Cloning, expression and characterization of D-aminoacylase from Achromobacter xylosoxidans subsp. denitrificans ATCC 15173.
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 high amino 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-28a with 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 and 50°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.
School of Life Science & Technology, China Pharmaceutical University, No. 24, Tong Jia Xiang, Nanjing 210009, PR China.
Wei
W
Wang
School of Life Science & Technology, China Pharmaceutical University, No. 24, Tong Jia Xiang, Nanjing 210009, PR China.
Huange
H
Xi
School of Life Science & Technology, China Pharmaceutical University, No. 24, Tong Jia Xiang, Nanjing 210009, PR China.
Qirui
Q
Bi
School of Life Science & Technology, China Pharmaceutical University, No. 24, Tong Jia Xiang, Nanjing 210009, PR China.
Ying
Y
Hu
School of Life Science & Technology, China Pharmaceutical University, No. 24, Tong Jia Xiang, Nanjing 210009, PR China.
Yang
Y
Zhang
School of Life Science & Technology, China Pharmaceutical University, No. 24, Tong Jia Xiang, Nanjing 210009, PR China. Electronic address: nimx_2000@yahoo.cn.
Mengxiang
M
Ni
D042441:Achromobacter denitrificans / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D000581:Amidohydrolases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D003001:Cloning, Molecular; D004795:Enzyme Stability; D015870:Gene Expression; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity
D001426:Bacterial Proteins; D011994:Recombinant Proteins; D000581:Amidohydrolases; C019672:aminoacylase I