Altering the substrate specificity of organophosphorus hydrolase for enhanced hydrolysis of chlorpyrifos

被引:88
作者
Cho, CMH
Mulchandani, A
Chen, W [1 ]
机构
[1] Univ Calif Riverside, Dept Chem Environm Engn, Riverside, CA 92521 USA
[2] Univ Calif Riverside, Environm Toxicol Program, Riverside, CA 92521 USA
关键词
D O I
10.1128/AEM.70.8.4681-4685.2004
中图分类号
Q81 [生物工程学(生物技术)]; Q93 [微生物学];
学科分类号
071005 ; 0836 ; 090102 ; 100705 ;
摘要
Chlorpyrifos is one of the most popular pesticides used for agriculture crop protection, and widespread contamination is a potential concern. However, chlorpyrifos is hydrolyzed almost 1,000-fold slower than the preferred substrate, paraoxon, by organophosphorus hydrolase (OPH), an enzyme that can degrade a broad range of organophosphate pesticides. We have recently demonstrated that directed evolution can be used to generate OPH variants with up to 25-fold improvement in hydrolysis of methyl parathion. The obvious question and challenge are whether similar success could be achieved with this poorly hydrolyzed substrate, chlorpyrifos. For this study, five improved variants were selected from two rounds of directed evolution based on the formation of clear haloes on Luria-Bertani plates overlaid with chlorpyrifos. One variant, B3561, exhibited a 725-fold increase in the k(cat)/K-m value for chlorpyrifos hydrolysis as well as enhanced hydrolysis rates for several other OP compounds tested. Considering that wild-type OPH hydrolyzes paraoxon at a rate close to the diffusion control limit, the 39-fold improvement in hydrolysis of paraoxon by B3561 suggests that this variant is one of the most efficient enzymes available to attack a wide spectrum of organophosphate nerve agents.
引用
收藏
页码:4681 / 4685
页数:5
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