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

研究了中度脱硫菌红红红球菌（Rhodococcus erythropolis）中2′-羟基联苯2-亚磺酸脱硫酶（DszB）的结构，并揭示了酶-底物复合物的结构。本文首次报道了二苯并噻吩（DBT）脱硫酶的三维结构。通过加入底物2'-羟基联苯2-亚磺酸盐，酶的结构发生了变化，并且他的60残基进入了催化中心。基于DszB的结构，对其进行定点诱变以改善酶的性能。结果发现，一些突变体酶的热稳定性高于野生型酶。菌株枯草芽孢杆菌WU-S2B是一种可在50℃下生长的嗜热脱硫菌。将参与DBT代谢的酶（BdsC、BdsA、BdsB）纯化至均匀性，构建高产大肠杆菌菌株，并对其酶学性质进行研究。由于我们测定了BdsC对几种芳香族化合物的活性，发现BdsC利用吲哚作为底物。结果表明，BdsC可以催化没有DBT骨架的化合物的反应，是脱硫酶的新功能。为了在高温下进行微生物脱硫，我们从嗜热菌株芽孢杆菌（Bacillus sp.DSM411）中纯化黄素还原酶。我们获得了相应的基因，并利用重组大肠杆菌菌株过量生产了该酶。产量是野生型菌株的440倍。证实了BdsC偶联反应的有效性。

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.