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

新型酶催化碳键形成和裂解的功能阐明及其应用

基本信息

批准号：
09650877
负责人：
IZUMI Yoshikazu
金额：
$ 2.05万
依托单位：
Tottori University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1997
资助国家：
日本
起止时间：
1997 至 1998
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09650877/
关键词：
biotin BioB bioB dibenzothiophen (DBT)DszC,A 脱硫菌 DszA DszC

项目摘要

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.

(1)生物素合成酶(1-1)生物素合成酶(Biob)的研究从高效表达BioB的球形芽孢杆菌无细胞提取转化子中高产率、高纯度地纯化了生物素合成酶(Biob)。利用纯化的Biob酶生物素类似物，发现在20 mM和10 mM的浓度下，生物素合成酶(TVA)和酸性霉素(ACM)完全参与合酶反应。通过对一株野生型无菌芽孢杆菌的无细胞提取液进行硫酸铵层析和柱层析，发现一种热稳定性的低分子量组分(LF)可以通过截留分子量为10,000的超膜，分子量组分(HF)是热标记的不透膜的分子量组分，二者单独对生物素合成酶反应都没有刺激活性，但在生物素合成酶的反应混合物中两者的活性w*被加在一起。(2)二苯并噻吩酶(DRI)、脱硫代谢酶(2-1)、DBT单加氧酶(DszC)和DBT单加氧酶(DszA)的纯化研究从DBT ck Rhodxoccus erympolis D-1中分离纯化了DBT单加氧酶(DszC)和DBT单加氧酶(DszA)。DszC和DszA被成功结晶。(2-2)催化DBT最后一步脱硫代谢的亚磺酸酶(DszB)也是从另一种DBT脱硫剂中分离得到的。