Development of dehalogenases by molecular evolution technology : Application of production of useful materials and bioremediation of environments

该项目的目的是开发可用于环境生物修复和生产有用化合物的脱卤酶。主要研究成果如下：1.研究成果。本研究以DL-2-氯丙酸酯为唯一碳源，从静冈滨松的三那湖分离到一株脱卤酶产生菌--甲基乳杆菌CPA1。我们从细胞提取液中纯化了DL-2-卤代酸脱卤酶，并对其基因进行了克隆和测序。该酶作用于D-氯丙酸酯和L-2-氯丙酸酯，催化卤离子的释放。氯乙酸酯和溴乙酸酯也是底物，但氟乙酸酯不是底物。该酶还作用于2-氯丙酰胺。我们分离到一株产氟乙酸酯脱卤酶的土壤细菌Burkholderia sp.FA1，该酶能催化氟乙酸酯的水解性脱氟。该酶对氟乙酸酯的作用远好于对氯乙酸酯和溴乙酸酯的作用。分离了该酶的编码基因，并测定了该酶的一级结构。我们分析了DL-2-卤酸脱卤酶的反应机理，发现水分子直接攻击底物的α-碳原子生成相应的2-羟基烷酸。这一机制与其他脱卤酶不同：在其他脱卤酶的反应中，酶的天冬氨酸残基攻击底物的α-碳原子产生酯中间体，该中间体随后被水分子水解。对L-2-卤代酸脱卤酶D10N突变酶的反应机理进行了分析，发现AsN10作为催化残基，β-氰基丙氨酸残基作为中间结构。

The aim of this project is to develop dehalogenases that can be used for bioremediation of environments and production of useful compounds. The research results are as follows.1. We isolated a dehalogenase-producing bacterium, Methylobacterium sp.CPA1, from Lake Sanaru, Hamamatsu, Shizuoka using DL-2-chloropropionate as the sole carbon source for screening. We purified DL-2-haloacid dehalogenase from the cell extract, and cloned and sequenced its gene. The enzyme acted on both D- and L-2-chloropropionate to catalyze the release of the halide ion. Chloroacetate and bromoacetate also served as the substrates, but fluoroacetate was not the substrate. The enzyme also acted on 2-chloropropionamide.2. We isolated a soil bacterium, Burkholderia sp.FA1, producing fluoroacetate dehalogenase, which catalyzes hydrolytic defluorination of fluoroacetate. The enzyme acted much better on fluoroacetate than on chloroacetate and bromoacetate. The gene coding for the enzyme was isolated, and the primary structure of the enzyme was determined.3. We analyzed the reaction mechanism of DL-2-haloacid dehalogenase, and revealed that a water molecule directly attacks the α-carbon atom of the substrate to produce the corresponding 2-hydroxyalkanoic acid. This mechanism is different from that of other dehalogenases : in the reactions of other dehalogenases, an aspartate residue of the enzyme attacks the α-carbon atom of the substrate to produce an ester intermediate, and this intermediate is subsequently hydrolyzed by a water molecule.4. We analyzed the reaction mechanism of L-2-haloacid dehalogenase D10N mutant enzyme, and found that Asn 10 functions as a catalytic residue and a β-cyanoalanine residue is produced as an intermediate structure.