Improvement of Functions of Novel Enzymes in the Desulfurization Metaoblism of Petroleum by Protein Engineering and Molecular Genetics

去除化石燃料中的多环含硫化合物是一件很困难的事情。二苯并硫(DBT)被认为是化石燃料中含有的一种典型的多环硫化合物。一些细菌，如红球红球菌D-1，利用DBT作为硫的唯一来源，而不通过硫特有的途径破坏其碳-碳主干。在该反应中，DBT被DszC氧化成DBT砜，DBT砜被DszA氧化成2-羟基联苯2-亚磺酸(HBPSi)，HBPSi被DszB还原成2-羟基联苯。黄素还原酶是DszC和DszA进行单加氧酶反应所必需的。本研究的研究结果概括如下：1.红城红曲霉Ka2-5-1及其与联苯-2-亚磺酸络合的突变体C27S的DszB进行了结晶，并进行了初步的X-射线晶体分析。2.在大肠杆菌中高效表达了与DszC偶联的耐热黄素还原酶基因DSM411，并对重组酶进行了纯化和鉴定。3.根据上述结果，DszB突变株Q65H表现出比亲本DszB酶更高的热稳定性和最适温度B)从中等嗜热菌枯草芽孢杆菌Wu-S2B中分离纯化了参与DBT脱硫的菌株，并对其进行了表征。

It is difficult to remove polycyclic sulfur compounds in fossil fuels. Dibenzothiophene(DBT) is considered as a model polycyclic sulfur compound contained in fossil fuels. Some bacteria such as Rhodococcus erythropolis D-1 utilize DBT as a sole source of sulfur without breaking its carbon-carbon backbone through the sulfur-specific pathway. In this pathway, DBT is oxidized to DBT sulfone via DBT sulfoxide by DszC,DBT sulfone is converted to 2-hydroxybiphenyl 2-sulfinic acid (HBPSi) by DszA, and HBPSi is desulfurized to 2-hydroxybiphenyl by DszB. Flavin reductase is necessary for monooxygenase reactions by DszC and DszA.The research results of this study are summarized as follows.1.DszB of R.erythropolis KA2-5-1 and its C27S mutant complexed with biphenyl-2-sulfinic acid were crystallized and preliminary X-ray crystallographic analyses were conducted.2.The gene of thermostable falvin reductase (Bfl) from Bacillus sp.DSM411, which coupled with DszC, was overproduced in Escherichia coli, and the recombinant enzymes were purified and characterized.3.Following the above 1, the mutant Q65H of DszB showed higher thermostability and optimal temperature than the parent DszB enzyme.4.Three enzymes (BdsC,A,B) involved in the DBT desulfurization were purified from the moderately thermophilic bacterium, Bacillus subtilis WU-S2B and characterized.