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

本研究的研究结果总结如下：(1)生物素合成酶的研究（1-1）生物素合成酶（biotin sythase, biotin sythase, BioB）是由能过表达BioB的球形芽孢杆菌（Bacillus sphaericus）的无细胞提取物转化中高产、高纯度纯化得到的。使用纯化的BioB酶生物素类似物，噻吩戊酸（TVA）和酸霉素（ACM）分别在20 mM和10 mM的浓度下完全发生合成酶反应。（1-2）发现了生物素合成酶反应必需的刺激因子。通过对一株野生型无细胞芽孢杆菌提取物的硫酸铵和柱层析，发现热稳定的低质量分数（LF）和热稳定的不通过膜的分子量分数（HF），分别对生物素合成酶反应没有刺激作用。但在生物素合酶的反应混合物中，将两者的活性w*加在一起。因为HF中可能存在刺激蛋白。(2)对二苯并噻吩（DRI）脱硫代谢酶（2-1）的纯化进行了研究，从一株红红球菌D-1中纯化出DBT单加氧酶（DszC）和DBT砜单加氧酶（DszA），发现它们的反应基本需要还原酶。DszC和DszA成功结晶。（2-2）催化DBT脱硫代谢最后一步的亚砜酶（Sulfinase, DszB）也通过各种层析从另一种DBT脱硫剂红球菌ka2 -5- 1中分离出来。

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.