Cloning, Expression And Characterization Of A Novel Epoxide Hydrolase From Vigna Radiata

Epoxide hydrolases (EHs) have attracted significant interest as biocatalysts of industrial potential. Recently, two novel interesting EHs that catalyze enantioconvergent hydrolysis of both (R)-and (S)-styrene epoxides to (R)-diols was found in Vigna radiate L. In this thesis, for the.first time, a novel gene, mbEH A, encoding one of the two EHs from Vigna radiate L, mbEH A, was obtained, cloned and highly expressed in Escherichia coli. Recombinant mbEH A was one-step purified and characterized.First, total RNA was isolated from seed, germ and sprouts of Vigna radiata by UNIQ-10 column, Trizol and modified Trizol methods, respectively. Modified Trizol method was selected for further use because of high yield, high purity and integrity of RNA. After RT-PCR, partial mbEH A was only amplified from RNA of Vigna radiata germ. Whole length mbEH A was obtained by rapid amplification of cDNA ends (RACE), which composed of a 960 bp open reading frame,117 bp 5'-upstream and 239 bp 3'-downstream non-coding regions, respectively. The three-dimensional (3D) structure of the deduced mbEH A was predicted and compared to that of potato EH.Next, recombinant expression plasmid pET-28a-mbEH A was successfully constructed and transformed into Escherichia coli BL21(DE3). Recombinant mbEH A was about 30% of total bacterial protein after IPTG induction, which had a molecular mass of 41 kDa and pI value of 6.02. Recombinant mbEH A was purified to 85% pure by one-step nickel chelating affinity chromatography. The recovery yield was over 85%. Its activity was increase by 500-fold compared to that of purified native enzyme using p-nitrostyrene oxide (pNSO) as substrate.Then, enzymatic properties assay showed that recombinant mbEH A can catalyze enantioconvergent hydrolysis of both (R)-and (S)-styrene epoxides to (R)-diols and display a enantiopreference to trans-pNSO; its pH and temperature optima were pH 7.0 and 25℃, the Km and Vmax were 2.05 mM and 14.8μmol·min-1·mg-1, respectively; it was stable in buffers within pH6-10 and showed high tolerance against methanol, Tween-40 and Triton X100, which can even strongly activate the enzyme.At last, cryoprotectants for recombinant mbEH in freeze-drying process were preliminarily screened. All cryoprotectants tested (trehalose, lactose, sucrose, mannitol and glycine) showed better protect at low concentration. The optima was 5% trehalose(92.4%) and 1% sucrose (92.1%). For large scale freeze-drying process,1%sucrose is better and more economic.