Research On Microbiol Conversion Of Vitamin D And The Conversion Mechanism

The active of vitamin D is essential to maintain normal metabolism of body,these actions have potential therapeutic applications for rickets,and immune modplation,regμlation of endocrine systems.To date,the researches on bio-conversion for vitamin D by microbial conversion.It reported that vitamin D was converted to the active of vitamin D with actinomycetes including Pseudonocardia sp,Streptomyces sp and others microorganisms which had activities to substitute hydrogen group on the molecμlar structure of vitamin D,however,the less studies a-broad showed the conversion mechanism,especially for Pseudonocardia sp.In domestic,it is almost empty about conversion mechanism of the active of vitamin D produced with Pseudonocardia sp.In order to fill the gap,this article had showed CGMCC 5098 screened by our group had capable of converting vitamin D to 25(OH)vitamin D and had studied its conversion mechanism.Firstly,vitamin D3,vitamin D2,10-keto-6-methoxy-3,5-cyclovitamin D2,la-19-Nor-1α-hydroxy vitamin D2 were converted to their active forms with CGMCC 5098 screened by our group,and these conversion products had been analyzed with TLC,NMR and mass spectrometry that 25-hydroxy-substituted products exsited.It is showed the strain CGMCC 5098 had capable of converting four vitamin D substrates to 25-hydroxy-substituted vitamin D.To determine the relationship between species strains CGMCC 5098,the strains had identified Psedunocardia autotrophica by morphological and molecular biological methods including microscopic observation and amplified 16s rDNA sequencing.Based on similar functional genes reported in NCBI database,this study had found and analyzed vitamin D hydroxylase gene highly conserved region,thus determining oxygen binding sites and two heme binding site.Then,the vitamin D hydroxylase gene highly conserved region was cloned and sequenced by PCR with degenerate primers designed.To clone the vitamin D hydroxylase gene and research on the conversion mechanism of this strain,we tried to clone the hydroxylase gene with four kinds of methods including LA PCR,reverse sequencing and other molecμlar biological methods,but failed to amplify the full length.It constructed an improved CTAB method for rapid extraction of Gram-positive Pesudonocardica strains gnomic DNA in order to meet the requirements of de nove high-throughput sequencing.This method is applicable to a variety of other biological molecules operations.Finally,our group had accomplished the de novo genome sequencing in collaboration with Microgen Biotechnology Company.The vitamin D hydroxylase gene was screened from genome and hydroxylase genes of genomic data and its encoding protein biological characteristics had been predicted with bioinformatics analytical methods.