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

本研究的研究成果归纳如下。(1)生物素合成酶的研究（1-1）从能过表达生物素合成酶（BioB）的球形芽孢杆菌（Bacillus sphaericus）的无细胞提取物中以高产率和高纯度纯化了生物素合成酶（BioB）。使用纯化的BioB酶生物素类似物，发现噻吩戊酸（TVA）和酸性霉素（ACM）分别在20 mM和10 mM的浓度下完全参与合成酶反应。(1-2)通过硫酸铵过滤和柱色谱法对无菌芽孢杆菌（Bacillusaphaericus）野生型菌株的无细胞提取物进行分离，得到了低分子量组分（LF），它是热稳定的，能通过截留分子量为10，000的超膜，分子量组分（HF），它是热不稳定的，不能通过超膜，LF和HF单独对生物素合酶反应均无刺激活性，但两者同时加入生物素合酶反应混合物中时，对生物素合酶反应均有刺激活性。（2）二苯并噻吩（DRI）脱硫代谢酶纯化的研究（2-1）从DBT ck Rhodxoccus erythmpolis D-1中分离纯化了DBT单加氧酶（DszC）和DBT砜单加氧酶（DszA），发现它们的反应基本上是还原酶。成功地结晶了DszC和DszA。(2-2)另外，还从另一株DBT菌株Rhod 2coccus eythmpolis KA 2 -5- 1中分离到了催化DBT脱硫代谢最后一步的亚硫酸酶DszB。

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.