Chemo-Enzymatic Approach For The Production Of Chiral Epichlorohydrin With Glycerin As Substrate

Chiral epichlorohydrin（ECH） is an important intermediate for organic synthesis and have found wide application in preparation of chemical with high optical purity.At present,biotechnological and chemical method are two main ways for resolution of ECH.The traditional chemical method using Salen-Co as catalyst has the drawback of high price and severe environmental pollution.On the contrary, enzymatic hydrolysis of racemic ECH to（S）-ECH is more ecofriendly and meets the demands of "green chemistry".With the rapid development of biodiesel in the world,the by-product glycerine is excessive in great quantity.Therefore,bioproduction of chiral epichlorohydrin from glycerine is of great potential for industrialization.A chemo-enzymatic approach for the preparation of ECH with glycerine as strating material was developed in the dissertation.In the organic synthesis step,racemic ECH was prepared from glycerine via dichloropropanol as intermedate.Firstly,the reaction conditions from glycerine to dichloropropanol were investigated.The optimal conditions were as follows:reaction temperature 115-120℃,n _glycerine:n_{glacial acetic acid}=1:0.26 and reaction time 11 h.Then,the reaction conditions from dichloropropanol to ECH was also optimized:reaction temperature 65℃,n_{dichloropropanol}:n_NaOH=1:1.36 and reaction time 12 min. Under these conditions,the yield of ECH is 90.0%.The dissertation was researched enzymatic resolution of raceme epichlorohydrin.A screening method was established by combination of chiral gas chromatography and enrichment technique with ECH as carbon source.Through this method,5 microorganisms capable of converting ECH to（S）-ECH were isolated from soil,among which,strain 09101 with the maximum epoxide hydrolase activity,was chosen as the best strain for further studies.Based on morphology,the ITS sequence and phylogeny,strain 09101 was identified as Aspergillus niger.The medium composition was optimized by single factors and response surface methodology（RSM）.The optimized medium composition was as follows （g/1）:starch 14.63,soybean cake 6.45,K₂HPO₄0.8,KH₂PO₄ 0.4,MgSO₄ 0.05.The optimum conditions for cell growth and formation of epoxide hydrolase were as follows:initial pH value,7.0;30℃;inoculum volume, 2%（v/v）;medium volumetric ratio,20%（100ml/500ml）.Under these conditions,a specific epoxide hydrolase activity of 103.76 U 1^-1 was achieved after cultivation for 4 d.Effects of reaction conditions on epoxide hydrolase activity and enantio selectivity were also investigated.Results indicated that optimal working pH of the epoxide hydrolase was 2.3（Water-Cyclohexane）.The epoxide hydrolase showed highest activity at 40℃,but its enantiomeric ratio decreased with the increase of temperature.From correlation linear equation of Arrhenius equation,thermodynamic parameter for A. niger.ZJB-09101 catalyzed hydrolysis of racemic ECH was determined (Ea =4.34 kJ mol^-1);And the half-life(t_1/2) of intracellular epoxide hydrolase at 40℃(t_1/2 12.99 h) was determined,suggesting that the thermostability of the epoxide hydrolase in A.niger.ZJB-09101 was low. Moreover,kinetics of the epoxide hydrolase catalyzed reaction were investigated and the kinetic constants were as follows:K_m=0.045mol L^-1, V_max=152.21μmol·min^-1·l^-1.