title: Recombinant production and characterization of an N-Acyl-D-amino acid amidohydrolase from Streptomyces sp. 64E6.
abstract: 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.
authors.affiliation: Department of Agricultural, Biological, and Environmental Sciences, Faculty of Agriculture, Tottori University, Tottori 680-8553, Japan. arima@muses.tottori-u.ac.jp
authors.forename: Jiro
authors.initials: J
authors.lastname: Arima
authors.forename: Yoshitaka
authors.initials: Y
authors.lastname: Isoda
authors.forename: Tadashi
authors.initials: T
authors.lastname: Hatanaka
authors.forename: Nobuhiro
authors.initials: N
authors.lastname: Mori
mesh_terms: D000581:Amidohydrolases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D004795:Enzyme Stability; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D016415:Sequence Alignment; D013302:Streptomyces / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D048170:Streptomyces lividans / Q000235:genetics / Q000378:metabolism
chemical_list: D000596:Amino Acids; D001426:Bacterial Proteins; D000581:Amidohydrolases