Production Of Epoxide Hydrolase And Its Application In Chiral Resolution Of Epichlorohydrin

Chiral epoxides are highly valuable intermediates,used for the synthesis of pharmaceutical drugs and agrochemicals.They have broad scope of market demand because of their applications.It has been reported that ECH can be produced by both chemical and biological synthesis.Chemical synthesis using Salen-Co as catalyst has the drawback of high price,low enantio-selectivity and severe environmental pollution.However,biological methods are of high enantio-selectivity and environmentally-friendly.Epichlorohydrin has been a situation of oversupply in the whole world for the present,which will bring the prize down.Therefore,utilizing the cheap racemic ECH to produce chiral ECH will have higher economic benefits and social returns.It can be the research frontier and hot topics.In this study,the microbial cell and immobilized enzyme were used as biocatalyst in their biosynthesis,which can design process routes for producing the chiral ECH.Research in characterization,preparation and application of biological catalysts.A.mediolanus ZJB09106 was isolated from soil by our lab,this paper investigated effects of reaction conditions on epoxide hydrolase activity first.The medium composition was optimized by single factors and orthogonal experiment.The optimized medium composition was as follows(g/1):sodium citrate 22.3 g/1.starch 10.0 g/1.yeast extraction 10.0 g/1.beef extraction 16.0 g/1.The optimum conditions for cell growth and formation of epoxide hydrolase were as follows:initial pH value,6.5;inoculum volume,3%(v/v);medium volumetric ratio,100ml/500ml.Under these conditions,a enzyme epoxide hydrolase activity of 396.08 U/l and biomass of 4.32 g/1 was achieved after cultivation for 32 h.In this paper,the epoxide hydrolase from A.mediolanus ZJB09106 was also purified and identified.The epoxide hydrolases(EH)had been purified to homogeneity in three steps:ultrasonic fragmentation,ammonium sulfate precipitation and ion-exchange column chromato-graphy.The purified enzyme had the molecular mass of about 28 KD,the purification fold of 12.4 times and 15.2%yield.The epoxide hydrolase had the homology with Haloalcohol Dehalogenase HheA from Arthrobacter sp.StrainAD2 and Haloalcohol Dehalogenase HheA from Corynebacterium sp.Strain N-1074 through the analysis of peptide mass fingerpringting.The first seven amino acid sequence was MRIALVT through the identification of the protein N end.Further,The use of enantioselective hydrolysis for preparing chiral epichlorohydrins was accomplished using immobilized crude epoxide hydrolase of recombinant Escherichia coli.The effects of reaction conditions on epoxide hydrolase activity and enantio-selectivity was also investigated.Results showed that hexane was selected as the best organic phase,the optimized conditions at pH 7.5,temperture 30?,water content was 1%,substrate concentration 20 mM were obtained through optimization of biocatalytic reaction conditions.(R)-epichlorohydrins with an enantio-purity of 100%ee and a yield of 10.7%were obtained within 16 h from 20 mM racemates.The residual enzyme activity of 40%was achieved after 7 cycles of reaction.Moreover,kinetics of the epoxide hydrolase catalyzed reaction were investigated and the kinetic constants were as follows:Vmax=2.331 mmolmin-1g-1,Km=76.28 mmol/l?...