棉铃虫酯酶突变体的构建、表达及酶促动力学特性
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...
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| Published in | 浙江大学学报（农业与生命科学版） Vol. 40; no. 1; pp. 16 - 24 |
|---|---|
| Main Author | |
| Format | Journal Article |
| Language | Chinese |
| Published |
西北农林科技大学无公害农药研究服务中心，陕西 杨凌712100
2014
西北农林科技大学植物保护学院，陕西 杨凌712100 |
| Subjects | |
| Online Access | Get full text |
| ISSN | 1008-9209 |
| DOI | 10.3785/j.issn.1008-9209.2012.04.062 |
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| Abstract | 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0.09～0.33μmol^-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. |
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| AbstractList | 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0.09～0.33μmol^-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. Q966; 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0.09～0.33μmol-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_FL | 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 Lepidopterapestslikecottonbollworm,H.armigeratodate.Hence,followingourpreviousstudiestheaimofthe 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 (6.5 52.1 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 A 127 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 . |
| Author | 李永强 吴美玲 马志卿 冯俊涛 张兴 |
| AuthorAffiliation | 西北农林科技大学无公害农药研究服务中心,陕西 杨凌712100 西北农林科技大学植物保护学院,陕西 杨凌712100 |
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| Author_FL | Wu Meiling Ma Zhiqing Zhang Xing Feng Juntao Li Yongqiang |
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| DOI | 10.3785/j.issn.1008-9209.2012.04.062 |
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| Keywords | 氨基酸突变 4-硝基苯基乙酸酯 酯酶 amino acid mutations 酶促动力学 α-naphthyl acetate enzymatic kinetics carboxylesterases para-nitrophenyl acetate α-乙酸萘酯 |
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| Notes | The cotton bollworm , Helicoverpa armigera （ Hubner）,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 Lepidopterapestslikecottonbollworm,H.armigeratodate.Hence,followingourpreviousstudiestheaimofthe current work was to eluc |
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| Publisher | 西北农林科技大学无公害农药研究服务中心，陕西 杨凌712100 西北农林科技大学植物保护学院，陕西 杨凌712100 |
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| Snippet | 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... Q966; 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... |
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| SubjectTerms | 4-硝基苯基乙酸酯 α-乙酸萘酯 氨基酸突变 酯酶 酶促动力学 |
| Title | 棉铃虫酯酶突变体的构建、表达及酶促动力学特性 |
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