Recombinant production and characterization of an N-Acyl-D-amino acid amidohydrolase from Streptomyces sp. 64E6.

N-Acyl-D-amino acid amidohydrolases (D-aminoacylases) are often used as tools for the optical resolution of D-amino acids, which are important products with applications in industries related to medicine and cosmetics. For this study, genes encoding D-aminoacylase were cloned from the genomes of Streptomyces spp. using sequence-based screening. They were expressed by Escherichia coli and Streptomyces lividans. Almost all of the cell-free extracts exhibit hydrolytic activity toward N-acetyl-(Ac-)D-Phe (0.05-6.32 μmol min(-1) mg(-1)) under conditions without CoCl2. Addition of 1 mM CoCl2 enhanced their activity. Among them, the highest activity was observed from cell-free extracts prepared from S. lividans that possess the D-aminoacylase gene of Streptomyces sp. 64E6 (specific activities were, respectively, 7.34 and 9.31 μmol min(-1) mg(-1) for N-Ac-D-Phe and N-Ac-D-Met hydrolysis). Furthermore, when using glycerol as a carbon source for cultivation, the recombinant enzyme from Streptomyces sp. 64E6 was produced in 4.2-fold greater quantities by S. lividans than when using glucose. D-Aminoacylase from Streptomyces sp. 64E6 showed optimum at pH 8.0-9.0. It was stable at pH 5.5-9.0 up to 30 °C. The enzyme hydrolyzed various N-acetyl-D-amino acids that have hydrophobic side chains. In addition, the activity toward N-chloroacetyl-D-Phe was 2.1-fold higher than that toward N-Ac-D-Phe, indicating that the structure of N-acylated portion of substrate altered the activity.