Express Of Recombinant Chiral Epoxide Hydrolase Engineering And Application In Production Of Chiral Epichlorohydrin

EHs were found in various sources including mammals,plants,insects and microorganisms,which catalyze the hydrolysis of epoxides to corresponding vicinal diols with high enantioselectivities and reaction rates.Recently,EH has received much attention and were promising for industrial applications.Most of the researches focus on wild strains screened by high-throught sceening method.There are still many problems,such as:low enantioselectivities and productivity.With the recent development in bioinformatics and gene engineering,it is possible to obtain novel EHs by analysis of putative EHs sequence and activity information.In this study,we synthesized an EH gene from Rhodosporidium toruloides CBS 14 and optimized its codons,the total length of nucleotide sequence was 1230 bp.Through the reaction of colony PCR and digestion analysis,the recombinant Escherichia coli BL21(DE3)/pET28b-EH was successfully constructed.Recombinant expression was induced by IPTG and analyzed by SDS-PAGE.The result indicated the molecular mass was about 46.3kDa,and specific activity is 14.45U/mg.In addition,we study the catalytic properties of recombinant EH.It showed that the optimal pH was 7.5,and the optimal temperature was 35?.The recombinant EH was stable at pH 7.5 and was sensitive to heat and the half-life(t1/2)of the purified enzyme at 25,35,45,and 55? were 2.820,0.917,0.592,and 0.319 h,respectively.The EH activity could be activated by Li+,and completely inhibited by Hg2+.The EH with Tween-20 at 5%(v/v)concentration exhibitd the highest hydrolysis activity.And the values of apparent Km and Vmax were calculated to be 0.5953 mol/1 and 0.0105 mol/(L*min).The time course of hydrolysis 1%(v/v)(R,S)-epichlorohydrin showed the enantiomeric excesses value of(R)-epichlorohydrin could be 100%after 40 min reaction.To pave foundation for the use of this EH in industrial application,the flask fermentation medium of recombinant E.coli producing epoxide hydrolase was optimized by using response surface methodology(RSM).A central composite design and RSM were applied to determine the optimal concentration of three significant variables:glucose,yeast extract and tryptone.The optimal culture medium is consist of glucose 5.2 g/l?yeast 5.0 g/l?tryptone 32.4 g/l?NaCl 10.0g/l,pH=7.0,allowed the EH activity yield to increase from 7.69U/ml to 29.49U/ml in flask-shaking batch fermentation.The optimal concentration of lacose was 1.0g/l.When the induction temperature was 28 ?,the optimal induction time was 9h.