Expression Of Cholesterol Oxidase Gene From Brevibacterium Sp. DGCDC-82

The significant functions of cholesterol oxidase (EC 1.1.3.6, COD) in food processing, medical assay and biological pest-resistance were recognized gradually and showed potentially use. This research is mainly focused on expression of Cholesterol oxidase gene from Brevibacterium sp. DGCDC-82 in Escherichia coli and expression of the gene in eukaryotes.The structure gene of Brevibacterium sp. DGCDC-82 Cholesterol oxidase with signal sequence was amplified from genomic DNA of Brevibacterium sp. DGCDC-82 by PCR technique. The recombinant vector, pET28a-COD(s+), was constructed by inserting the amplified fragment(about 1.5kb)into the expression vector pET28a(+). The DNA fragment encoding mature peptide of COD was amplified from pET28a-COD(s+) and was then inserted into pET28a(+) to construct pET28a-COD(s-).Both recombinant plasmids were transformed into E.coli BL21(DE3) respectively. Promoted and controlled by T7lac promoter, the fusion protein containing His-tag and the peptide of COD with or without signal peptide could be expressed within the cell of recombinant E.coli. The active recombinant enzyme was tested after the two recombinants induced by IPTG.. The expression products of full gene and mature enzyme gene were analyzed by SDS-PAGE indicating that about 55kD protein was obtained, which accounted for about 50% of the cell total protein.The mature enzyme gene of COD from Brevibacterium sp. DGCDC-82 was inserted into a prokaryotic expression vector pTrc99a. The vector was controlled by trp/lac promoter. A recombinant plasmid pTrc99a-COD was obtained and then transformed into the expression host JM109. The influences of induction conditions such as IPTG concentration, the induction temperature and time of induction on the expression of the recombinant protein were investigated. Under optimal condition, the enzyme activity reached 700U/L.The enzymatic analysis of the cholesterol oxidase revealed that its optimal pH and temperature was 7.5 and 40℃, respectively.The structure gene of COD without signal sequence was into the site EcoR I,Not I of pPIC9K, a secreting expression vector of Pichia pastoris. The plasmid pPIC9K-COD was linear with Sal I and transform P.pastoris GS115. Inducting with methanol, The active COD enzyme expressed from the recombinant P.pastoris was found only exsisting in the supernatant of the crude cell extract, but not secreted into culture medium.