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

A novel enzyme, N-acylamino acid racemase (NAAR) which catalyzes the interc onversion of the enantiomers of N-acylamino acid, but does not act on amino acids, has been found in the actinomycetes Streptomyces atrutus Y-53 and A mycolatopsis sp. TS-1-60, isolated from soil. These strains also produced L - and D-aminoacylases simultaneously. Furthermore, another 13 strains of ac tinomycetes with NAAR activity were observed from the type culture collecti on of the Institute for Fermentation. Osaka (IFO). Thermostable N-acylamino acid racemase From Amycolatopsis sp. TS-1-60, a ra re actinomycete strain selected for its ability to grow on agar plates incu bated at 40 degreesC, was purified to homogeneity and characterized. The en zyme was stable at 55 degreesC 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 additi on, this enzyme also catalyzed the racemization of the dipeptide L-alanyl-L -methionine. The optically active amino acids, N-alkyl-amino acids and ethy l ester derivatives of N-acetyl-D and L-methionine, however, were not racem ized. Enzyme activity was markedly enhanced by the addition of divalent met al ions such as Co2+, Mn2+ and Fe2+ and was inhibited by the addition of ED TA and PCMB. The NAAR gene from Amycolatopsis sp. TS-1-60, consists of an open reading f rame of 1104 nucleotides. which specifies a 368-amino acid protein with a m olecular weight of 39,411. No significant sequence homology was found betwe en 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 a mino acid sequences of mandelate racemase and NAAR showed that NAAR has par tial 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 expre ssion plasmid was 1100-fold more than that produced by Amycolatopsis sp. TS -1-60. Bioreactors for the production of optically active amino acids were constru cted 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-me thionine by these bioreactors. (C) 2001 Elsevier Science B.V. All rights re served.