三唑磷水解酶基因的克隆及水解产物的确定

Cloning of triazophos hydrolase gene and the determination of the hydrolysate.

利用鸟枪法从三唑磷降解菌株mp-4(Ochrobactrum sp.)中克隆了三唑磷水解酶基因tpd,序列分析发现,该基因与甲基对硫磷水解酶基因mpd的同源性为98%,其结构基因含有996个碱基,除终止密码子外编码331个氨基酸,但该基因编码的酶比mpd编码的酶底物范围更广,不仅能水解甲基对硫磷,而且能高效水解三唑磷.利用克隆到的tpd基因编码的水解酶水解三唑磷,将水解反应产物进行液相色谱-质谱联机分析,发现该水解产物分子量为161,经理论分析确定该水解产物为1-苯基-3-羟基-1,2,4-三唑.

A triazophos degrading-bacterium strain mp-4（Ochrobactrum sp.） was isolated and stored. Triazophos hydrolase tpd gene was cloned by shotgun cloning method from mp-4. The tpd gene shared 98% homology with parathion-methyl hydrolase gene mpd, and its structure gene had 996 base pairs, coding 331 amino acid resides and a stop codon. The enzyme contained gene tpd could hydrolyze parathionmethyl and triazophos efficiently. The hydrolyzed pruduct of triazophos was determined as 1-benzene-3-hydroxy -1,2,4-triazol by its moleuler weight of 161 Dalton by LC-MS.