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.

已经阐明了2'-羟基二苯基2-硫酸脱硫酸盐脱硫酶（DSZB）的rhodoccus erythropolis（中等脱硫化细菌）的结构，也已被阐明，并且还揭示了酶 - 底物复合物的结构。这是关于Dibenzothiophene（DBT）脱硫酶中三维结构的第一个报告。酶结构通过转换底物，2'-羟基苯基2-硫酸酯而变化，他的60居住地转移到了催化中心。基于DSZB的结构，进行了定向的诱变以改善酶特性。发现某些突变酶的热稳定性高于野生型酶。菌株，枯草芽孢杆菌WU-S2B是嗜热脱硫细菌，可长达50℃。将参与DBT代谢的酶（BDSC，BDSA，BDSB）纯化为均匀性，构建了过量产生的大肠杆菌菌株，并研究了其酶特性。由于我们估计了BDSC对几种芳族化合物的活性，并发现BDSC利用Indole作为底物。该结果表明，BDSC催化没有DBT骨架的化合物的反应，这是脱硫酶的新功能。为了在较高温度下执行微生物脱硫，我们从嗜热菌株中纯化黄素含量减少了细菌菌株。我们获得了相应的基因，并用重组大肠杆菌菌株过度生产酶。生产率比野生型菌株高440倍。确认了与BDSC结合的有效反应。