Active Expression Of VD₃ Hydroxylase,Electron Transfer Chain And Application In Catalytic Synthesis Of 25-hydroxy VD₃

Vitamin D₃（VD₃）is a fat-soluble vitamin,as an important hormone precursor which possesses extensive physiological activity.If C1 or C25located in the nuclear structure of VD₃ converted into hydroxylated form,it became the active form of VD₃,which is significantly invovled in the maintainence of basic physological activity in human being.Biotransformation of active VD₃ by selective hydroxylase has the advantages of mild reaction conditions,the stereo specificity,as well as environmental friendly,all this merits contribute to a good prospect of industrialization.VD₃ hydroxylase originate from a wide range of species,including Pseudonocardia,Streptomyces griseolus and a mammalian CYP450 hydroxylase,some of which have been actively expressed in Escherichia coli cells.This study aims to establish the biotransformation route of active VD₃（25（OH）VD₃）.According to the earlier reports,VD₃hydroxylase（Vdh）was cloned from Pseudonocardia autotrophica as well as its oxidoreductase FdR and redox protein Fdx with comparatviely higher coupling efficiency.Active expression of these three kinds of protein are confirmed by the study on enzyme purification and electron transfer efficiency in vitro.A bioconversion system with VD3 as a substrate to catalytic synthesis of 25（OH）VD3 was further constructed,preliminary study focus on the influence of coenzyme a cycle of bacteria in multi enzyme system on transformation efficiency,the research contents are as follows:According to the previous studies,the sequence of Vdh gene from Pseudonocardia autotrophica strain was artificially synthesized and cloned into the epression vector pET28b-Vdh.As one member of the cytochrome CYP450 monooxygenase superfamily,the process of VD₃selective hydroxylation catalyzed by enzyme Vdh needs coenzyme NADH or NADPH as the electron donor,and this process needs protein oxidation and oxidation reductase component to serve as the electron transport chain through a cascade of redox process of selective hydroxylation of VD₃.We chose ferredoxin reductase（FdR）and ferredoxin（Fdx）with high coupling efficiency to serve as electron transfer chain,and use the ribosome binding sequence（RBS）to achieve tandem expression of the two genes,we constructed the recombinant plasmid pET28b-Fdr-Fdx.Then the expression condition were optimized in engineered Escherichia coli cells with pET28b-Vdh,pET28b-Fdr-Fdx plasmid respectively:and the best induction temperature were respectively 20,24℃,and the addition of inducer for IPTG were 0.25and 0.5 mM respectively,the induction time is 12 h.The biological function of Vdh and the electron transport chain Fd R-Fdx were further studied.Based on the structural characteristics of CYP450 enzyme,the specific absorption peaks at 450nm was shown in reduced Vdh enzyme for CO difference spectroscopy,which confirmed that the heme domain is the correctly folded.In addition,in the study of electron transfer chain biofunction,reducing activity of FdR is further verified by using DCPIP as the artificial electron acceptor,and cytochrome C was used as electron acceptor to prove that Fdx can be coupled with FdR.According to the specific absorption peak of Vdh enzyme at 450nm wavelength,which is reduced by the electron from coenzyme NADH coupled with the electron transfer chain Fd R-Fdx,the results demonstrated that the lelctron transfer chain was successfully construncted in vitro.All the results above confirmed that Vdh and its electron transfer chain of FdR-Fdx in E.coli cells the activity expressed.Using VD₃ as the substrate,the conversion system was constructed using the crude enzyme liquid of Vdh and Fdr-Fdx to catalytic synthesis of 25（OH）VD₃,the optimum catalytic reaction conditions were as following:temperature 30℃,reaction pH is 7.5,reaction time was 12 h,the substrate concentration was 100 uM,containing 160 mM sodium acetate buffer,80 mM glucose,100 mM NaCl and the best extraction solvent is ethyl acetate,the highest conversion rate of VD₃ reached to12.15%.In order to further increase the conversion rate of VD₃,we constructed a tricistronic enzymes expression system for substrate VD₃hydroxylation in Escherichia coli.By means of transforming plasmid to pET32b、pET20b,the conversion rate of VD₃ increases to 21.11%;In addition,we chose glucose dehydroxylase（GDH）to realize the coenzyme NADH recycling,a double plasmids expression system of pCDFDuet-Vdh-GDH and pET28b-Fd R-Fdx was constructed and conversion rate of VD₃ hydroxylation within the 12 h was increased to 27.31%.