1995 Fiscal Year Final Research Report Summary
Development of stable cholesterol oxidase used for diagnosis
| Project/Area Number |
05555223
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| Research Category |
Grant-in-Aid for Developmental Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Research Field |
生物・生体工学
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| Research Institution | Osaka University (1995)
Hiroshima University (1993-1994) |
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Principal Investigator |
MUROOKA Yoshikatsu Osaka University, Faculty of Engineering, Professor, 工学部, 教授 (60029882)
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| Co-Investigator(Kenkyū-buntansha) |
SOGABE Yukihiro Toyoboseki Co., Ltd.Institute for Biotechnology Senior Researcher, バイオ研究所, 主席部員
HIRAYAMA Noriaki Tokai University, Faculty of Developmental Engineering, Professor, 開発工学部, 教授 (70238393)
YAMASHITA Mitsuo Osaka University, Faculty of Engineering, Assistant Professor, 工学部, 助手 (40220347)
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| Project Period (FY) |
1993 – 1995
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| Keywords | Cholesterol / Cholesterol oxidase / Thermostable enzyme / コンピュータグラフィックス / 臨床検査用酵素 / 酵素の耐熱性 |
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| Research Abstract |
For the purpose of development of stable cholesterol oxidase which can be used for determination of cholesterol in specimen. The enzyme has to be stable for long term since the enzyme are exporting to Europe. We design the cholesterol oxidase from Streptomyces sp. to overproduce in Escherichia coli and change the active site and predicted site of thermal stability of the enzyme in which the sites were deduced by computer graphic. For three years, we have devoted the following research work and obtained fruitful results.
1.We made modeling and constructed the structure-function by comparison of cholesterol oxidases between the enzymes from Streptomyces and Brevibacterium sterolicum.
2.Amino acids of the predicted sites concerned with enzymatic active site and FAD binding site were substituted with other amino acids by site-directed mutagenesis. The active site was determined. Two new functional enzymes that can catalyze each oxidation or isomerization of cholesterol were constructed.
3.By comparison with two enzymes from Streptomyces and Brevibcterium, we deduced the predicted sites concerned with thermal stability. By site-directed mutagenesis of the predicted site, a highly stable enzyme was constructed.
4.The genes encoded cholesterol degradating enzymes were cloned and analyzed. The genes encoded ketosteroid-DELTA^1-dehydrogenase, ketosteroid-DELTA^5-isomerase and the regulator were found.
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[Publications] Molnar, I., Hayashi, N., Choi, K.-P., Yamamoto, H., Yamashita, M.and Murooka, Y.: "Bacterial cholesterol oxidases are able to act as flavoprotein-linked ketosteroid monooxidases that catalyse hydroxylation of cholesterol to 4-cholesten-6-ol-3-one." Molec.Microbiol.7 (3). 419-428 (1993)
Description
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[Publications] Wilmanska, D., Dziadek, J., Sajduda, A., Miczarek, K., Jawoes-ki, A.and Murooka, Y.: "Identification of cholesterol oxidase from fast-growing mycobac-terial strains and Rhodococcus sp." J.Ferment.Bioeng.79 (2). 119-124 (1995)
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[Publications] Rho, J.-H., Suzuki, H., Kumagai, H., Yamashita, M., Azakami, H.and Murooka, Y.: "Crystallization and preliminary X-ray analysis of copper amine oxidase from Escherichia coli" Journal of Molecular Biology. 58. 635-637 (1994)
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[Publications] Murooka, Y., Choi, K.-P., Molnar, I., Nomura, N., Cho, H.-J.and Yamashita, M.: "Recent advances in studies on Streptomyces cholesterol oxidase and gene cluster involved in steroid catabol-ism in prokaryotes." Actinomycetorogia. (in press). (1996)
Description
「研究成果報告書概要(欧文)」より
