Elucidation of functions of novel enzymes catalyzing the formation and the cleavage of cabond and their application

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The research results of this study are summarized as follows.(1) Studies on biotin synthase(1-1) Biotin sythase (BioB) was purified with a high yield and with a high purity from cell-free extract transformant of Bacillus sphaericus which could overexpress BioB.Using the purified BioB enzyme biotin analogs, thiohene valeric acid (TVA) and acidomycin (ACM), were found to completely in synthase reaction at a concentration of 20 mM and 10 mM, respectively.(1-2) Stimulatory factors essential for biotin synthase reactions were found. Through ammonium sulfate fi* and column chromatographies of cell-free extract of a wild type strain of Bacillus aphaericus, a low weight fraction (LF), which was heat-stable and passed through ultramembrane of cut-off MW=10,000, molecular weight fraction (HF), which was heat-lable and did not pass through the membrane, wen* Neither of LFand HF alone showed stimulatory activity toward biotin synthase reaction, but activity w* both of them were added together in the reaction mixutre of biotin synthase. Since mon* stimulatory proteins may exist in the HF.further purification of HF was considered to be necessary(2) Studies on the purification of enzymes of dibenzothiophene (DRI) desulfurization metabolism(2-1) DBT monoxygenase (DszC) and DBT sulfone monooxygenase (DszA) were purified from a DBT ck Rhodxoccus eryrhmpolis D-1 and their reactions were found to essentially rerpuire reductase. I DszC and DszA were successfully ciystallized.(2-2) Sulfinase (DszB) which catalyzes the last step of DBT desulfurization metabolism, was also purl degree from another DBT desulfurizing Rhod2coccus eythmpolis KA2-5-l by various chromatoraphies.