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

中温脱硫菌红球红球菌D-1能够脱除微生物脱硫的模型化合物二苯并噻吩(DBT)生成2-羟基联苯(2-HBP)。DBT脱硫酶体系由DszC、DszA、DszB攻击DBT及其代谢产物组成。此外，黄素还原酶(FR)对DszC和DszA的活性也是必不可少的。本研究对FR和DszB进行了研究，从红城红曲霉D-1中分离纯化了FR。该酶为四聚体，亚基相对分子质量为22 kDa。该酶具有严格的底物专一性和较高的热稳定性。重组菌产FR，比活力提高275倍。首先，从红城红曲霉D-1菌株中分离纯化DszB。但由于酶的不稳定性，无法制备出纯酶。接下来，构建了重组菌株。由于使用表达载体时形成了包涵体，DszB基因与分子伴侣基因groELgroES共表达。从重组大肠杆菌中制备纯化的酶。结果表明，DszB在不含任何其他蛋白质组分和辅因子的情况下，断裂了芳香族化合物的C-S键。

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.