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

Abstract

A multi-enzyme system composed of glutamate racemase, thermostable D- amino acid aminotransferase, glutamate dehydrogenase and formate dehydrogenase was employed for the production of aromatic D-amino acids, D- phenylalanine and D-tyrosine, from the corresponding α-keto acids, phenylpyruvate and hydroxyphenylpyruvate, respectively. The optimal concentration of ammonium formate for the production of these D-amino acids was found in the range of 0.25-1.0 M. The optimal concentration of α-keto acid was determined to be 50 mM, above which the productivity greatly decreased. To keep the concentration of α-keto acid around this concentration, α-keto acid was intermittently fed into the multi-enzyme system during the production period. By running the multi-enzyme system for 35 h, 48 gl-1 of D-phenylalanine and 60 gl-1 of D-tyrosine were produced with 100% of optical purity from the equimolar amounts of phenylpyruvate and hydroxyphenylpyruvate, respectively. The production levels of both aromatic D-amino acids were demonstrated to be dependent on the stability of glutamate racemase.ope