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

点击翻译按钮获取中文摘要

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.