title: Cloning and characterization of an epoxide hydrolase from Cupriavidus metallidurans-CH34.
abstract: A putative epoxide hydrolase-encoding gene was identified from the genome sequence of Cupriavidus metallidurans CH34. The gene was cloned and overexpressed in Escherichia coli with His(6)-tag at its N-terminus. The epoxide hydrolase (CMEH) was purified to near homogeneity and was found to be a homodimer, with subunit molecular weight of 36 kDa. The CMEH had broad substrate specificity as it could hydrolyze 13 epoxides, out of 15 substrates tested. CMEH had high specific activity with 1,2-epoxyoctane, 1,2-epoxyhexane, styrene oxide (SO) and was also found to be active with meso-epoxides. The enzyme had optimum pH and temperature of 7.5 and 37°C respectively, with racemic SO. Biotransformation of 80 mM SO with recombinant whole E. coli cells expressing CMEH led to 56% ee(P) of (R)-diol with 77.23% conversion in 30 min. The enzyme could hydrolyze (R)-SO, ∼2-fold faster than (S)-SO, though it accepted both (R)- and (S)-SO with similar affinity as K(m)(R) and K(m)(S) of CMEH were 2.05±0.42 and 2.11±0.16 mM, respectively. However, the k(cat)(R) and k(cat)(S) for the two enantiomers of SO were 4.80 and 3.34 s(-1), respectively. The wide substrate spectrum exhibited by CMEH combined with the fast conversion rate makes it a robust biocatalyst for industrial use. Regioselectivity studies with enantiopure (R)- and (S)-SO revealed that with slightly altered regioselectivity, CMEH has a high potential to synthesize an enantiopure (R)-PED, through an enantioconvergent hydrolytic process.
authors.affiliation: Department of Biochemistry, Panjab University, Chandigarh, India.
authors.forename: Ranjai
authors.initials: R
authors.lastname: Kumar
authors.forename: Shadil Ibrahim
authors.initials: SI
authors.lastname: Wani
authors.forename: Nar Singh
authors.initials: NS
authors.lastname: Chauhan
authors.forename: Rakesh
authors.initials: R
authors.lastname: Sharma
authors.forename: Dipti
authors.initials: D
authors.lastname: Sareen
mesh_terms: D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D049921:Cupriavidus / Q000201:enzymology* / Q000235:genetics; D004851:Epoxide Hydrolases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D004852:Epoxy Compounds / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D008969:Molecular Sequence Data; D009795:Octanes / Q000378:metabolism; D016415:Sequence Alignment; D013379:Substrate Specificity
chemical_list: C487864:1,2-epoxyoctane; D004852:Epoxy Compounds; D009795:Octanes; C419870:1,2-epoxyhexane; C013690:styrene oxide; D004851:Epoxide Hydrolases