Studies on the expression of cholesterol oxidase gene from Arthrobacter simplex in Streptomyces lividans

• Main Authors: Yi-Fang Cheng, 鄭伊芳
• Other Authors: Wen-Hsiung Liu, Dr.
• Format: Others
• Language: zh-TW
• Published: 2000
• Online Access: http://ndltd.ncl.edu.tw/handle/60589596633882046270

碩士 === 國立臺灣大學 === 農業化學研究所 === 88 === Abstract In this study, a host-vector system of Streptomyces was established with host (S. lividans TK64) and expression vector (pIJ702). The transformation frequency was 8.3×104 colonies/g DNA. At the same time, the expression of cholesterol oxidase gene from Arthrobacter simplex USA18 in Streptomyces was investigated for the expression of Arthrobacter cholesterol oxidase gene in different host system. The sequence of cholesterol oxidase gene (2.7 kb) from Arthrobacter was determined again. The results show the total length of cholesterol oxidase gene was 810 bps and the deduced amino acids was 270, with a calculated molecular weight of 28,135 Dalton. The overall G+C content of cholesterol oxidase gene was 68%. There is a high similarity in the results of deduced amino acid sequence of dehydrogenase alignmented with molecular biological database. A recombinant plasmid pCOI-1 constructed by pUC19 and Arthrobacter cholesterol oxidase gene (2.7 kb) was used as a template for the amplification of cholesterol oxidase gene and cholesterol oxidase structure gene by PCR. The hybrid plasmids named pIJ702CL (6.9 kb) and pIJ702CS (6.535kb) were constructed individually by PCR product 〔(1.3kb) and (0.935kb)〕 and expression vector pIJ702. Then pIJ702CL and pIJ702CS were transformed, respectively, into S. lividans TK64, and the transformation frequencies were 102 colonies/g DNA. Recombinant transformants contained cholesterol oxidase gene or cholesterol oxidase structure gene were isolated by resistance of thiostrepton and white colony characters. Recombinant plasmids contained cholesterol oxidase gene or cholesterol oxidase structure gene were extracted from ten white transformants, and digested with restriction endonucleases BglII and SacI. The cholesterol oxidase gene and the cholesterol oxidase structure gene constructed in pIJ702CL and pIJ702CS, respectively, were identified by electrophoresis analysis. Cultivation of S. lividans TK64 and recombinant transformants were carried out at 30℃ for 7 days, and the extracellular and intracellular crude extracts were prepared from the culture per day. It was found that no obviously difference between host and recombinant transformants in protein electrophoresis analysis and cholesterol oxidase activity analysis.