| Budget Amount *help |
¥3,800,000 (Direct Cost: ¥3,800,000)
Fiscal Year 2006: ¥700,000 (Direct Cost: ¥700,000)
Fiscal Year 2005: ¥700,000 (Direct Cost: ¥700,000)
Fiscal Year 2004: ¥2,400,000 (Direct Cost: ¥2,400,000)
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| Research Abstract |
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.
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