Cloning, Expression and Characterization of Enantioselective Epoxide Hydrolases from Marine Microorganisms

Abstract

Genes encoding putative EHases were identified by analyzing open reading frames (ORFs) of several marine microorganisms whose genome sequences are available in public. The three EHases - sEEH, nEEH, and rEEH – from three different marine microorganisms were cloned and expressed in E. coli. The recombinant EHases were purified by metal affinity chromatography and further characterized. The EHases were highly soluble. Optimal activities of sEEH, nEEH and rEEH occurred at 45 °C, 35°C and 40°C, respectively and the enzymes were very active at neutral pHs. Especially, the purified rEEH was highly enantioselective toward styrene oxide. The enzyme could preferentially hydrolyze (R)-styrene oxide with ee value of 99% and more than 40% yield. Km and kcat of rEEH toward (R)-styrene oxide were calculated as 5.2 ± 0.3 mM and 31.78 s-1, respectively, while Km and kcat of rEEH toward (S)-styrene oxide were 4.1± 0.3mM and 7.94 s-1. sEEH and nEEH also preferentially hydrolyzed (R)-styrene oxide with ee value of 99%, however, the yield of enantiopure (S)-styrene oxide was around 10%. Km and kcat of sEEH and nEEH toward (R) or (S)-styrene oxide supported the observation. sEEH, nEEH and rEEH could hydrolyze other racemic epoxide substrates. The application to chiral resolution of racemic epoxide substrate by sEEH, nEEH, and rEEH is under progress.