Cloning and characterization of an epoxide hydrolase from Novosphingobium aromaticivorans.
A gene encoding a putative epoxide hydrolase (EHase) was identified by analyzing an open reading frame of the genome sequence of Novosphingobium aromaticivorans, retaining the conserved catalytic residues such as the catalytic triad (Asp177, Glu328, and His355) and the oxyanion hole. The enantioselective EHase gene (neh) was cloned, and the recombinant EHase could be purified to apparent homogeneity by one step of metal affinity chromatography and further characterized. The purified N. aromaticivorans enantioselective epoxide hydrolase (NEH) showed enantioselective hydrolysis toward styrene oxide, glycidyl phenyl ether, epoxybutane, and epichlorohydrin. The optimal EHase activity toward styrene oxide occurred at pH 6.5 and 45 degrees C. The purified NEH could preferentially hydrolyze (R)-styrene oxide with enantiomeric excess of more than 99% and 11.7% yield after 20-min incubation at an optimal condition. The enantioselective hydrolysis of styrene oxide was also confirmed by the analysis of the vicinal diol, 1-phenyl-1,2-ethanediol. The hydrolyzing rates of the purified NEH toward epoxide substrates were not affected by as high as 100 mM racemic styrene oxide.
Woo
Jung-Hee
JH
Marine Biotechnology Research Centre, Korea Ocean Research & Development Institute, P.O. Box 29, Ansan, 425-600, South Korea.
Kang
Ji-Hyun
JH
Kang
Sung Gyun
SG
Hwang
Young-Ok
YO
Kim
Sang-Jin
SJ
D020816:Amino Acid Motifs; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D016384:Consensus Sequence; D004269:DNA, Bacterial / Q000032:analysis; D004851:Epoxide Hydrolases / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D004926:Escherichia coli / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007218:Industrial Microbiology; D008969:Molecular Sequence Data; D010802:Phylogeny; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D017422:Sequence Analysis, DNA; D042301:Sphingomonadaceae / Q000201:enzymology*; D013379:Substrate Specificity; D013696:Temperature
D004269:DNA, Bacterial; D011993:Recombinant Fusion Proteins; D004851:Epoxide Hydrolases