中国媒介生物学及控制杂志

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利用正交试验法建立德国小蠊羧酸酯酶的最佳反应体系

魏绪强1,2;钱坤2;曾晓芃2; 高希武1   

  1. 1中国农业大学农学与生物技术学院昆虫系 北京100094;2北京市疾病预防控制中心消毒与有害生物防制所 北京100013
  • 出版日期:2008-08-20 发布日期:2008-08-20

Establishment of optimization reaction condition of carboxylesterase from German cockroach(Blattella germanica) by orthogonal matrix method

WEI Xu-qiang1;2; QIAN Kun; ZENG Xiao-peng; GAO Xi-wu   

  1. 1 Department of Entomology, China Agricultural University, Beijing 100094, China 2 Department of Disinfection and Vector Control, Beijing Center for Disease Control and Prevention, Beijing 100013, China
  • Online:2008-08-20 Published:2008-08-20

摘要: 目的 为了系统地研究德国小蠊羧酸酯酶(CarE)的性质,确定其抗药性的发生和发展及其抗性水平,建立适宜的快速、准确的酶活性检测方法。方法 用正交试验方法研究酶液浓度、底物浓度、反应体系的pH值、反应温度、反应时间5个因素对德国小蠊CarE活性的影响,并从试验组合中选出最优的条件组合。对正交试验结果进行极差和方差分析。结果 实验结果表明,影响德国小蠊CarE比活力的5个因素中各因素水平之间差异有统计学意义。A因素(酶浓度)的最优水平是酶的浓度为0.35只/ml;B因素(底物浓度)的最优水平是底物浓度为0.6mmol/L;C因素(反应时间)的最优水平是反应时间为5min;D因素(反应温度)的最优水平是酶反应温度为40℃;E因素(反应体系的pH值)的最优水平是酶液反应体系的pH值为6.5。极差和方差分析结果表明A4B3C1D4E2是最优组合,利用此组合测得的CarE比活力最高为37.286μmol/(mgpro.min)。各因素对试验结果影响的顺序为:反应温度>反应时间>底物浓度>酶浓度>pH值。结论 确定了测定德国小蠊CarE活性的最佳反应条件是:酶浓度为0.35只/ml;底物浓度为0.6mmol/L;反应时间为5min;反应温度为40℃;酶反应体系的pH值为6.5。

关键词: 德国小蠊, 羧酸酯酶, 正交试验, 方差分析, 最佳条件

Abstract: Objective To confirm the resistance status of Blattella germanica through studying its carboxylesterases(CarE) characteristics systemically and establish a quick, accurate and simple detection method. Methods The optimal concentrations of CarE and substrate, pH, temperature and reaction time of the reaction system, were determined by orthogonal matrix methods. And the optimal group was selected in the experiment. The data was analyzed with range analysis and analysis of variance. Results The results revealed that there were significant difference among various levels of the selected five factors which affected specific activity of CarE from Blattella germanica. The optimal levels of A (CarE concentration), B (substrate concentration), C (reation time), D (reation temperature) and E (pH of reation system) were A4 (0.35 head/ml), B3 (0.6 mmol/L), C1 (5 minutes), D4 (40 ℃) and E2 (pH 6.5), respectively. The results of range analysis and variance analysis indicated that the optimal combination was A4B3C1D4E2, and the maximum specific activity of CarE was 37.286 μmol/(mg pro·min) determined under the optimal conditions. The effect of factors on CarE specific activity were in the order of temperature>time>substrate>enzyme concentration>pH. Conclusion The optimal condition were: 0.35 head/ml, 0.6 mmol/L substrate, 5 mintues, 40 ℃, respectively.