Screening And Site-directed Mutagenesis For Halohydrin Dehalogenase And Its Application In Production Of Epichlorohydrin

Epichlorohydrin(ECH)is an important industrial raw material which can be applied to the synthesis of epoxy resins,epichlorohydrin rubber,coatings,adhesives,reinforced materials,casting materials and electronic laminates.Optically active ECH is a key C3 chiral synthon for the preparation of many chiral pharmaceutical intermediates.ECH was synthesized by two main approaches including high temperature chlorination of propylene and allyl acetate,both of them are dependent on oil resources,requiring highly costs and leding to serious environmental pollution.In recent years,a synthetic route which combination of chemical processing and biological dehalogenation using glycerol as the raw material was developed to synthesis of epichlorohydrin and become a hot topic.In this paper,we mainly investigated screening and site-directed mutagenesis of halohydrin dehalogenase,purification and characterization of halohydrin dehalogenase and using the resting cells in the production of ECH.Firstly,a high-throughput screening model was established based on pH dependent color change of bromothmol blue and combination of gas chromatography analysis(GC)was to select the purpose microbes.As a result,two strains producing halohydrin dehalogenase were acquired,one was wild strain ZJB11071 and another one was genetic engineering strain ZJB09258.When the concentration of 1,3-DCP was reached to 20 mM and after 6 h reaction,the conversion rate were 42.3%and 67.4%,respectively.Through morphological,physiological and biochemical characteristics ZJB 11071 was identified as Bacillus cereus.Due to the genetic engineering strain ZJB09258 has higher activity and substrate tolerance than the wild strain ZJB11071,so we select ZJB09258 as the biocatalyst for further research.Secondly,because the activiy and the level of substrate tolerance of HheC were low,so we analysis of the interaction between the substrate and the enzyme through homology modeling and molecular docking,the mutations were selected.We composed the proposed mutation halohydrin dehalogenase genes and using Escherichia coli BL21(DE3)as the host cell and pET-28b(+)as the expression vector to construct the recombinant engineered bacteria E.coli BL21(DE3)/pET-28b-HHDH.The mutation halohydrin dehalogenase(Mut-HHDH)and the original halohydrin dehalogenase(Wt-HHDH)were isolated and studied their enzymatic properties.The results showed that the specific activity of the Mut-HHDH was enhanced to 26-fold.The Mut-HHDH showed 3-fold extension of half-life at 45? than that of Wt-HHDH.The value of kcat was 18-fold compared to the Wt-HHDH.Finally,we studied the process of biotransformation 1,3-DCP to ECH by resting cells E.coli BL21(DE3)/pET-28b-HHDH.The reaction conditions of resting cells were optimized,the results were as follows:pH 8.4,45? for temperature,cell concentration was 8 g/L.Under optimized reaction conditions,60 mM of 1,3-DCP could afford 43.2 mM ECH after 1 h reaction.At the same time,we investigated the effect of products and substrate concentration on the enzyme.The results showed that,when the concentration of substrate at the range of 0-180 mM,there was no substrate inhibition.While product ECH and Cl" were very obviouly affecting the activity of the enzyme.In order to relieve the inhibition of Cl",we select calcined hydrotalcite(Mg3Al-CLDH)as an adsorbent and to study the ability of removal Cl-which was generated during dehalogenation.The results showed that during the reaction,added Mg3Al-CLDH 0.03 g/mL,60 mM of 1,3-DCP could afford 54.3 mM ECH after 1 h reaction(conversion was 94.5%).The conversion rate was increased from 78.1%to 94.5%and the concentration of ECH was increased from 43.2 mM to 54.3 mM compared to without addition of Mg3Al-CLDH.