Basic Study of Chemical-enzymatic Synthesis of Chiral Epichlorohydrin from Glycerol

Author:Jin Huo Xi

Supervisor:zheng yu guo


Degree Year:2013





Enantiopure epichlorohydrin,a key chiral synthon and building block,are used to prepare many chiral pharmaceuticals such as atorvastatin,atenolol,carnitine,and pheromone.A large number of byproduct glycerol were produced in the production of biodiesel,resulting in significant decline of the glycerol price.Therefore,it is an economic and promising business for producing materials with high value such as chiral epichlorohydrin from glycerol.This paper investigated the chemical halogenation of glycerol,enzymatic dehalogenation of dichloropropanol,and asymmetric resolution of racemic epichlorohydrin catalyzed by enzymes,realizing the process of production of chiral epichlorohydrin from glycerol and laying the foundations for its application in industrial production.The recombinant Escherichia coli expressing haloalcohol dehalogenase HheC was constructed based on the gene sequence reported in previous paper.High activity of HheC was obtained after optimization of inducer concentration,induction temperature,and induction time.The optimum expression condition was as follows:IPTG 0.5 mM,induction temperature 28 ℃,induction time 7 h.Under these conditions,the activity of HheC achieved 216 U/L.In addition,the results of enzymatic properties showed that the highest activity of HheC was obtained at pH 8.0 and 50 ℃,respectively.The half-life of HheC at 30 ℃ was 108 h,but only 5.3 h was observed at 50 ℃.The racemic epichlorohydrin was produced from glycerol by combining chemical halogenation with enzymatic dehalogenation.The reaction of halogenation was performed in a specially designed reactor and the water formed was absorbed by the excess desiccant in the reaction mixtures.The optimum reaction condition was as follows:10%(w/w)isobutyric acid,5%(w/w)desiccant,reaction temperature 110 ℃,reaction time 8-9 h.Under these conditions,the yield of dichloropropanol achieved 93%.Afterward,the dehalogenation of dichloropropanol catalyzed by HheC was investigated and the results indicated that the conversion rate of dichloropropanol and yield of epichlorohydrin were both increased by increasing of reaction temperature.However,the epichlorohydrin was hard to be accumulated at high temperature due to its dramatic spontaneous hydrolysis.Therefore,two-phase reaction system was used to decrease the spontaneous hydrolysis of epichlorohydrin.Consequently,the yield of epichlorohydrin achieved 75.6%in two-phase reaction system containing 40%(v/v)cyclohexane.A mutant with high activity of epoxide hydrolase(EH),A.niger ZJB-09173,was obtained by 60Co γ and UV mutagenesis.Compared with the original strain A.niger ZJB-09103,the EH activity of mutant increased by 167%but the growth rate of mycelia decreased.In addition,smaller mycelia and broom sporangium of A.niger ZJB-09173 were observed.Afterward,the medium composition and condition for EH formation in A.niger ZJB-09173 were optimized by single factors.The optimal medium composition and culture condition were as follows:glucose 18 g/L,soybean cake 10 g/L,KH2PO4 0.4 g/L,K2HPO4 0.8 g/L,MgS04 0.2 g/L,initial pH 6.0,temperature 30 ℃.Under these conditions,9.5 g CDW/L of biomass and 330 U/L of EH activity were obtained after 4.5 d cultivation.Compared with that before optimization of medium composition and culture condition,the EH activity increased by 74.6%.The enantioselective hydrolysis of racemic epichlorohydrin using whole cells of A.niger ZJB-09173 in organic solvents was investigated.It was indicated that EH from A.niger ZJB-09173 exhibited the excellent enantioselectivity in cyclohexane at 30 ℃.The substrate inhibition,rather than product inhibition of catalysis,was observed in the hydrolysis of racemic epichlorohydrin.Furthermore,high concentration of product would improve the EH activity of A.niger ZJB-09173.Based on these findings,the method of continuous feeding of substrate was used to reduce the substrate inhibition.Finally,16.4%of(S)-epichlorohydrin with>98%enantiomeric excess(ee)from 128 mM of racemic epichlorohydrin was obtained.If the dry cells of A.niger ZJB-09173 were taken as the catalyst,the substrate concentration and yield of(S)-epichlorohydrin achieved 153.6 mM and 18.5%,respectively.The recombinant E.coli expressing the EH was constructed by synthesis of the EH gene sequence reported in previous paper.Afterward,the production of(R)-epichlorohydrin catalyzed by this recombinant EH was investigated.The ee of(R)-epichlorohydrin reached 99%from 25.6 mM racemic epichlorohydrin with a yield of 42.7%at pH 8.0 and 30 ℃.Inhibition studies revealed that the conversion of(R)-enantiomer was strongly inhibited by(S)-enantiomer(Ki =1.4 mM),whereas the conversion of(S)-enantiomer was slightly inhibited by(R)-enantiomer(Ki=10.9 mM).The product(S)-3-chloro-1,2-propanediol was found to be noncompetitive inhibition for the conversion of(S)-epichlorohydrin(Ki=203.6 mM).The highest concentration of substrate was 320 mM by one-time feeding of substrate but 448 mM by continuous feeding of substrate and 512 mM by two-phase reaction system.The cells of recombinant E.coli harbouring EH were immobilized by expanded perlite in its growth process.When 4.5 g of perlites were added into 50 ml medium,the immobilization rate and remaining activity of immobilized cells were 65%and 85%,respectively.As a result,the stability and product-resistance were greatly improved compared with those of free cells.Finally,the recombinant E.coli cells harbouring halohydrin dehalogenase were also immobilized by the same method and the production of(R)-epichlorohydrin from 1,3-dichloro-2-propanol by two-step biocatalysis using the immobilized cells in two-phase system was investigated.Consequently,≥99%ee of(R)-epichlorohydrin with yield of 25.1%was obtained in a specially designed reactor.In addition,it was indicated that reducing the flow rate of air in the first reactor and increasing the amount of cells in the second reactor were favorable to improve the ee and yield of(R)-epichlorohydrin.