Biochemical characterization and transcriptional analysis of the epoxide hydrolase from white-rot fungus Phanerochaete chrysosporium.
The white-rot basidiomycetes Phanerochaete chrysosporium is a model fungus used to investigate the secondary metabolism and lignin degradation. Genomic sequencing reveals the presence of at least 18 genes encoding putative epoxide hydrolases (EHs). One cDNA encoding EH (designated as PchEHA) was cloned and expressed in Escherichia coli. Transcriptional analysis demonstrated that the transcripts of PchEHA could be detected under the ligninolytic and nonligninolytic conditions as well as amended with anthracene. The recombinant enzyme exhibits broad hydrolytic activity toward several racemic epoxides including styrene oxide, epichlorohydrin, and 1,2-epoxybutane, but with different specificity. Using racemic styrene oxide as the substrate, the optimal pH and temperature are pH 9.0 and 40 degrees C, respectively. The enzyme is not sensitive to EDTA, and is inhibited by H2O2, and several metal ions including Zn(2+), Cd(2+), and Hg(2+) at various extents. Several organic cosolvents including acetone, dimethylsulfoxide, formamide, glycerol and ethanol at 10% (v/v) cause slight or no inhibition of the hydrolytic reaction. More importantly, the recombinant enzyme displays distinct enantioselective preference to several chiral epoxides. The enzyme showed good enantioselectivity toward chiral styrene oxide with preferential hydrolysis of (R)-enantiomer. PchEHA is likely a novel soluble EH based on the sequence analysis and catalytic properties, and is a great potential biocatalyst for the preparation of enantiopure styrene oxide in racemic kinetic resolution.
Li
Nian
N
Sichuan Key Laboratory of Molecular Biology and Biotechnology, College of Life Sciences, Sichuan University, Chengdu, China.
Zhang
Yizheng
Y
Feng
Hong
H
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D003116:Color; D004851:Epoxide Hydrolases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004852:Epoxy Compounds / Q000737:chemistry / Q000378:metabolism; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D008969:Molecular Sequence Data; D020075:Phanerochaete / Q000201:enzymology*; D010802:Phylogeny; D012333:RNA, Messenger / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017421:Sequence Analysis; D013237:Stereoisomerism; D013379:Substrate Specificity; D013696:Temperature; D014158:Transcription, Genetic
D004852:Epoxy Compounds; D012333:RNA, Messenger; D011994:Recombinant Proteins; D004851:Epoxide Hydrolases