production of aromatic D-amino acids, D-phenylalanine an d D-tyrosine, from the corresponding alpha-keto acids, phenylpyruvate and h ydroxyphenylpyruvate, respectively. The optimal concentration of ammonium f ormate for the production of these D-amino acids was found in the range of 0.25-1.0 M. The optimal concentration of alpha-keto acid was determined to be 50 mM, above which the productivity greatly decreased. To keep the conce ntration of alpha-keto acid around this concentration, alpha-keto acid was intermittently fed into the multi-enzyme system during the production perio d. By running the multi-enzyme system for 35 h, 48 gl(-1) of D-phenylalanin e and 60 gl(-1) of D-tyrosine were produced with 100% of optical purity fro m the equimolar amounts of phenylpyruvate and hydroxyphenylpyruvate, respec tively. The production levels of both aromatic D-amino acids were demonstra ted to be dependent on the stability of glutamate racemase. (C) 1999 Elsevi er Science B.V. All rights reserved.