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Conversion of Organofluorine Compounds with Microbial Enzymes : Mechanistic Analysis of the Enzyme Reactions and Their Application to Production of Useful Compounds and Bioremediation of Environments

用微生物酶转化有机氟化合物：酶反应的机理分析及其在有用化合物生产和环境生物修复中的应用

基本信息

批准号：
17360397
负责人：
KURIHARA Tatsuo
金额：
$ 10.78万
依托单位：
Kyoto University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2005
资助国家：
日本
起止时间：
2005 至 2007
项目状态：
已结题

项目摘要

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.

1.氟乙酸脱卤酶催化氟乙酸水解脱卤生成乙醇酸。酶的D104亲核攻击底物的α-碳原子以释放氟离子并形成酯中间体，其中底物衍生部分与酶共价结合。中间体被H271活化的水分子水解，释放乙醇酸盐并再生D104。野生型酶的X射线晶体学分析，D104 N突变体酶与氟乙酸（米氏复合物），和11271 A突变体酶与氯乙酸（酯中间体）复合支持拟议的反应机制。基质的羧酸酯基与H149、W150、Y212、R105和R108结合，氟原子与R108结合。我们发现W150 F突变体酶不作用于氟乙酸，而突变体对氯乙酸的活性与野生型酶相似，表明W150是脱氟所特异性需要的.我们通过以1，1，1-三氯-2，2，2-三氟乙烷（氟利昂113 a）作为电子受体的富集培养获得了一株属于硫杆菌属的厌氧细菌。以反氯乙烯为电子受体进行富集培养，获得了一株厌氧细菌，其16 SrRNA序列与未培养细菌的16 SrRNA序列有98%的同源性.我们构建了一个重组大肠杆菌菌株生产2-氯丙烯酸酯还原酶从伯克霍尔德菌属物种WS和葡萄糖脱氢酶，这是一个NADPH再生酶。利用重组E.大肠杆菌细胞。该方法在产物收率方面上级常规方法（光学拆分）。