Study On Recombinant Expression Of VD₃ Hydroxylase And VD₃ Hydroxylation

25-hydroxyvitamin D₃ is a important metabolite of vitamin D₃,which can not only treat vitamin D₃ deficiency,coronary heart disease,chronic kidney disease,but also can be used as an indicator of cough variant asthma,sjogren syndrome,and has a wide range of physiological activity and medicinal value.The vdh gene was first discovered in a rare actinomycetes called Pseudonocardia autotrophica,which has the ability to hydroxyl VD₃ into 25-OH VD₃.In this study,a recombinant strain was constructed via heterologous expression of VD₃ hydroxylase(Vdh)and was further applied for the cellular synthesis of 25-OH VD₃.In order to further improve the conversion rate of biosynthesis 25-OH VD₃,Adx and Adr were cloned into the genome of Bacillus subtilis WB600.Finally,the effects of three conversion methods on the conversion rate of 25-OH VD₃ were compared,which provided a new idea for the preparation and production of food-grade 25-OH VD₃.The main contents and results are described as follows:(1)The vdh gene derived from Pseudonocardia sp.was synthesized according to the sequence in NCBI,and the recombinant strain Escherichia coli BL21(DE3)/p TIG-vdh was constructed.SDS-PAGE analysis showed that Vdh was soluble in E.coli and the protein size was about 46 k Da.After purification on nickel column,the in vitro activity of Vdh was evaluated by CO differential spectroscopy.The results showed that the enzyme activity of lysates reached 0.98 nmol·g^-1 after culture at 25?for 12 h.(2)The recombinant B.subtilis WB600/p MA5-vdh was constructed,and the 25-OH VD₃was synthesized by its cellular catalysis system.The yield of 25-OH VD₃ was improved by optimizing the whole-cell transformation conditions,such as substrate concentration,cosolvent,salt ion,carbon source,temperature,time,rotational speed and p H.The results showed that under the conditions of substrate concentration 0.1 g·L^-1,18 g·L^-1 glucose,22.5 g·L^-1 2-hydroxypropyl-?-cyclodextrin,2 g·L^-1 Na Cl and K₂HPO₄ for 48 h,the conversion rate of 25-OH VD₃ was 10.36%when the initial p H was 7,the reaction temperature was 30?,and the rotation speed was 200 r·min^-1.(3)In order to further improve the conversion rate of 25-OH VD₃,Adx and Adr were cloned and expressed as electron transport chains.Adx and Adr were successfully expressed in the p43 promoter with the size of about 14 k Da and 55 k Da.The strain B.subtilis WB600-AA/p MA5-vdh was successfully constructed by tapping p43-adx-adr into B.subtilis WB600,which could be used for the exploration of later whole cell transformation experiments.(4)VD₃ was transformed by in vitro transformation,static cell transformation and growth cell transformation.Among them,in vitro conversion method consumes the shortest time,but the pretreatment is complicated,and the Vdh and NADH in the system are limited,resulting in the final yield of 25-OH VD₃ is 13.14 mg·L^-1.The yield of cell transformation by static culture was the lowest,about 1.19 mg·L^-1.The yield of the growth cell transformation method was as high as 17.95 mg·L^-1,which was the best method among the three transformation methods.