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

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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.