Elucidation and Application of Enzyme System and Genes Involved in Microbial Desulfurization

微生物脱硫酶系统及基因的阐明及应用

• 批准号: 12660078
• 负责人: OHSHITO Takashi
• 金额: $ 2.56万
• 依托单位: Tottori University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2000
• 资助国家: 日本
• 起止时间: 2000 至 2002
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-12660078/
• 关键词: Microbial Desulfurization; Rhodococcus; Dibenzothiophene; Microbial desulfurization; Bacillus; Molecular chaperon; Desulfurization; Flavin reductase

The mesophilic desulfurizing bacterium, Rhodococcus erythropolis D-1, has an ability of desulfurizing dibenzothiophene (DBT), the model compound for the microbial desulfurization, to form 2-hydoxybiphenyl (2-HBP). The enzyme system of DBT desulfurization consists of DszC, DszA, and DszB attacking DBT and its metabolites. Moreover, flavin reductase (FR) is essential for the activities of DszC and DszA. In this study, FR and DszB were studied.FR was purified from R. erythropolis D-1 to homogeneity. The enzyme was tetramer, and the molecular mass of subunit was 22kDa. The enzyme had a strict substrate specificity and showed a high thermostability. The recombinant Escherichia coli strain overproduced FR, and the specific activity increased higher 275 times than the wild strain.At first, the purification of DszB was tried form R. erythropolis D-1. But the pure enzyme could not be prepared because of the instability. Next, the recombinant strain was constructed. Because the inclusion body was formed when the expression vector was used, the DszB gene was coexpressed with the molecular chaperon genes, groELgroES. The purified enzyme was prepared from the recombinant E. coli. It was demonstrated that DszB cleaved the C-S bond of aromatic compound without any other proteinic component and cofactor.

嗜温细菌红串红球菌D-1具有降解微生物脱硫模型化合物二苯并噻吩（DBT）生成2-羟基联苯（2-HBP）的能力。DBT脱硫酶系由DszC、DszA和DszB组成，它们分别作用于DBT及其代谢产物。此外，黄素还原酶（FR）对于DszC和DszA的活性是必需的。本研究对FR和DszB进行了研究。erythropolis D-1至均一。该酶为四聚体，亚基分子量为22 kDa。该酶具有严格的底物特异性和较高的热稳定性。重组大肠杆菌能高效表达FR，比活比野生菌提高了275倍。红串菌D-1。但由于酶的不稳定性，不能制备纯酶。接着，构建重组菌株。由于使用表达载体时形成包涵体，因此DszB基因与分子伴侣基因groELgroES共表达。从重组E.杆菌结果表明，DszB在没有任何其他蛋白质组分和辅因子的情况下切断芳香族化合物的C-S键。

项目成果

T.Ohshiro, Y.Izumi: "Desulfurization of fossil fuels"Encyclopedia of Environmental Microbiology. 1041-1051 (2002)

T.Ohshiro，Y.Izumi：“化石燃料脱硫”环境微生物学百科全书。

O. Yoshikawa, T. Ohshiro, Y. Nishina, and Y. Izumi: "Enhancement and stabilization of desulfurization activity of Rhodococcus erythropolis KA2-5-1 by feeding ethanol and sulfur components"J. Biosci. Bioeng.. 94. 447-452 (2002)

O. Yoshikawa、T. Ohshiro、Y. Nishina 和 Y. Izumi：“通过添加乙醇和硫成分增强和稳定红平红球菌 KA2-5-1 的脱硫活性”J。

O.Yoshikawa, Y.Ishii, K.Koizumi, T.Ohshiro, Y.Izumi, K.Maruhashi: "Enhancement and stabilization of desulfurization activity Rhodococcus erythropolis KA2-5-1 by feeding ethanol and sulfur components"J.Biosci.Bioeng.. 94. 447-452 (2002)

O.Yoshikawa、Y.Ishii、K.Koizumi、T.Ohshiro、Y.Izumi、K.Maruhashi：“通过添加乙醇和硫成分增强和稳定红平红球菌 KA2-5-1 的脱硫活性”J.Biosci.Bioeng

N.NaKayama, T.Matsubara, T.Ohshiro, Y.Moroto, Y.Kawata, K.Koizumi, Y.Hirakawa, M.Suzuki, K.Maruhashi, Y.Izumi, R.Kurane: "A novel enzyme, 2'-hydroxybiphenyl-2-sulfinate desulfinase (DszB), from a dibenzothiophene-desulfurizing bacterium Rhodococcus erythr

N.NaKayama、T.Matsubara、T.Ohshiro、Y.Moroto、Y.Kawata、K.Koizumi、Y.Hirakawa、M.Suzuki、K.Maruhashi、Y.Izumi、R.Kurane：“一种新型酶，2

T.Ohshiro and Y.Izumi: "Purification, characterization and crystallization of enzymes for dibenzothiophene desulfurization"Bioseparation. 9. 185-188 (2000)

T.Ohshiro 和 Y.Izumi：“二苯并噻吩脱硫酶的纯化、表征和结晶”生物分离。