Altering the substrate specificity of organophosphorus hydrolase for enhanced hydrolysis of chlorpyrifos
Altering the substrate specificity of organophosphorus hydrolase for enhanced hydrolysis of chlorpyrifos
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DOI：
10.1128/aem.70.8.4681-4685.2004
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发表时间：
2004-08-01
影响因子：
4.4
通讯作者：
Chen, W
中科院分区：
文献类型：
--
作者：
Cho, CMH;Mulchandani, A;Chen, W
毒死蜱是用于农业作物保护的最受欢迎的农药之一，广泛的污染是一个潜在的问题。然而，毒死蜱被有机磷水解酶（OPH）水解的速度几乎比首选底物对氧磷慢1,000倍，OPH是一种可以降解多种有机磷农药的酶。我们最近已经证明，定向进化可用于产生OPH变体，其在甲基异戊二醇水解方面具有高达25倍的改进。显而易见的问题和挑战是，用这种水解不良的底物毒死蜱是否能取得类似的成功。在这项研究中，五个改进的变体是从两轮定向进化的基础上形成的清晰的晕覆盖毒死蜱的Luria-Bertani板。一种变体B3561表现出毒死蜱水解的k（cat）/K-m值增加了725倍，并且测试的几种其他OP化合物的水解速率增强。考虑到野生型OPH以接近扩散控制极限的速率水解对氧磷，B3561对对氧磷水解的39倍改善表明该变体是可用于攻击广谱有机磷酸盐神经毒剂的最有效的酶之一。
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.