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浙江大学学报（农业与生命科学版）
浙江大学学报(农业与生命科学版)  2014, Vol. 40 Issue (1): 16-24    DOI: 10.3785/j.issn.1008-9209.2012.04.062
生物科学与技术     
棉铃虫酯酶突变体的构建、表达及酶促动力学特性
李永强1,2, 吴美玲1，2， 马志卿1,2, 冯俊涛1,2, 张兴1,2*
（1.西北农林科技大学无公害农药研究服务中心,陕西 杨凌712100；2.西北农林科技大学植物保护学院，陕西 杨凌712100）
Construction, expression and activities of mutant carboxylesterases from Helicoverpa armigera
Li Yongqiang1,2, Wu Meiling1,2， Ma Zhiqing1,2,   Feng   Juntao1,2, Zhang Xing1,2*
(1. Research and Development Centre of Biorational Pesticides, Northwest A & F University, Yangling, Shaanxi 712100, China; 2. College of Plant Protection, Northwest A & F University, Yangling, Shaanxi 712100, China)
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摘要： 在前期研究的基础上，基于定点突变技术在棉铃虫酯酶001F和001G的A127位和F238位分别人工构建由丙氨酸（A）变为天冬氨酸（D）、苯丙氨酸（F）变为亮氨酸（L）的单、双位点突变体，利用杆状病毒载体表达系统对其进行异源真核表达，采用酶标仪测定表达产物对α-乙酸萘酯和4-硝基苯基乙酸酯的酶促动力学参数。结果表明：酯酶A127位突变降低了酯酶的酶促水解活性，其对底物的亲和力明显减弱（反应常数Km值变大），速率常数kcat/Km值仅为突变前的1/20～1/90；F238位突变对酯酶的酶促水解作用影响相对较小，其kcat/Km值为突变前的1/2～1/7；双位点突变体（A127D/F238L）几乎全部失去了对底物的酶促水解活性，其亲和力明显下降（Km值升高至突变前的4~30倍），kcat/Km值为0.09～0.33 μmol-1·s-1·L，仅为突变前的1/70～1/370。表明突变对酯酶活性有明显影响，A127位和F238位氨基酸残基是与酶催化功能相关的重要位点，这将为研究酯酶在棉铃虫代谢抗性中的作用提供一定的参考。
Abstract: The cotton bollworm, Helicoverpa armigera  (Hübner), is a major pest of many agricultural crops around the world, and many of the classes of chemical insecticides are widely used for its control such as organophosphates (OPs), synthetic pyrethroids (SPs), and so on. Currently H.armigera has developed serious resistance to OPs and SPs all over the world. Carboxylesterases (CarEs) are a multi-gene family of enzymes that hydrolyze a diverse range of carboxylesters and are frequently implicated in the resistance of insects. Gene mutations of CarEs are a major mechanism of insects for the development of resistance to OPs in the OP-resistant Diptera pests like Musca domestica and Lucilia cuprina. It involves the substitution of a single amino acid within the active site of the esterase which convert it to an OP hydrolyase. However, no resembled mutation in nature was reported in the Lepidoptera pests like cotton bollworm, H. armigera  to date. Hence, following our previous studies the aim of the current work was to elucidate the effects of specific point mutations of the carboxylesterases from H. armigera on the kinetic properties of the enzymes.
Two CarEs, 001F and 001G from H. armigera, were induced to mutate at positions 127 (A→D) or 238 (F→L) using a site-directed mutagenesis technique. They were then expressed with the baculovirus expression vector system (BEVS). The kinetic assays  with α-naphthyl acetate (α-NA) and para-nitrophenyl acetate (p-NA) were carried out for all mutants using a spectrophotometer.
The results showed that the A127D mutations had dramatically reduced the hydrolytic activities of the CarEs toward the two substrates. The mutants all showed lower affinities to the substrates as the Km values were at least 1.6-fold higher than those of the wild type enzymes, and the kcat values (6.552.1 s-1) were decreased obviously, which were between 4- and 20-fold lower than those of the wild type enzymes. What’s more, the rate constants (kcat/Km values) of the A127D mutants  (0.12 and 1.2 μmol-1·s-1·L)  were also substantially declined, which were about between 20- and 90-fold lower than those of the wild type enzymes. By contrast, the F238L mutations had relatively less effects on the kinetic properties of the enzymes than the A127D mutations. The kcat values of these mutants were slightly lower than those of the wild type enzymes with the exception of the 001F F238L against α-NA, and the kcat/Km values were just between 1.5- and 7-fold lower than those of the wild type enzymes. For the double-mutation at both sites, however, they had remarkable effects on the hydrolytic activities toward the substrates. These mutants showed obviously poor affinities to the substrates as the Km values were between 4- and 30-fold higher compared to the the wild type CarEs. The kcat values of these mutants were between 10- and 15-fold lower than those of the wild type CarEs, and they nearly nullified all the hydrolytic activities shown by the very lower rate constants, which were only between 0.09 and 0.33 μmol-1·s-1·L, and between 70- to 370-fold lower than those of the wild type CarEs.
These results indicate  that the mutation at positions 127 (A→D) and 238 (F→L) in CarEs has  marked effects on their enzymatic activities, and it also strongly  suggests  that the positions A127 and F238 are two important active sites in enzymes involving in the catalytic function. Hence, this study can provide useful data for further understanding the functions of the CarEs in metabolic resistance of cotton bollworm,  H. armigera  in future.
出版日期: 2014-01-20
CLC:  Q 966  
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李永强1
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冯俊涛1
张兴1
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引用本文:

李永强1,2, 吴美玲1，2， 马志卿1,2, 冯俊涛1,2, 张兴1,2*. 棉铃虫酯酶突变体的构建、表达及酶促动力学特性[J]. 浙江大学学报(农业与生命科学版), 2014, 40(1): 16-24.

Li Yongqiang1,2, Wu Meiling1,2， Ma Zhiqing1,2, Feng Juntao1,2, Zhang Xing1,2*. Construction, expression and activities of mutant carboxylesterases from Helicoverpa armigera. Journal of Zhejiang University (Agriculture and Life Sciences), 2014, 40(1): 16-24.

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http://www.zjujournals.com/agr/CN/10.3785/j.issn.1008-9209.2012.04.062    或    http://www.zjujournals.com/agr/CN/Y2014/V40/I1/16

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