Studies on the breeding of cholesterol oxidase producing bacterium

• Main Authors: Hsieh,Chia-Wen, 謝佳雯
• Other Authors: Liu,Wen-Hsiung
• Format: Others
• Language: zh-TW
• Published: 1993
• Online Access: http://ndltd.ncl.edu.tw/handle/17668085597108133945

碩士 === 國立臺灣大學 === 農業化學系 === 81 === The application of cholesterol assay kit for detecting the limited actuvity of cholesterol oxidase was investigated. It was found that the optimal compositions were as follows:Na- axide 2mM, Na-cholate 1mM,4-1ntipyrine 0.82mM, phenol 14mM, cholesterol 10mM,Na-phosphate pH 7.2 100mM, Triton X-100 0.5%, peroxidase 3000 U/L. After the cell membrane were destructed by the treatment of chloroform vaper, the cholesterol oxidase producing bacteria can be directly detected on the plate by 0.6% gel-form kit reagent. BamHI fragments of A.simplex U- S-3011 chromosomal DNA were ligated into the BamHI site of pUC19 and expressed in E.coli DH5.alpha.. Three clonies with cholesterol oxidase activity were selected from the gene library by means of the kit reagent plate assay. The three recombinant plasmids were identified as pCOS-1(4.4kb), pCOS-2(4.35kb) and pCOS-3(4.5kb),respectively. The conversion activity of cholesterol into cholestenone with cell-free extracts of these transformants could also be detected by TLC and HPLC analysis. According the the southern blotting analysis, it was confirmed that the three recombinant plasmids showed homogenous sequence with A.simplex U-S-3011 chromosomal DNA.