Studies On 3-Ketosteroid-1,2-Dehydrogenase Of Mycobacterium Sp.M1

The selective side-chain cleavage of stigmosterol to androsta-1-ene-3, 17-dione(AD) and androsta-1,4-diene-3,17-dione(ADD) by Mycobacterium sp.M1 was investigated.3-ketosteroid-1-dehydrogenase activities in Mycobacterium sp. M1 were studied during the transformation of AD by the cells and cell extracts. The chromosome DNA of Mycobacterium sp. was extracted, and a pair of degenerate PCR primers was designed. The partial conserved gene of 3-ketosteriod-1-dehydrogenase in Mycobacterium sp.M1 was obtained by PCR with designed primers and cloned into Escherichia coli. The sequence of aim gene was menstruated. According to the result of sequence, pUC19-MK plasmid for gene knock out was constructed. Positive clones was screened after transform plasmid pUC19-MK into Mycobacterium sp.M1. The mainly researching contents were as follows:1. Stigmosterol were cleaved effectively into AD and ADD through cleaving the side chain of sterols by Mycobacterium sp.M1. AD(D) yield rate was about 60%, the ratio of AD to ADD is 1:5.2. Distinct 3-ketosteroid-1-dehydrogenase activities in Mycobacterium sp.M1 were found during the transformation of AD to ADD by the cells and cell extracts. The cell extracts of Mycobacterium sp. M1 had the ability to convert stigmosterol to ADD effectively, ADD yield rate was about 20%.3. Using quick DNA extraction method to obtain the minimal quantities of the chromosome DNA of Mycobacterium sp M1.Operate the AlignX software to contrasting five exited genes of 3-ketosteriod-1-dehydrogenase from Arthrobacter simplex, Pseudomonas testosteroni, Nocardia opacus, Rhodococcus rhodochrous, Rhodococcus erythropolis. A pair of degenerate PCR primers was designed according to the high conserved amino acid sequences and nucleotide sequences of five known genes. The partial gene of 3-ketosteriod-1-dehydrogenase in Mycobacterium sp.M1 was obtained through PCR.4. The aim gene was connected with pUCm-T plasmid and the recombinant plasmid was transformed into Escherichia coli JM109. The aim gene was sequenced. According to the sequence of aim gene, the gene of kanamycin resistance was insert into the aim gene. pUC19-MK plasmid used to gene knock out was constructed on the basis of pUC19 plasmid.5. Transform pUC19-MK into Mycobacterium sp.Ml by electro-transformation, 21 transformants were screened by kanamycin resistance firstly. Screen the homologous recombinant clones through fermentation of these transformants.