Cloning of the N-acylamino acid racemase gene from Amycolatopsis azurea and biochemical characterization of the gene product

A gene encoding N-acylamino acid racemase (NAAAR) from Amycolatopsis azurea CCRC13413 was cloned. An analysis of its sequence revealed an open reading frame encoding 368 amino acid residues. The deduced amino acid sequence of NAAAR showed an 89% identity with that of Amycolatopsis sp. TS-1–60. The N-acylamino acid racemase gene (aaar) was subcloned into expression vector pET-17b, and was transformed into E. coli BL21 (DE3). A 41-kDa protein band was present on the SDS-PAGE gel from the intracellular soluble part of E. coli. The recombinant NAAAR produced in E. coli was purified by Toyopearl DEAE-650M and Sephacryl S-200 h chromatography. The molecular weight of NAAAR, determined by gel-filtration chromatography, was 320 kDa, indicating that NAAAR was composed of eight identical subunits. NAAAR had maximal activity at 40°C and pH 7.4. The enzyme activity was enhanced notably by Co2+ and Mn2+. Substrate specificity revealed that N-acetyl-D-methionine, N-acetyl- L-methionine, N-acetyl-D-tyrosine, N-acetyl-L-valine, and N-chloroacetyl -D-phenylalanine were the effective substrates. For the substrate N- acetyl-D-methionine, Km was 27.8 mM with a Vmax of 0.07 μmol−1 min−1 and a kcat/Km of 311.4 M−1 s−1. Our data revealed that this N-acylamino acid racemase has a broad substrate range. In addition, we procured the distribution of sequence-conserved regions from known NAAARs and putative NAAARs in this study.