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Elucidation of properties of novel enzymes catalyzing the formation and the cleavage of carbon-sulfur bond in microooganisms

阐明微生物中催化碳硫键形成和断裂的新型酶的特性

基本信息

批准号：
11660091
负责人：
IZUMI Yoshikazu
金额：
$ 1.98万
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：
1999
资助国家：
日本
起止时间：
1999 至 2000
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-11660091/
关键词：
biotin BioB bioB dibenzothiophen (DBT)DszC,A フラビンレダクターゼ DszA ビオチン BioB bioB

项目摘要

The research results of this study are summarized as follows.(1) Studies on biotin synthase(1-1) Biotin sythase (BioB), the enzyme responsible for the last step of biotin biosynthesis by microorganisms, is known to supply a sulfur atom in the Fe-S cluster of the enzyme. It was purified with a high yield and with a high purity from cell-free extract of a bioB transformant of Bacillus sphaericus which could overproduce BioB.Using the purified BioB enzyme preparation, biotin analogs, thiophene valeric acid (TVA) and acidomycin (ACM), were found to completely inhibit biotin 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 fractionation and column chromatographies of cell-free extract of a wild type strain of B.sphaericus, a low-molecular weight fraction (LF), which was heat-stable and passed through ultramembrane of cutoff MW=10,000, and a high molecular weight fraction (HF), which was heat-lable and did not pass through the membrane, were obtained. Neither of LF and HF alone showed stimulatory activity toward biotin synthase reaction, but activity was obtained when both of them were added together in the reaction mixutre of biotin synthase. Since more than two 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 (DBT) desulfurization metabolism(2-1) DBT monoxygenase (DszC) and DBT sulfone monooxygenase (DszA) were purified from a DBT desulfurizing Rhodococcus erythropolis D-1 and their reactions were found to essentially require reductase. In addition, DszC and DszA were successfully crystallized.(2-2) Sulfinase (DszB) which catalyzes the last step of DBT desulfurization metabolism, was also purified to a high degree from another DBT desulfurizing Rhodococcus erythropolis KA2-5-1 by various column chromatoraphies.

本研究的研究成果归纳如下。(1)生物素合成酶的研究（1-1）生物素合成酶（BioB）是微生物生物素合成的最后一步酶，已知在该酶的Fe-S簇中提供硫原子。它是纯化的高产率和高纯度的无细胞提取物的bioB的球形芽孢杆菌，可以过度生产BioB。使用纯化的BioB酶制剂，生物素类似物，噻吩戊酸（TVA）和酸性霉素（ACM），被发现完全抑制生物素合成酶反应，分别在20 mM和10 mM的浓度。(1-2)通过对球形B.sphaericus野生型菌株的无细胞提取物进行硫酸铵分级分离和柱层析，得到热稳定的并通过截留MW= 10，000的超膜的低分子量级分（LF）和热不稳定的且不通过膜的高分子量级分（HF），获得了LF和HF单独对生物素合成酶反应均无刺激活性，但当两者一起加入生物素合成酶反应混合物中时，获得了活性。（2）二苯并噻吩（DBT）脱硫代谢酶的纯化研究（2-1）从DBT发酵菌红串红球菌（Rhodococcus erythropolis）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上需要还原酶。此外，成功地结晶了DszC和DszA。(2-2)催化DBT脱硫代谢最后一步的亚硫酸酶（DszB）也通过多种柱层析从另一株DBT脱硫红平红球菌KA 2 -5-1中高度纯化。