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

Vitamin D₃?VD₃?,as an important precursor of hormone,is essential for human and animal growth and reproduction.If hydroxylation occurs at C1 alpha or C25 in the parent nuclear structure of VD₃,it can form VD₃ with physiological activity,which has the characteristics of regulating the balance of calcium and phosphorus in the body,improving immunity,inducing cell proliferation and differentiation,and has great application value.With the increasing demand of human being for active VD₃,and the use of chemical synthesis to produce active VD₃ has many synthetic steps,low yield,high cost,serious environmental pollution and other shortcomings,which are not suitable for large-scale industrial production.Therefore,scholars at home and abroad have turned their attention to the use of microbial transformation method to produce active VD₃,which has the advantages of mild reaction conditions,less environmental pollution,high stereoselective,etc.,and the use of selective hydroxylase to achieve the biological transformation of active VD₃ has a wider application prospect.Therefore,in this study,the enzyme gene CYP10A1 with the function of hydroxylation of VD₃ and the electron transfer chain gene from acinetobacter with good compatibility?fdx-fdr?were selected to be heterogenically expressed in escherichia coli by means of genetic engineering.By constructing a multi-enzyme system of bacteria and carrying out whole-cell catalytic experiment,the problem of intracellular enzyme instability was overcome,and the efficiency of catalytic hydroxylation of VD₃ was improved,which laid a foundation for the synthesis of VD₃ with physiological activity.The main research contents are as follows:?1?In this paper,engineered bacteria containing hydroxylase gene CYP10A1 and electron transport chain gene fdx-fdr were successfully constructed,and the three key enzymes CYP10A1,Fdx and Fdr were all activated.?2?Three key enzymes were isolated and purified.The activity of Fdr was investigated by DCPIP,and it was verified that Fdr could receive electrons from coenzyme NAD?P?H.Cytochromes C was used to investigate the conjugation of Fdr with Fdx and Fdx could be conjugated with Fdr to transfer the electrons of coenzyme NADH to Fdx via electron transport chain.The electron transport chain?Fdx/Fdr?prefers NADH.?3?The detection method of VD₃ and its hydroxylation products was determined,and the catalytic hydroxylation reaction system of VD₃ was successfully established.It was verified that CYP10A1 had hydroxylation function,and the combination of CYP10A1 and electron transport chain fdx-fdr could hydroxylation substrate VD₃,so as to synthesize VD₃ with physiological activity.?4?Successfully built contains both CYP10A1 hydroxylase gene,and electron transport chain gene Fdx-Fdr genetic engineering bacteria,establish a system of more than a bacterial enzyme reaction,and three proteases are soluble expression is achieved,verified the building a successful engineering bacteria can be hydroxylation catalytic substrate VD₃,synthetic VD₃with physiological activity,for direct use of genetic engineering bacteria for whole-cell catalysis synthesis VD₃ do matting with physiological activity.?5?In a multi-enzyme system of bacteria,whole-cell catalytic VD₃ experiment was carried out to explore the effects of cosolvent,coenzyme and biocatalyst load?the concentration of reconstituted cells?on its hydroxylation efficiency,so as to provide a basis for industrial production of synthetic VD₃ with physiological activities.The results showed that the cosolvent DMSO could promote the hydroxylation of VD₃ to a certain extent.When the addition amount was 1%,the output of the two active products reached 1.581 mg/L and0.348 mg/L.Secondly,the addition of coenzyme NADH had a certain effect on the hydroxylation of VD₃.The yield of 25?OH?VD₃ reached 1.660 mg/L when the addition amount was 1mM,and the yield of 1 alpha,25?OH?₂VD₃ was 0.492 mg/L.Therefore,it is very important to ensure adequate NADH for catalytic synthesis of bioactive VD₃.The optimal addition amount of recombinant cells as a biocatronic catalyst was 35g/L,when the production of 25?OH?VD₃ was the highest,and the production of 1 alpha,25?OH?₂VD₃ was also high,reaching 2.491 mg/L and 0.698 mg/L,respectively.