Cloning and sequencing of a gene encoding D-aminoacylase from Alcaligenes xylosoxydans subsp. xylosoxydans A-6 and expression of the gene in Escherichia coli.
The gene encoding the D-aminoacylase of Alcaligenes xylosoxydans subsp. xylosoxydans A-6 (Alcaligenes A-6) was cloned and its complete nucleotide sequence was identified. The D-aminoacylase structural gene consists of 1452 nucleotides and encodes 484 amino acid residues. The molecular weight of D-aminoacylase was calculated to be 51,918. This value agreed well with the apparent molecular weight of 52,000 found for the purified enzyme from Alcaligenes A-6 by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE). The N-terminal amino acid sequence (NH2-SQSDSQPFDLLRAG-) predicted by the nucleotide sequence exactly matched those of the purified D-aminoacylase both from Alcaligenes A-6 and from cloned Escherichia coli (E. coli), with the exception of the removal of the N-terminal methionine processed after translation. The purified recombinant enzyme showed almost the same enzymatic properties as the native enzyme from Alcaligenes A-6. Alcaligenes A-6 D-aminoacylase showed 25-29% homology with L-aminoacylases from Bacillus stearothermophilus, porcine and humans.
Wakayama
M
M
Department of Applied Chemistry, Faculty of Engineering, Oita University, Japan.
Katsuno
Y
Y
Hayashi
S
S
Miyamoto
Y
Y
Sakai
K
K
Moriguchi
M
M
D000421:Alcaligenes / Q000201:enzymology / Q000235:genetics*; D000581:Amidohydrolases / Q000235:genetics* / Q000302:isolation & purification; D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004269:DNA, Bacterial; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification
D004269:DNA, Bacterial; D011994:Recombinant Proteins; D000581:Amidohydrolases; C019672:aminoacylase I