Characterization and functional improvement of novel enzymes involved in the desulfi dibenzothiophene in microorganisms

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Organic sulfur compounds are found in fossil fuels, the combustion of which causes serious environmental problems, such as acid rain. At the refinery, hydrodesulfurization is currently performed to remove sulfur compounds form fossil fuels. However, it is difficult to remove polycyclic sulfur compounds. As legislative limits on sulfur emissions have become tighter, the need to remove polycyclic sulfur compounds form fuel has become more pressing. Dibenzothiophene (DBT) is considered as a model polycyclic sulfur compound contained in fossil fuels. We have reported that 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 monooxygen … More ase reactions by DszC and DszA.The research results of this study are summarized as follows.(1) We purified and characterized the flavin reductase from R. erythropolis D-1 grown in a medium containing DBT as the sole source of sulfur. The purified flavin reductase contains no chromogenic cofactors and was found to have a molecular mass of 86 kDa and four identical 22 kDa subunits. The purified enzyme was characterized: 1) NADH-dependent reduction of flavin mononucleotide (FMN), the Km values for NADH and FMN (208 and 10.8 μM. respectively), the optimal temperature and optimal pH (35^。C and 6.0, respectively), and the heat stability (30% activity retaining at 80^。C for 30 minutes). The flavin reductase gene was amplified with primers designed by using a DszD of R. erythropolis IGTS8, and the enzyme was overexpressed in Escherichiacoli. The specific activity in crude extracts of the overexpressed strain was about 275-fold that of the wild-type strain.(2) DszB of a DBT-desulfurizing bacterium Rhodococcus erythropolis KA2-5-1 was overproduced in Escherick coexpression with chaperonin genes, groEL/groES. The recombinant DszB was purified to homogeneity and character was thought to be a novel enzyme (HBPSi desulfinase) in that it could specifically cleave the carbon-sulfurbond of H2-HBP and sulfite ion without the aid of any other proteinic components and coenzymes. Less