浙江大学学报（农业与生命科学版）
浙江大學學報（農業與生命科學版）
절강대학학보（농업여생명과학판）
JOURNAL OF ZHEJIANG UNIVERSITY(AGRICULTURE & LIFE SCIENCES)
2014年
1期
16-24
,共9页
李永强%吴美玲%马志卿%冯俊涛%张兴
李永彊%吳美玲%馬誌卿%馮俊濤%張興
리영강%오미령%마지경%풍준도%장흥
酯酶%氨基酸突变%α-乙酸萘酯%4-硝基苯基乙酸酯%酶促动力学
酯酶%氨基痠突變%α-乙痠萘酯%4-硝基苯基乙痠酯%酶促動力學
지매%안기산돌변%α-을산내지%4-초기분기을산지%매촉동역학
carboxylesterases%amino acid mutations%α-naphthyl acetate%para-nitrophenyl acetate%enzymatic kinetics
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.
在前期研究的基礎上,基于定點突變技術在棉鈴蟲酯酶001F和001G的A127位和F238位分彆人工構建由丙氨痠(A)變為天鼕氨痠(D)、苯丙氨痠(F)變為亮氨痠(L)的單、雙位點突變體,利用桿狀病毒載體錶達繫統對其進行異源真覈錶達,採用酶標儀測定錶達產物對α-乙痠萘酯和4-硝基苯基乙痠酯的酶促動力學參數.結果錶明：酯酶A127位突變降低瞭酯酶的酶促水解活性,其對底物的親和力明顯減弱(反應常數Km值變大),速率常數kcat/Km值僅為突變前的1/20～1/90；F238位突變對酯酶的酶促水解作用影響相對較小,其kcat/Km值為突變前的1/2～1/7；雙位點突變體(A127D/F238L)幾乎全部失去瞭對底物的酶促水解活性,其親和力明顯下降(Km值升高至突變前的4～30倍), kcat/Km值為0.09～0.33μmol-1. s-1. L,僅為突變前的1/70～1/370.錶明突變對酯酶活性有明顯影響, A127位和F238位氨基痠殘基是與酶催化功能相關的重要位點,這將為研究酯酶在棉鈴蟲代謝抗性中的作用提供一定的參攷.
재전기연구적기출상,기우정점돌변기술재면령충지매001F화001G적A127위화F238위분별인공구건유병안산(A)변위천동안산(D)、분병안산(F)변위량안산(L)적단、쌍위점돌변체,이용간상병독재체표체계통대기진행이원진핵표체,채용매표의측정표체산물대α-을산내지화4-초기분기을산지적매촉동역학삼수.결과표명：지매A127위돌변강저료지매적매촉수해활성,기대저물적친화력명현감약(반응상수Km치변대),속솔상수kcat/Km치부위돌변전적1/20～1/90；F238위돌변대지매적매촉수해작용영향상대교소,기kcat/Km치위돌변전적1/2～1/7；쌍위점돌변체(A127D/F238L)궤호전부실거료대저물적매촉수해활성,기친화력명현하강(Km치승고지돌변전적4～30배), kcat/Km치위0.09～0.33μmol-1. s-1. L,부위돌변전적1/70～1/370.표명돌변대지매활성유명현영향, A127위화F238위안기산잔기시여매최화공능상관적중요위점,저장위연구지매재면령충대사항성중적작용제공일정적삼고.
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 .
