棉铃虫羧酸酯酶表达及解毒作用研究
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摘要
羧酸酯酶是昆虫对有机磷、拟除虫菊酯、氨基甲酸酯等杀虫剂进行解毒作用的重要代谢酶系。为研究羧酸酯酶在棉铃虫有机磷、拟除虫菊酯代谢抗性中的作用和角色以及解毒酶在环境修复中的应用，本文采用杆状病毒表达载体系统（BEVS）对澳洲棉铃虫GR品系的9个羧酸酯酶基因进行了异源真核表达，从分子和生化水平上研究了其对有机磷、拟除虫菊酯类杀虫剂的酶促解毒特性；并利用氨基酸定点突变技术人工构建了1～2个位点突变的18个羧酸酯酶突变体，研究了突变对有机磷及拟除虫菊酯类杀虫剂的解毒活性的影响；在此基础上初步构建了解毒酶工程菌，旨在为环境污染修复奠定基础。主要研究结果如下：
1.棉铃虫羧酸酯酶在昆虫杆状病毒表达载体系统中的表达及酶活分析
从棉铃虫GR品系的cDNA文库中得到9个羧酸酯酶基因，氨基酸序列比对表明其均具有酯酶活性必须的催化三联体残基（S-E-H），以及酯酶家族的特征基序（G-x-S-x-G）和酶活相关的氧阴离子洞氨基酸残基（G-G-A），表明均为有酶促活性的酯酶基因。酯酶与杆状病毒线型DNA重组后，转染草地夜蛾Sf9细胞系，表达和制备了具有天然酶活性的酯酶蛋白产物。Native-PAGE和α-乙酸萘酯检测表明，酯酶条带主要分布在相对迁移率（Rm值）为0.27～0.35和0.39～0.48之间的两个区域，很可能是与棉铃虫代谢抗性相关酯酶。紫外读板机测定结果进一步表明，酯酶对α-乙酸萘酯底物的亲和系数（Km值）在3.3μmol/L～671μmol/L之间，速率常数（kcat/Km值）在0.3μmol~(-1)·S-1·L～95μmol~(-1)·S~(-1)·L之间。酯酶对α-乙酸萘酯的酶促水解活性普遍高于4-硝基苯基乙酸酯。
2.棉铃虫羧酸酯酶对有机磷的解毒活性
采用荧光法根据酯酶与2种有机磷底物的酶促反应特点，测定了表达产物中的酯酶浓度及解毒活性，结果表明8个酯酶对二甲基和二乙基有机磷（dMUP和dECP）均有一定代谢活性，催化反应常数（kcat值）在1.5×10-3min~(-1)～15.8×10-3min~(-1)之间，与对照铜绿蝇酯酶E3（kcat=3.5×10-3min~(-1)）的代谢活性处在同一水平范围。棉铃虫酯酶对模式羧酸酯底物的水解活性与其对有机磷杀虫剂的解毒活性无直接相关性。
3.棉铃虫羧酸酯酶对氰戊菊酯和氯氰菊酯异构体的解毒活性
通过荧光仪和高效液相色谱（HPLC）仪测定了其对氰戊菊酯（类型I）1种异构体2(S)-α(S)及4种异构体类似物的解毒活性，结果表明4个酯酶（001B、001C、001D、001J）对异构体2(R)-α(R)和2(R)-α(S)类似物的酶促解毒活性较高；其中酯酶001G对顺式氰戊菊酯杀虫剂异构体2(S)-α(S)的解毒活性为13.19μmol·min~(-1)·μmol~(-1)，其余酯酶解毒活性均较低。酯酶对氯氰菊酯8种异构体（类型II）及异构体类似物的测定结果表明，其中4个酯酶（001B、001C、001D和001J）对氯氰菊酯的2种反式异构体1(S) trans及类似物的解毒活性最高（17μmol·min~(-1)·μmol~(-1)～43μmol·min~(-1)·μmol~(-1)），但对杀虫毒性最高的异构体1(R)cis-α(S)及其类似物的解毒活性却相对很低；另外4个酯酶对氯氰菊酯异构体及类似物代谢作用极弱甚至难以检测到。
4.棉铃虫羧酸酯酶突变体的构建及对模式羧酸酯的酶促动力学特性
基于氨基酸序列比对和分析，采用定点突变技术构建了在棉铃虫酯酶氧阴离子洞和酰基化口袋内1～2个氨基酸位点突变的18个酯酶突变体。酶促动力学分析表明，酯酶氧阴离子洞丙氨酸（或甘氨酸）变为天冬氨酸（A/G→D）的突变体对α-乙酸萘酯的亲和力普遍降低（Km值升高），速率常数（kcat/Km值）也降低至突变前的1/20～1/437；酰基化口袋的苯丙氨酸变为亮氨酸的突变（F→L）同样降低了酯酶对羧酸酯底物的亲和力和水解活性，但与氧阴离子洞突变相比对酶的水解活性影响相对较小，速率常数最低降至突变前的1/5；酯酶在上述两个位点同时突变的双位点突变体(A/G→D+F→L)则几乎完全失去了对底物的酶促水解活性。上述酯酶突变也显著降低了酯酶对另一种模式羧酸酯底物4-硝基苯基乙酸酯的酶促水解活性。
5.羧酸酯酶突变对有机磷的解毒活性影响
酯酶单双位点的突变对有机磷的解毒活性也产生了较大的影响，部分突变体提高了对有机磷的解毒活性，相反部分突变体则降低了解毒活性。氧阴离子洞突变的部分突变体增强了对有机磷底物的解毒作用，尤其是001CA127D对二乙基有机磷dECP的kcat值高达32.8×10-3min~(-1)，是突变前的7倍。酯酶酰基化口袋突变的5个突变体，对分子结构较小的二甲基有机磷dMUP的kcat值均提高到突变前的2倍以上，解毒活性均高于分子结构较大的二乙基有机磷dECP，表现出对dMUP的代谢偏爱性。双位点突变中多数酯酶对2种有机磷底物的代谢活性有增强作用。
6.羧酸酯酶突变对拟除虫菊酯异构体解毒活性影响
通过荧光仪对类型I和类型II拟除虫菊酯异构体解毒作用测定结果表明，棉铃虫酯酶氧阴离子洞和酰基化口袋突变体中，仅有001BF236L、001DF235L和001GF238L对氰戊菊酯4个异构体类似物有代谢活性，与突变前相比是降低的。采用HPLC测定结果表明，只有001BF236L对氰戊菊酯2(S)-α(S)异构体解毒活性提高到突变前的4倍，其它突变体的解毒活性均很低，与突变前相比明显降低。
5个氧阴离子洞突变体酯酶对氯氰菊酯的8种异构体的解毒活性均极低，这与未检测到其对类似物的代谢活性是一致的。5个酯酶酰基化口袋突变体中001BF236L、001DF235L和001JF236L对2种反式氯氰菊酯异构体1(S)trans的解毒活性与突变前相比几乎都是增强的。特别是001DF235L和001JF236L对1(S)trans-α(R)的解毒活性分别为43μmol·min~(-1)·μmol~(-1)和51μmol·min~(-1)·μmol~(-1)，接近突变前的2倍。其中001DF235L对杀虫毒性较强的顺式异构体1(R)cis-α(S)的解毒活性也较高，为15.6μmol·min~(-1)·μmol~(-1)。三个双位点突变体中，均未检测到对氯氰菊酯的8种异构体类似物的代谢活性，但却检测到个别酯酶对部分异构体有较低的代谢作用。
7.羧酸酯酶解毒酶工程菌构建和表达
采用原核生物表达系统，将去掉N端信号肽的001C、001C~(A127D)、001F和001F~(A127D+F238L)，以及001I和001I~(G130D)与PETMCS III载体重组，转入大肠杆菌（Ecoli.BL21）菌株中，构建解毒酶工程菌。在温度为18℃、28℃和37℃条件下分别表达后，SDS凝胶电泳检测结果表明在温度为37℃时酯酶表达量最大；但酯酶表达产物主要以无活性的非水溶性形式存在，水溶性酯酶（有活性）占的比例非常低。提高活性酯酶表达量及解毒活性等相关工作尚需继续深入，但目前的工作为进一步的解毒酶工程菌研究奠定了前期基础。
综上所述，文中通过对来源于澳洲棉铃虫敏感品系（GR）的9个羧酸酯酶基因进行氨基酸序列分析和异源真核表达产物的代谢活性检测，从分子水平和生化水平两方面证实其中8个羧酸酯酶分别对2种有机磷和2种拟除虫菊酯类杀虫剂具有一定代谢解毒活性。通过人工定点突变和代谢活性检测，表明氧阴离子洞和酰基化口袋位点突变对酯酶的活性有明显影响，部分酯酶增强了对二甲基和二乙基有机磷、类型II拟除虫菊酯的解毒活性。去掉N端信号肽的部分酯酶在大肠杆菌中表达后，具天然酶活的酯酶表达水平较低，这为进一步构建高效解毒酶工程菌奠定了一定前期基础。上述结果，预示着羧酸酯酶突变在今后棉铃虫对有机磷和拟除虫菊酯类杀虫剂的代谢抗性发展中可能起着重要作用；同时也表明通过人工突变修饰可以提高棉铃虫羧酸酯酶的解毒活性。
Carboxylesterases(CCEs) are a major class of enzymes involved in the detoxification oforganophosphates (OPs), synthetic pyrethroids (SPs) and carbamates (CBs) in insects. Theaim of this research is to elucidate the role of CCEs from H. armigera in the development ofmetabolic resistance to these chemical insecticides. Furthermore, identification of pesticidesdegrading enzymes from cotton bollworm will have its application in bioremedation ofcontaminated environment. Nine CCEs genes from GR strain of H. armigera weresuccessfully expressed in the Sf9cells using the Baculovirus Expression Vector System(BEVS). The hydrolysis activities of eight CCEs against OPs and SPs were measured andanalyzed so further in term with molecular and biochemical methods. In addition, a total18mutants of CCEs were maken using the site-directed mutagenesis technique, the kineticefficiency of the CCEs and the effects of the mutations on the catalytic activities toward OPsand SPs were studied. Lastly, following above the gentic engineering bacteria strain E.coliBL21were constructed for potential hydrolytic degradation of insecticides. The significancesof this research were showed as below:
1. Baculovirus expression and esterase activity assay of CCEs in vitro
Nine CCEs genes were cloned from the cDNA library of the H. armigera GR strain.Alignment of anmino acid sequences showed that they all contain the critical motifs in theactive centre: the catalytic triad (S-E-H), the conservative pentapeptide (GxSxG) and the highconservative oxyanion hole (G-G-A). It has shown they should have hydrolytic activitiestoward some insecticides. Baculovirus expression system was used to produce native folded(active form) protein of CCEs. The expressed CCEs were analyzed by native-PAGE andstained with artificial substrate α-naphthyl acetate (α-NA), the resulting stained bands (closelytogether) indicated that the relative mobility (Rm) of all CCEs fell in two zones (0.27～0.36and0.37～0.48), which thus has shown most CCEs are probabaly associated with metabolicresistance. The kinetic assay with α-NA and p-NA were carried out for all CCEs using amicroplate reader. Eight of nine CCEs showed hihger affinity to α-NA and their Km valuewere between3.3μmol/L and671μmol/L. The rate constants (kcat/Km value) varied from0.3μmol~(-1)·S-1·L to95μmol~(-1)·S-1·L. The eight CCEs showed relatively lower hydrolyticactivities against para-nitrophyl acetate (p-NA) compared with those of α-NA.
2. Hydrolysis of organophosphrous insecticides by CCEs expression in vitro
The molar concentration of esterase protein in the Sf9cell extracts and the OP hydrolysiswere determined fluorometrically by using diethyl-and dimethyl-phosphates (dECP anddMUP) in term with the enzymatic reaction. The results showed the kinetic constants (kcatvalue) varied from1.3×10-3min~(-1)to15.8×10-3min~(-1)and resembled those of the E3controlfrom Lucilia cuprina. Although the eight CCEs had hydrolytic activities toward OPs, noclosed correlations between the OPs hydrolysis and the carboxylesterase activity were found.
3. Hydrolysis of individual isomers of fenvalerate and cypermethrin insecticides byCCEs
The catalytic activities of the CCEs toward individual SP insecticides and analogs weremeasured with fluorometric and HPLC assays repectively. The reuslts showed four of eightCCEs (001B,001C,001D and001J) had relatively higher hydrolytic activities toward twoisomers2(R)-α(R) and2(R)-α(S) of fenvalerate analogs, and001G had some activities againstesfenvalerate2(S)-α(S)(13.19μmol·min~(-1)·μmol~(-1)). In contrast with001G others hadnegligible activities. The same four CCEs also were observed higher activities toward thelight insecticidal1S trans cypermethrin isomers (17μmol·min~(-1)·μmol~(-1)~43μmol·min~(-1)·μmol~(-1))as well as the corresponding analogs. But their hydrolytic activities for the most insecticidalisomer1(R)cis-α(S) of cypermethrin and its analog were relatively lower. Other four CCEsshowed even lower or undetectable activities toward the isomers of cypermethrin.
4. Construction of mutant CCEs and their kinetic properties for model substrates
One or two amino acids were introduced to mutate in oxyanion hole (A/G to D) and acylbinding pocket (F to L) in the CCEs using the site-directed mutagenesis technique to make18mutant enzymes. The results of kinetic assay with a-NA showed that the single point mutationin oxyanion hole had dramatically reduced the esterase activities. The constants Km values ofthese mutants were generally higher than that of the wild type enzymes and the rate constantskcat/Km were between20-and437-fold lower than that of the wild type enzymes. By contrast,the single point mutations in acyl binding pocket had relatively less effects on the kinceticefficiencies of CCEs than that of the A to D mutants. The kcat/Km values were between1.5-and5-fold lower than that of the wild type enzymes. The double point mutations at both sitesnearly nullified the carboxylesterase activities. In line with the pattern of kinetic assay witha-NA, the mutations also markedly reduced the activities toward p-NA.
5. The effects on hydrolysis of OPs by two point mutations of CCEs
The amino acid mutations had marked effects on the hydrolytic activities of CCEstoward dimethyl-and diethyl-phosphates. Some mutants increased hydrolytic activites, but others reduced the hydrolysis. Among the CCEs modified in oxyanion hole, some obviouslyenhanced the hydrolysis of OPs, in particular, the kcat value for dECP of001CA127Dwas32.8×10-3min~(-1). By contrast, the F to L mutations in acyl binding pocket showed enhancedhydrolytic activites for relatively smaller substrate dimethylphosphates, the kcat values wereat least1.5-fold higher than that of the wild type enzyme, which were also higher comparedwith the relatively larger substrate diethylphosphates. It’s shown the dimethylphosphates wasas the preferred substrate in the hydrlolysis. The A to D mutations combined with F to Lmutations almost all enhanced the hydrolysis of OPs to some extent.
6. The effects of mutations on the hydrolytic activities of CCEs against individual SPsinsecticides
The catalytic activities toward individual SP insecticides and analogs of the mutant CCEswere also measured with fluorometric and HPLC assays. Among all the mutants, just threemutants (001B~(F236L),001D~(F235L)and001G~(F238L)) showed some hydrolytic activities against the4isomers of envalerate analogs, but the hydrolysis were lower compared to those of the wildtype CCEs with the exception of the001BF236L.001BF236Lshowed an enhancement ofhydrolysis of2(S)-α(S) isomer analog compared to its wild type. In line with that thismutation increased the activity about4-fold higher for the most insecticidal2(S)-α(S) isomer.Others reduced the hydrolytic activities toward this most insecticidal isomer.
The CCEs modified in oxyanion hole had undetectable activities toward any of thecypermethrin analogs, and also showed lowest activities toward most of the isomers ofcypermethrin insecticides. By contrast, three of five CCEs modified in the acyl binding pocket(001B~(F236L),001D~(F235L)and001J~(F236L)) showed enhancement of hydrolytic activities toward thelight insecticidal1(S) trans isomers of cypermethrin insecticide than that of the wild typeCCEs. The turnover for the1(S) trans-α(R) isomer were20μmol·min~(-1)·μmol~(-1),43μmol·min~(-1)·μmol~(-1)and51μmol·min~(-1)·μmol~(-1)respectiviely. In particular, the turnover for themost insecticidal1(R) cis-α(S) isomer by001D~(F236L)and001J~(F238)Lwere9.3μmol·min~(-1)·μmol~(-1)and15.6μmol·min~(-1)·μmol~(-1)respectively. All three double point mutations in CCEs had verylower or nearly nullifiled the activities toward both the cypermethrin analogs and the isomers.
7. Construction of genetic engineering bacteria with degradation CCEs and itsexpression
The N-terminal signal peptide sequence were removed from three CCEs and their codingregions were amplification with new designed primers. They were cloned into an expressionvector PETMCS III and transferred into the E.coli BL21cells. The CCEs genes wereexpressed in E.coli BL21at18,28and37degree respectively to yield the CCEs protein. The CCEs expressing BL21(DE3) cells were lysed and the soluble and insoluble proteins wereseparated. Both supernatants and precipitates of cell harvests from the cell cultures wereanalyzed by SDS-PAGE. The results showed that the higher expression level was found whenthe cells were grown at37degree. However, nearly all the over-expressed CCEs wereexpressed as insoluble protein (in precipitates) and very little enzyme were in solubale andactive. Future work will focus on improving correct folding of CCEs in E.coli BL21cellsbefore they can be applied for removing pesticide contamination in the environment. Currentresults provided useful data for further devolopment of degradation enzymes using genticlymicrobial engineering.
In summary, CCEs from H. armigera GR strain contain the active sites of typicalhydrolase. All CCEs were successfully expressed in Baculovirus Expression Vector System.Their hydrolytic activities were assayed and the results showed eight CCEs had somehydrolysis activities to some extent toward OPs and SPs insecticides. The kinetic results ofthe substitution of amino acids in oxyanion hole or acyl binding pocket had significant effectson the the enzymatic activities, in which some really showed enhancement of the hydrolysisof OP and the type II SP insecticides. The results demonstrated that the CCEs hydrolaseactivity toward insecticides could be enhanced through mutations at around enzyme activesites. It’s thus shown the mutation in active site of CCEs will play important role for thedeveolepment of OP and SP insecticides resistance in H. armigera in future. The results alsoindicated there are great potentials for using the CCEs of H. armigera to develop geneticengineering bacterial for detoxificaiton in contaminated enviroment.
1.棉铃虫羧酸酯酶在昆虫杆状病毒表达载体系统中的表达及酶活分析
从棉铃虫GR品系的cDNA文库中得到9个羧酸酯酶基因，氨基酸序列比对表明其均具有酯酶活性必须的催化三联体残基（S-E-H），以及酯酶家族的特征基序（G-x-S-x-G）和酶活相关的氧阴离子洞氨基酸残基（G-G-A），表明均为有酶促活性的酯酶基因。酯酶与杆状病毒线型DNA重组后，转染草地夜蛾Sf9细胞系，表达和制备了具有天然酶活性的酯酶蛋白产物。Native-PAGE和α-乙酸萘酯检测表明，酯酶条带主要分布在相对迁移率（Rm值）为0.27～0.35和0.39～0.48之间的两个区域，很可能是与棉铃虫代谢抗性相关酯酶。紫外读板机测定结果进一步表明，酯酶对α-乙酸萘酯底物的亲和系数（Km值）在3.3μmol/L～671μmol/L之间，速率常数（kcat/Km值）在0.3μmol~(-1)·S-1·L～95μmol~(-1)·S~(-1)·L之间。酯酶对α-乙酸萘酯的酶促水解活性普遍高于4-硝基苯基乙酸酯。
2.棉铃虫羧酸酯酶对有机磷的解毒活性
采用荧光法根据酯酶与2种有机磷底物的酶促反应特点，测定了表达产物中的酯酶浓度及解毒活性，结果表明8个酯酶对二甲基和二乙基有机磷（dMUP和dECP）均有一定代谢活性，催化反应常数（kcat值）在1.5×10-3min~(-1)～15.8×10-3min~(-1)之间，与对照铜绿蝇酯酶E3（kcat=3.5×10-3min~(-1)）的代谢活性处在同一水平范围。棉铃虫酯酶对模式羧酸酯底物的水解活性与其对有机磷杀虫剂的解毒活性无直接相关性。
3.棉铃虫羧酸酯酶对氰戊菊酯和氯氰菊酯异构体的解毒活性
通过荧光仪和高效液相色谱（HPLC）仪测定了其对氰戊菊酯（类型I）1种异构体2(S)-α(S)及4种异构体类似物的解毒活性，结果表明4个酯酶（001B、001C、001D、001J）对异构体2(R)-α(R)和2(R)-α(S)类似物的酶促解毒活性较高；其中酯酶001G对顺式氰戊菊酯杀虫剂异构体2(S)-α(S)的解毒活性为13.19μmol·min~(-1)·μmol~(-1)，其余酯酶解毒活性均较低。酯酶对氯氰菊酯8种异构体（类型II）及异构体类似物的测定结果表明，其中4个酯酶（001B、001C、001D和001J）对氯氰菊酯的2种反式异构体1(S) trans及类似物的解毒活性最高（17μmol·min~(-1)·μmol~(-1)～43μmol·min~(-1)·μmol~(-1)），但对杀虫毒性最高的异构体1(R)cis-α(S)及其类似物的解毒活性却相对很低；另外4个酯酶对氯氰菊酯异构体及类似物代谢作用极弱甚至难以检测到。
4.棉铃虫羧酸酯酶突变体的构建及对模式羧酸酯的酶促动力学特性
基于氨基酸序列比对和分析，采用定点突变技术构建了在棉铃虫酯酶氧阴离子洞和酰基化口袋内1～2个氨基酸位点突变的18个酯酶突变体。酶促动力学分析表明，酯酶氧阴离子洞丙氨酸（或甘氨酸）变为天冬氨酸（A/G→D）的突变体对α-乙酸萘酯的亲和力普遍降低（Km值升高），速率常数（kcat/Km值）也降低至突变前的1/20～1/437；酰基化口袋的苯丙氨酸变为亮氨酸的突变（F→L）同样降低了酯酶对羧酸酯底物的亲和力和水解活性，但与氧阴离子洞突变相比对酶的水解活性影响相对较小，速率常数最低降至突变前的1/5；酯酶在上述两个位点同时突变的双位点突变体(A/G→D+F→L)则几乎完全失去了对底物的酶促水解活性。上述酯酶突变也显著降低了酯酶对另一种模式羧酸酯底物4-硝基苯基乙酸酯的酶促水解活性。
5.羧酸酯酶突变对有机磷的解毒活性影响
酯酶单双位点的突变对有机磷的解毒活性也产生了较大的影响，部分突变体提高了对有机磷的解毒活性，相反部分突变体则降低了解毒活性。氧阴离子洞突变的部分突变体增强了对有机磷底物的解毒作用，尤其是001CA127D对二乙基有机磷dECP的kcat值高达32.8×10-3min~(-1)，是突变前的7倍。酯酶酰基化口袋突变的5个突变体，对分子结构较小的二甲基有机磷dMUP的kcat值均提高到突变前的2倍以上，解毒活性均高于分子结构较大的二乙基有机磷dECP，表现出对dMUP的代谢偏爱性。双位点突变中多数酯酶对2种有机磷底物的代谢活性有增强作用。
6.羧酸酯酶突变对拟除虫菊酯异构体解毒活性影响
通过荧光仪对类型I和类型II拟除虫菊酯异构体解毒作用测定结果表明，棉铃虫酯酶氧阴离子洞和酰基化口袋突变体中，仅有001BF236L、001DF235L和001GF238L对氰戊菊酯4个异构体类似物有代谢活性，与突变前相比是降低的。采用HPLC测定结果表明，只有001BF236L对氰戊菊酯2(S)-α(S)异构体解毒活性提高到突变前的4倍，其它突变体的解毒活性均很低，与突变前相比明显降低。
5个氧阴离子洞突变体酯酶对氯氰菊酯的8种异构体的解毒活性均极低，这与未检测到其对类似物的代谢活性是一致的。5个酯酶酰基化口袋突变体中001BF236L、001DF235L和001JF236L对2种反式氯氰菊酯异构体1(S)trans的解毒活性与突变前相比几乎都是增强的。特别是001DF235L和001JF236L对1(S)trans-α(R)的解毒活性分别为43μmol·min~(-1)·μmol~(-1)和51μmol·min~(-1)·μmol~(-1)，接近突变前的2倍。其中001DF235L对杀虫毒性较强的顺式异构体1(R)cis-α(S)的解毒活性也较高，为15.6μmol·min~(-1)·μmol~(-1)。三个双位点突变体中，均未检测到对氯氰菊酯的8种异构体类似物的代谢活性，但却检测到个别酯酶对部分异构体有较低的代谢作用。
7.羧酸酯酶解毒酶工程菌构建和表达
采用原核生物表达系统，将去掉N端信号肽的001C、001C~(A127D)、001F和001F~(A127D+F238L)，以及001I和001I~(G130D)与PETMCS III载体重组，转入大肠杆菌（Ecoli.BL21）菌株中，构建解毒酶工程菌。在温度为18℃、28℃和37℃条件下分别表达后，SDS凝胶电泳检测结果表明在温度为37℃时酯酶表达量最大；但酯酶表达产物主要以无活性的非水溶性形式存在，水溶性酯酶（有活性）占的比例非常低。提高活性酯酶表达量及解毒活性等相关工作尚需继续深入，但目前的工作为进一步的解毒酶工程菌研究奠定了前期基础。
综上所述，文中通过对来源于澳洲棉铃虫敏感品系（GR）的9个羧酸酯酶基因进行氨基酸序列分析和异源真核表达产物的代谢活性检测，从分子水平和生化水平两方面证实其中8个羧酸酯酶分别对2种有机磷和2种拟除虫菊酯类杀虫剂具有一定代谢解毒活性。通过人工定点突变和代谢活性检测，表明氧阴离子洞和酰基化口袋位点突变对酯酶的活性有明显影响，部分酯酶增强了对二甲基和二乙基有机磷、类型II拟除虫菊酯的解毒活性。去掉N端信号肽的部分酯酶在大肠杆菌中表达后，具天然酶活的酯酶表达水平较低，这为进一步构建高效解毒酶工程菌奠定了一定前期基础。上述结果，预示着羧酸酯酶突变在今后棉铃虫对有机磷和拟除虫菊酯类杀虫剂的代谢抗性发展中可能起着重要作用；同时也表明通过人工突变修饰可以提高棉铃虫羧酸酯酶的解毒活性。
Carboxylesterases(CCEs) are a major class of enzymes involved in the detoxification oforganophosphates (OPs), synthetic pyrethroids (SPs) and carbamates (CBs) in insects. Theaim of this research is to elucidate the role of CCEs from H. armigera in the development ofmetabolic resistance to these chemical insecticides. Furthermore, identification of pesticidesdegrading enzymes from cotton bollworm will have its application in bioremedation ofcontaminated environment. Nine CCEs genes from GR strain of H. armigera weresuccessfully expressed in the Sf9cells using the Baculovirus Expression Vector System(BEVS). The hydrolysis activities of eight CCEs against OPs and SPs were measured andanalyzed so further in term with molecular and biochemical methods. In addition, a total18mutants of CCEs were maken using the site-directed mutagenesis technique, the kineticefficiency of the CCEs and the effects of the mutations on the catalytic activities toward OPsand SPs were studied. Lastly, following above the gentic engineering bacteria strain E.coliBL21were constructed for potential hydrolytic degradation of insecticides. The significancesof this research were showed as below:
1. Baculovirus expression and esterase activity assay of CCEs in vitro
Nine CCEs genes were cloned from the cDNA library of the H. armigera GR strain.Alignment of anmino acid sequences showed that they all contain the critical motifs in theactive centre: the catalytic triad (S-E-H), the conservative pentapeptide (GxSxG) and the highconservative oxyanion hole (G-G-A). It has shown they should have hydrolytic activitiestoward some insecticides. Baculovirus expression system was used to produce native folded(active form) protein of CCEs. The expressed CCEs were analyzed by native-PAGE andstained with artificial substrate α-naphthyl acetate (α-NA), the resulting stained bands (closelytogether) indicated that the relative mobility (Rm) of all CCEs fell in two zones (0.27～0.36and0.37～0.48), which thus has shown most CCEs are probabaly associated with metabolicresistance. The kinetic assay with α-NA and p-NA were carried out for all CCEs using amicroplate reader. Eight of nine CCEs showed hihger affinity to α-NA and their Km valuewere between3.3μmol/L and671μmol/L. The rate constants (kcat/Km value) varied from0.3μmol~(-1)·S-1·L to95μmol~(-1)·S-1·L. The eight CCEs showed relatively lower hydrolyticactivities against para-nitrophyl acetate (p-NA) compared with those of α-NA.
2. Hydrolysis of organophosphrous insecticides by CCEs expression in vitro
The molar concentration of esterase protein in the Sf9cell extracts and the OP hydrolysiswere determined fluorometrically by using diethyl-and dimethyl-phosphates (dECP anddMUP) in term with the enzymatic reaction. The results showed the kinetic constants (kcatvalue) varied from1.3×10-3min~(-1)to15.8×10-3min~(-1)and resembled those of the E3controlfrom Lucilia cuprina. Although the eight CCEs had hydrolytic activities toward OPs, noclosed correlations between the OPs hydrolysis and the carboxylesterase activity were found.
3. Hydrolysis of individual isomers of fenvalerate and cypermethrin insecticides byCCEs
The catalytic activities of the CCEs toward individual SP insecticides and analogs weremeasured with fluorometric and HPLC assays repectively. The reuslts showed four of eightCCEs (001B,001C,001D and001J) had relatively higher hydrolytic activities toward twoisomers2(R)-α(R) and2(R)-α(S) of fenvalerate analogs, and001G had some activities againstesfenvalerate2(S)-α(S)(13.19μmol·min~(-1)·μmol~(-1)). In contrast with001G others hadnegligible activities. The same four CCEs also were observed higher activities toward thelight insecticidal1S trans cypermethrin isomers (17μmol·min~(-1)·μmol~(-1)~43μmol·min~(-1)·μmol~(-1))as well as the corresponding analogs. But their hydrolytic activities for the most insecticidalisomer1(R)cis-α(S) of cypermethrin and its analog were relatively lower. Other four CCEsshowed even lower or undetectable activities toward the isomers of cypermethrin.
4. Construction of mutant CCEs and their kinetic properties for model substrates
One or two amino acids were introduced to mutate in oxyanion hole (A/G to D) and acylbinding pocket (F to L) in the CCEs using the site-directed mutagenesis technique to make18mutant enzymes. The results of kinetic assay with a-NA showed that the single point mutationin oxyanion hole had dramatically reduced the esterase activities. The constants Km values ofthese mutants were generally higher than that of the wild type enzymes and the rate constantskcat/Km were between20-and437-fold lower than that of the wild type enzymes. By contrast,the single point mutations in acyl binding pocket had relatively less effects on the kinceticefficiencies of CCEs than that of the A to D mutants. The kcat/Km values were between1.5-and5-fold lower than that of the wild type enzymes. The double point mutations at both sitesnearly nullified the carboxylesterase activities. In line with the pattern of kinetic assay witha-NA, the mutations also markedly reduced the activities toward p-NA.
5. The effects on hydrolysis of OPs by two point mutations of CCEs
The amino acid mutations had marked effects on the hydrolytic activities of CCEstoward dimethyl-and diethyl-phosphates. Some mutants increased hydrolytic activites, but others reduced the hydrolysis. Among the CCEs modified in oxyanion hole, some obviouslyenhanced the hydrolysis of OPs, in particular, the kcat value for dECP of001CA127Dwas32.8×10-3min~(-1). By contrast, the F to L mutations in acyl binding pocket showed enhancedhydrolytic activites for relatively smaller substrate dimethylphosphates, the kcat values wereat least1.5-fold higher than that of the wild type enzyme, which were also higher comparedwith the relatively larger substrate diethylphosphates. It’s shown the dimethylphosphates wasas the preferred substrate in the hydrlolysis. The A to D mutations combined with F to Lmutations almost all enhanced the hydrolysis of OPs to some extent.
6. The effects of mutations on the hydrolytic activities of CCEs against individual SPsinsecticides
The catalytic activities toward individual SP insecticides and analogs of the mutant CCEswere also measured with fluorometric and HPLC assays. Among all the mutants, just threemutants (001B~(F236L),001D~(F235L)and001G~(F238L)) showed some hydrolytic activities against the4isomers of envalerate analogs, but the hydrolysis were lower compared to those of the wildtype CCEs with the exception of the001BF236L.001BF236Lshowed an enhancement ofhydrolysis of2(S)-α(S) isomer analog compared to its wild type. In line with that thismutation increased the activity about4-fold higher for the most insecticidal2(S)-α(S) isomer.Others reduced the hydrolytic activities toward this most insecticidal isomer.
The CCEs modified in oxyanion hole had undetectable activities toward any of thecypermethrin analogs, and also showed lowest activities toward most of the isomers ofcypermethrin insecticides. By contrast, three of five CCEs modified in the acyl binding pocket(001B~(F236L),001D~(F235L)and001J~(F236L)) showed enhancement of hydrolytic activities toward thelight insecticidal1(S) trans isomers of cypermethrin insecticide than that of the wild typeCCEs. The turnover for the1(S) trans-α(R) isomer were20μmol·min~(-1)·μmol~(-1),43μmol·min~(-1)·μmol~(-1)and51μmol·min~(-1)·μmol~(-1)respectiviely. In particular, the turnover for themost insecticidal1(R) cis-α(S) isomer by001D~(F236L)and001J~(F238)Lwere9.3μmol·min~(-1)·μmol~(-1)and15.6μmol·min~(-1)·μmol~(-1)respectively. All three double point mutations in CCEs had verylower or nearly nullifiled the activities toward both the cypermethrin analogs and the isomers.
7. Construction of genetic engineering bacteria with degradation CCEs and itsexpression
The N-terminal signal peptide sequence were removed from three CCEs and their codingregions were amplification with new designed primers. They were cloned into an expressionvector PETMCS III and transferred into the E.coli BL21cells. The CCEs genes wereexpressed in E.coli BL21at18,28and37degree respectively to yield the CCEs protein. The CCEs expressing BL21(DE3) cells were lysed and the soluble and insoluble proteins wereseparated. Both supernatants and precipitates of cell harvests from the cell cultures wereanalyzed by SDS-PAGE. The results showed that the higher expression level was found whenthe cells were grown at37degree. However, nearly all the over-expressed CCEs wereexpressed as insoluble protein (in precipitates) and very little enzyme were in solubale andactive. Future work will focus on improving correct folding of CCEs in E.coli BL21cellsbefore they can be applied for removing pesticide contamination in the environment. Currentresults provided useful data for further devolopment of degradation enzymes using genticlymicrobial engineering.
In summary, CCEs from H. armigera GR strain contain the active sites of typicalhydrolase. All CCEs were successfully expressed in Baculovirus Expression Vector System.Their hydrolytic activities were assayed and the results showed eight CCEs had somehydrolysis activities to some extent toward OPs and SPs insecticides. The kinetic results ofthe substitution of amino acids in oxyanion hole or acyl binding pocket had significant effectson the the enzymatic activities, in which some really showed enhancement of the hydrolysisof OP and the type II SP insecticides. The results demonstrated that the CCEs hydrolaseactivity toward insecticides could be enhanced through mutations at around enzyme activesites. It’s thus shown the mutation in active site of CCEs will play important role for thedeveolepment of OP and SP insecticides resistance in H. armigera in future. The results alsoindicated there are great potentials for using the CCEs of H. armigera to develop geneticengineering bacterial for detoxificaiton in contaminated enviroment.
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