粘虫中肠α-淀粉酶活性测定方法的参数优化  

Parametric optimization on the sensitive determination of midgut α-amylase in larvae of Mythimna separata Walker

作　　者：孔玉萍[1] 黄青春[1] 刘曼慧[1] 丰俊[1] 刘扬[1] 

机构地区：[1]华东理工大学药学院上海市化学生物学重点实验室,上海200237

出　　处：《昆虫学报》2007年第10期981-988,共8页Acta Entomologica Sinica

基　　金：国家重点基础研究发展规划"973"项目(2003CB114402);国家自然科学基金项目(30400295)

摘　　要：针对粘虫Mythimna separata中肠α-淀粉酶筛选了11种不同参数组合的3,5-二硝基水杨酸活性测定方法,并对其中最适组合的各个参数进行了优化。结果表明:在离体测定条件下,粘虫中肠α-淀粉酶活性的最优化测定参数为0.03mol/L磷酸盐缓冲液(pH8.0,含有55mmol/L NaCl)、温度45℃、吸收波长480nm。Ca2+对α-淀粉酶活性具有抑制作用。该优化法能够显著降低粘虫、德国小蠊Blattella germanica、黄粉虫Tenebrio molitor、淡色库蚊Culexpipiens pallens和家蝇Musca domestica等昆虫α-淀粉酶的米氏常数Km值,且粘虫和德国小蠊α-淀粉酶的Vmax值增大,但黄粉虫、淡色库蚊和家蝇α-淀粉酶的Vmax值均明显减小。结果说明,在该优化体系下,粘虫α-淀粉酶与底物的亲和力增强,最大反应速度增大,测定酶活性的准确性和灵敏度显著提高;同时该优化体系也可作为测定德国小蠊α-淀粉酶活性的优化方法,但不适合作为黄粉虫、淡色库蚊和家蝇α-淀粉酶的最优化测定方法。Parametric optimization on the activity determination of midgut α-amylase in larvae of Mythimna separata Walker was investigated by screening the 3, 5-dinitrosalicylic acid methods with eleven combinations of parameters. The results showed that the in vitro determined optimal parameters were 0.03 mol/L phosphate buffer （pH 8.0） containing 55 mmol/L NaCl, reaction temperature 45 ℃ and absorbance wavelength 480 nm. Ca^2＋ inhibited the activity of α-amylase. Moreover, the optimal method significantly decreased Km value of α- amylase from M. separata, BlatteUa germanica, Tenebrio molitor, Culex pipiens pallens and Musca domestica larvae, and increased Vmax value of α-amylase from M. separate and B. germanica, but strongly decreased Vmax values of α-amylase from T. molitor, C. pipiens pallens and M. domestica larvae. The study suggested that the optimal method not only enhanced the affinity and the maximum reaction velocity of α- amylase with its substrate, but also improved the accuracy and the sensitivity on the activity of α-amylase from M. separata larvae in assays, whereas it was not the optimal method for determining the activity of α-amylase from T. molitor, C. pipiens pallens and M. domestica larvae.

关 键 词：粘虫 Α-淀粉酶 参数优化 动力学常数 

分 类 号：Q966[生物学—昆虫学]