Property of midgut α-amylase from Mythimna separata (Lepidoptera: Noctuidae) larvae and its responses to potential inhibitors in vitro

Abstract

Midgut α-amylase is an important digestive enzyme involved in larval energy metabolism and carbohydrate assimilation. In this article, the properties of midgut α-amylase from the Oriental armyworm, Mythimna separata (Lepidoptera: Noctuidae), larvae were characterized, and its in vitro responses to chemical inhibitors were also determined. The kinetic parameters Km and Vmax of midgut α-amylase were 0.064 M, 4.81 U mg pro-1 in phosphate buffer, and 0.128 M, 1.96 U mg pro-1 in barbiturate-acetate buffer; α-amylase activity linearly increased as starch concentration increased. α-Amylase activity was not influenced by amino acids such as Pro, Met, Try, His, Ala, and Phe but was strongly activated by antioxidants. Reduced glutathione, 1,4-dithiothreitol, β-mercaptoethanol, and ascorbic acid improved the activity of α-amylase about 2.06, 3.46, 3.37, and 6.38 times, respectively, relative to the control. Ethylenediaminetetraacetic acid, sodium dodecyl sulfonate, and N-bromosuccinimide (NBS) strongly inhibited α-amylase. α-, β-, and γ-cyclodextrin were not the preferred substrates for α-amylase. Kinetic analysis showed that IC50 value of NBS against α-amylase was 1.52 (60.26) μM, and the mode of action of NBS with Ki as 2.53 (0.35) μM was a mixed-type inhibition that indicated a combination of partial competitive and pure noncompetitive inhibition. The midgutα-amylase of armyworm larvae may be a potential target for novel insecticide development and pest control.