Identification and cloning of a gene encoding dichloromethane dehalogenase from a methylotrophic bacterium, Bacillus circulans WZ-12 CCTCC M 207006.
The gene dehalA encoding a novel dichloromethane dehalogenases (DehalA), has been cloned from Bacillus circulans WZ-12 CCTCC M 207006. The open reading frame of dehalA, spanning 864 bp, encoded a 288-amino acid protein that showed 85.76% identity to the dichloromethane dehalogenases of Hyphomicrobium sp. GJ21 with several commonly conserved sequences. These sequences could not be found in putative dichloromethane (DCM) dehalogenases reported from other bacteria and fungi. DehalA was expressed in Escherichia coli BL21 (DE3) from a pET28b(+) expression system and purified. The subunit molecular mass of the recombinant DehalA as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis was approximately 33 kDa. Subsequent enzymatic characterization revealed that DehalA was most active in a acidic pH range at 30 degrees , which was quite different from that observed from a facultative bacterium dichloromethane dehalogenases of Methylophilus sp. strain DM11. The Michaelis-Menten constant of DCM dehalogenase was markedly lower than that of standard DCM dehalogenases.
Wu
Shijin
S
College of Biological and Environmental Engineering, Zhejiang University of Technology, 310032 Hangzhou, People's Republic of China. Wujan28@zjut.edu.cn
Zhang
Huaxing
H
Yu
Xiang
X
Chen
Jianmeng
J
D000595:Amino Acid Sequence; D001407:Bacillus / Q000201:enzymology* / Q000235:genetics* / Q000302:isolation & purification; D001483:Base Sequence; D001709:Biotechnology; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial; D006863:Hydrogen-Ion Concentration; D020582:Hyphomicrobium / Q000201:enzymology / Q000235:genetics; D007700:Kinetics; D008190:Lyases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D020580:Methylobacterium / Q000201:enzymology / Q000235:genetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity; D013696:Temperature; D014871:Water Microbiology
D004269:DNA, Bacterial; D011994:Recombinant Proteins; D008190:Lyases; C045224:dichloromethane dehalogenase