Cloning, expression and characterization of D-aminoacylase from Achromobacter xylosoxidans subsp. denitrificans ATCC 15173

摘要

D-Aminoacylase catalyzes the conversion of N-acyl-D-amino acids to D-amino acids and fatty acids. The aim of this study was to identify the D-aminoacylase gene from Achromobacter xylosoxidans subsp. denitrificans ATCC 15173 and investigate the biochemical characterization of the enzyme. A previously uncharacterized D-aminoacylase gene (ADdan) from this organism was cloned and sequenced. The open reading frame (ORF) of ADdan was 1467 bp in size encoding a 488-amino acid polypeptide. ADdan, with a highamino acid similarity to N-acyl-D-aspartate amidohydrolase from Alcaligenes A6. showed relatively low sequence similarities to other characterized D-aminoacylases. The recombinant ADdan protein was expressed in Escherichia coli BL21 (DE3) using pET-28awith a T7 promoter. The enzyme was purified in a single chromatographic step using nickel affinity gel column. The molecular mass of the expressed protein, calculated by SDS-PAGE, was about 52 kDa. The purified ADdan showed optimal activity at pH 8.0 and50 C, and was stable at pH 6.0-8.0 and up to 45 C. Its activity was inhibited by Cu~(2+), Fe~(2+), Ca~(2+) Mn~(2+), Ni~(2+), Zn~(2+) and Hg~(2+), whereas Mg~(2+) had no significant influence on this recombinant D-aminoacylase. This is the first report on the characterization of D-aminoacylase with activity towards both N-acyl derivatives of neutral D-amino acids and N-acyl-D-aspartate. The characteristics of ADdan could prove to be of interest in industrial production of D-amino acids.