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期 刊 ：植物保护学报 2012年

关键词：酯酶；氨基酸突变；有机磷；顺式氰戊菊酯；代谢活性；

Keywords:esterase, amino acid mutation, organophosphate, esfenvalerate, metabolic activity,

摘 要 ：为研究棉铃虫酯酶对有机磷和拟除虫菊酯类杀虫剂的代谢活性,在前期对酯酶001F和001G酶活性研究的基础上,进一步采用荧光法和高效液相色谱法测定了酯酶及其突变体(A127位和F238位突变)对二甲基-4甲基伞形酮磷酸酯(dMUP)、二乙基-7羟基香豆素磷酸酯(dECP)及顺式氰戊菊酯(2S, αS)的酶促代谢活性。酯酶001F和001G对有机磷的酶促反应常数(kcat值)在2.0 × 10^-3/min~5.2 × 10^-3/min之间,后者对顺式氰戊菊酯的代谢活性为10.19 μmol·min^-1·μmol^-1;001F的突变提高了对有机磷的代谢活性,其中001F^A127D和001F^F238L对dMUP的kcat值至少是突变前的2倍;001G的突变却降低了对dECP的代谢活性,但001G^F238L对dMUP的活性有所提高。此外,突变体中001F^A127D对顺式氰戊菊酯的活性与突变前接近,而001G^F238L则降低到突变前的1/2,其它突变体未检测到活性。结果表明,酯酶001F和001G对杀虫剂有代谢活性,A127位和F238位的突变对酶活性有显著影响。

Abstract:In order to understand the metabolic activities of esterases in Helicoverpa armigera toward organophosphorus (OPs) and pyrethroid insecticides, the catalytic activities of two esterases (001F and 001G) and their mutants (mutated at positions A127 and F238) toward three kinds of insecticides (i.e., dimethyl- and diethyl- phosphates (dMUP and dECP) and esfenvalerate (2S, αS) ) were measured with fluorometric and HPLC assays. The results showed the enzymatic constants (kcat) of 001F and 001G for OPs were between 2.0 × 10^-3/min and 5.2 × 10^-3/min, and 001G also had some metabolic activities toward esfenvalerate (10.19 μmol·min^-1·μmol^-1). The mutants of 001F showed enhanced activities for OPs, the kcat values of both 001F^A127D and 001F^F238L for dMUP (4.5×10^-3 /min and 6.1×10^-3/min respectively) were at least 2-fold higher than that of the wild type enzyme. However, the mutants of 001G obviously reduced the activities against dECP, and only 001G^F238L slightly improved the metabolic activities against dMUP. Among the mutants, the activity of 001F^A127D toward esfenvalerate resembled its wild type enzyme, but the activity of 001G^F238L toward esfenvalerate was only about half that of its wild type enzyme, and all others mutants showed undetectable activities. The 001F and 001G had metabolic activities against OPs and pyrethroids, and mutation at both positions A127 and F238 in esterases had marked effects on their catalytic activities.

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