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Research of Enzymes Involved in Metabolism of Organic Sulfur Compounds -Improvement by Protein Engineering and Search of Novel Functions-

有机硫化合物代谢相关酶的研究-蛋白质工程改良及新功能探索-

基本信息

批准号：
16580058
负责人：
OHSHIRO Takashi
金额：
$ 2.43万
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：
2004
资助国家：
日本
起止时间：
2004 至 2006
项目状态：
已结题

项目摘要

The structure of 2'-hydroxybiphenyl 2-sulfinate desulfinase (DszB) from Rhodococcus erythropolis, the moderately desulfurization bacterium, has been elucidated, and that of the enzyme-substrate complex has been also revealed. This is the first report about the three dimensional structure among dibenzothiophene (DBT) desulfurizing enzymes. The enzyme structure changed by incorporating the substrate, 2'-hydroxybiphenyl 2-sulfinate, and His 60 residue moved into the catalytic center. Based upon the structure of DszB, the site-directed mutagenesis was performed to improve the enzyme property. It was found that some mutant enzymes had higher thermal stability than the wild-type enzyme.The strain, Bacillus subtilis WU-S2B, is the thermophilic desulfurizing bacterium, which could grow up to 50℃. The enzymes (BdsC, BdsA, BdsB) involved in DBT metabolism were purified to homogeneity, the overproducing E.coli strains were constructed, and their enzymatic properties were investigated. Since we estimated the activity of BdsC toward several aromatic compounds and found out that BdsC utilize indole as a substrate. This result shows that BdsC catalyzes the reactions of compounds without the DBT skeleton, and it is the new function of the desulfurizing enzymes.In order to perform the microbial desulfurization at higher temperature, we purified flavin reductase from the thermophilic strain, Bacillus sp.DSM411. We obtained the corresponding gene and overproduce the enzyme with the recombinant E.coli strain. The productivity was 440 fold higher than the wild-type strain. The efficient reaction coupled with BdsC was confirmed.

本文报道了红平红球菌（Rhodococcus erythropolis）的2 ′-羟基联苯-2-亚磺酸酯酰化酶（DszB）的结构，以及酶-底物复合物的结构。这是首次报道二苯并噻吩（DBT）还原酶的三维结构。酶的结构发生了变化，通过纳入底物，2 '-羟基联苯2-亚磺酸酯，和His 60残基移动到催化中心。根据DszB的结构，进行定点突变，以提高酶的性质。结果表明，突变菌株的热稳定性比野生型酶高，其中枯草芽孢杆菌WU-S2 B是一株嗜热芽孢杆菌，可在50℃下生长。对DBT代谢相关酶（BdsC、BdsA、BdsB）进行了纯化，构建了高产菌株，并对其酶学性质进行了研究。由于我们测定了BdsC对几种芳香族化合物的活性，发现BdsC以吲哚为底物。这一结果表明，BdsC催化不含DBT骨架的化合物的反应，这是脱硫酶的新功能。为了在更高温度下进行微生物脱硫，我们从嗜热菌株Bacillussp. DSM 411中纯化了黄素还原酶。我们获得了相应的基因，并利用重组菌株进行了高产。产率比野生型菌株高440倍。证实了与BdsC偶联的有效反应。