Preliminary Studies On Strain Screening For Vitamin D₃ Hydroxylation And Subsequent Biotransformation In Ionic Liquid/Aqueous Biphase System

Hydroxyvitamin D3 can maintain the dynamic balance of calcium and phosphorus in the body,and regulate the immune system in vivo and the proliferation and differentiation of cells.It is widely used in medicine,food and feed industries.At present,vitamin D3 is commonly produced with chemical synthesis and biocatalysis.Chemical synthesis is complex in separation and purification,high cost,and polluted,so it is difficult to adapt to the needs of the market.Compared with chemical synthesis,biocatalysis has the advantages of mild reaction conditions,fast reaction rate,high specificity,less pollutants and low energy consumption.Moreover,ionic liquid based biphasic system may be favorite for the biocatalytic production of vitamin D3.In this work,two strains were screened to utilize vitamin D3 as sole carbon source in the enriched soil samples.According to morphological identification,they were preliminarily identified as Bacillus sp.(HDZJU1-11)and Streptomyces sp.(HDZJU1-12).Further transformation experiment showed that the strains had the ability of hydroxylation of vitamin D3 to some extent.Further testes were carried out to use different ionic liquids to form a biphasic system,and Bacillus HDZJU1-11 was used as a biocatalyst to catalyze the hydroxylation of vitamin D3 in the constructed system.By measuring the solubility and distribution coefficient of substrates in different ionic liquids and analyzing the biocompatibility of Bacillus to different ionic liquids,[BMIm][PF6],[BMIm][NTf2],and[OMIm][NTf2]were selected to form suitable biphase systems.The conditions of the conversion reaction were then optimized.When the strain age was 12 hours,the phase ratio was 4:1,and the water phase pH was 7.5,there were the best transformation effects.With the increase of substrate concentration,the conversion and yield decreased(calculated by weight).Under the optimal transformation condition,the highest concentration of 25-hydroxyvitamin D3 was 0.38 mg/mL,and the yield was 76.1%.Finally,the hydroxylases P450001 and P450002 gene from Streptomyces HDZJU1-12 with the ability of hydroxylating vitamin D3 was cloned into Escherichia coli by genetic engineering,respectively.After the expression of the above enzymes in E.coli BL21(DE3)/pET28a-p450001 and E.coli BL21(DE3)/pET28a-p450002,vitamin D3 was added as the substrate to perform a conversion reaction.The results showed that P450001 could hydroxylate vitamin D3 as the product 25-hydroxyvitamin D3 effectively,and the same result could be observed in P450002.