Abstract: Parametric optimization on the activity determination of midgut α-amylase in larvae of Mythimna separate 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. Ca²⁺ inhibited the activity of α-amylase. Moreover,  the optimal method significantly decreased Km value of α-amylase from M. separate, Blattella germanica,  Tenebrio molitor,  Culex pipiens pallens and Musca domestica larvae,  and increased V_max  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. separate 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.

Key words: Mythimna separata, α-amylase, parametric optimization, kinetic constant