Discovery and application of a new enzyme N-acylamino acid racemase

Abstract

A novel enzyme, N-acylamino acid racemase (NAAR) which catalyzes the interconversion of the enantiomers of N-acylamino acid, but does not act on amino acids, has been found in the actinomycetes Streptomyces atratus Y-53 and Amycolatopsis sp. TS-1-60, isolated from soil. These strains also produced l- and d-aminoacylases simultaneously. Furthermore, another 13 strains of actinomycetes with NAAR activity were observed from the type culture collection of the Institute for Fermentation, Osaka (IFO). Thermostable N-acylamino acid racemase from Amycolatopsis sp. TS-1-60, a rare actinomycete strain selected for its ability to grow on agar plates incubated at 40°C, was purified to homogeneity and characterized. The enzyme was stable at 55°C for 30 min and catalyzed the racemization of optically active N-acylamino acids such as N-acetyl d- or l-methionine, N-acetyl- l-valine, N-acetyl- l-tyrosine and N-chloroacetyl- l-valine. In addition, this enzyme also catalyzed the racemization of the dipeptide l-alanyl- l-methionine. The optically active amino acids, N-alkyl-amino acids and ethyl ester derivatives of N-acetyl- d and l-methionine, however, were not racemized. Enzyme activity was markedly enhanced by the addition of divalent metal ions such as Co 2+, Mn 2+ and Fe 2+ and was inhibited by the addition of EDTA and PCMB. The NAAR gene from Amycolatopsis sp. TS-1-60, consists of an open reading frame of 1104 nucleotides, which specifies a 368-amino acid protein with a molecular weight of 39,411. No significant sequence homology was found between the DNA sequence or the deduced amino acid sequence of NAAR and those of known racemases and epimerases in data bases. However, comparison of the amino acid sequences of mandelate racemase and NAAR showed that NAAR has partial homology with the catalytic and metal ion binding sites of that enzyme. The amount of NAAR produced by an E. coli transformant hosting a T7 expression plasmid was 1100-fold more than that produced by Amycolatopsis sp. TS-1-60. Bioreactors for the production of optically active amino acids were constructed with DEAE Toyopearl-immobilized NAAR and d- or l-aminoacylase. d- or l-Methionine was continuously produced with a high yield from N-acetyl dl-methionine by these bioreactors.