| Epichlorohydrin is an important organic intermediate.As a promising enantiopure epoxide,(R)-and(S)-epichlorohydrin are important C3 chiral building blocks for applications in medical,pesticide,chemical,material and many other industries.In recent years,because of maturing technology and the adaptation of a large-scale synthesis of epichlorohydrin from glycerol,which is a by-product of biodiesel,the market price of epichlorohydrin has fallen.Therefore,one of the most economic ways for preparing chiral epichlorohydrin was the kinetic resolution of racemic epichlorohydrin.In this thesis,epoxide hydrolase asymmetric bioresolution of epichlorohydrin for preparing(R)-epichlorohydrin were studied.A recombinant Escherichia coli BL21(DE3)/pET28b-EH strain expressing epoxide hydrolase from Agrobacterium radiobacter AD1 was constructed in our lab in the earlier study.Firstly,the conditions of enzyme production and bio transformation by the recombinant strain expressing epoxide hydrolases were studied.Through single factor experiment,the optimum medium composition and culture conditions were as follows:tryptone 10 g/L,yeast extract 7.5 g/L,sodium chloride 10 g/L and natural pH.The inducer was IPTG and the concentration was 0.15 mM,induced at 28 ℃ for 10 h.Under the conditions,the epoxide hydrolase activity was the best.On the other hand,the optimum catalytic conditions were determined as follows:30 ℃,phosphate buffer pH 8.0,bacteria concentration 15 g/L.Under the optimal condition,asymmetric bioresolution 369.9 mM of epichlorohydrin could afford yield of 32.6%(R)-epichlorohydrin in 99.2%e.e.within 50 min.The biocatalytic resolution of(R,S)-epichlorohydrin to(R)-epichlorohydrin by whole cells of recombinant Escherichia coli expressing epoxide hydrolase activity in organic solvent/buffer biphasic systems was studied.Isooctane was found to be the best organic solvent.The optimum values of volume ratio of the organic phase to the aqueous phase,reaction temperature,pH value,substrate concentration,cell concentration and reaction time were 7:3,30 ℃,8.0,574 mM,70 g/L wet cell and 45 min,respectively.Under the optimized conditions,the best yield of 37.5%,99.3%of product e.e.and the average productivity of 0.286 M/h)were obtained in a stirred reactor(1-L scale).The results demonstrated that the bioconversion was more efficient in isooctane/buffer biphasic systems than that in monophasic aqueous system,the substrate concentration and the average productivity of(R)-epichlorohydrin were improved 55.2%and 98.6%,respectively.Finally,the recombinant E.coli cells were immobilized to improve stability,the calcium alginate was found to be the best immobilization carrier.Other immobilization conditions such as concentration of sodium alginate and CaCl2,calcification time,cell embedding capacity and immobilized cell diameter were determined and optimized.The optimized immobilization conditions were follows.Sodium alginate 2.0%,CaCl22%,calcified 10 h,cell loading 50 g/L,particle diameter 2.7 mm.Under the optimized reaction conditions,the stability of temperature and pH of immobilized cells were significantly improved compared to reaction by the free cells.Using bioresolution of(R,S)-epichlorohydrin for preparing(R)-epichlorohydrin by immobilized cell,the best yield and product e.e.were 36.3%and 100%.The immobilized cell repeated four batches remains more than 81%of the residual activity and showed excellent operation stability. |
