|Budget Amount *help
¥16,840,000 (Direct Cost: ¥15,700,000、Indirect Cost: ¥1,140,000)
Fiscal Year 2007: ¥4,940,000 (Direct Cost: ¥3,800,000、Indirect Cost: ¥1,140,000)
Fiscal Year 2006: ¥4,600,000 (Direct Cost: ¥4,600,000)
Fiscal Year 2005: ¥7,300,000 (Direct Cost: ¥7,300,000)
1. Fluoroacetate dehalogenase catalyzes the hydrolytic defluorination of fluoroacetate to produce glycolate. D104 of the enzyme nucleophilically attacks the α-carbon atom of the substrate to release the fluoride ion and form an ester intermediate, in which the substrate-derived moiety is covalently bound to the enzyme. The intermediate is hydrolyzed by a water molecule activated by H271 to release glycolate and regenerate D104. X-ray crystallographic analysis of the wild-type enzyme, the D104N mutant enzyme complexed with fluoroacetate (Michaelis complex), and the 11271A mutant enzyme complexed with chloroacetate (ester intermediate) supported the proposed reaction mechanism. The carboxylate group of the substrate was bound to H149, W150, Y212, R105, and R108, and the fluorine atom was bound to R108. We found that the W150F mutant enzyme does not act on fluoroacetate, whereas the activity of the mutant toward chloroacetate was similar to that of the wild-type enzyme, indicating that W150 is specifically required for the defluorination.
2. We obtained an anaerobic bacterium belonging to the genus Sulfurospirillum by enrichment culture with 1,1,1-trichloro-2,2,2-trifluoroethane (Freon 113a) as an electron acceptor. By enrichment culture with retrachlorcethene as an electron acceptor, we obtained an anaerobic bacterium whose 16S rRNA sequence shows 98% identity with that of an uncultured bacterium.
3. We constructed a recombinant Escherichia coli strain producing 2-chloroacrylate reductase from Burkholderia sp. WS and glucose dehydrogenase, which serves as an NADPH regeneration enzyme. (S)-2-Chloropropionate, a precursor for the synthesis of herbicides, was produced by asymmetric reduction of 2-chloroacrylate by using the recombinant E. coli cells. This method was superior to the conventional method (optical resolution) in the yield of the product.