Purification and Characterization of Two Dimeric -amylases from Digestive Tract in the Tropical House Cricket Gryllodes sigillatus (Orthoptera: Gryllidae)

Two �-amylases (Amy A1 and Amy A2) were isolated from the digestive tract of the tropical house cricket Gryllodes sigillatus using a combination of ammonium sulphate saturation, gel filtration chromatography, anion-exchange chromatography and hydrophobic interaction chromatography. Amy A1 showed an optimum pH of 6.6, whereas for Amy A2, the optimum pH was 7.0. Both �-amylases exhibited optimum temperature at 55 °C. Amy A1 was found to be stable at pH 6.0 to 7.6 and up to 50 °C. Amy A2 was found to be stable at pH 6.6 to 7.6 and also up to 50 °C. The two enzymes retained over 95 % of their maximum activity at 37 °C after 3h. Whilst Amy A1 had a molecular mass of 85-89 kDa, the molecular mass of Amy A2 was 66-72 kDa. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) indicated that Amy A1 and Amy A2 were each composed of two different subunits indicating heterodimeric structures. Amy A1 and Amy A2 exhibited a high affinity towards soluble starch with Km values of 0.29 and 0.5 mg/ml, respectively. The two �-amylase activities were stimulated by Ca 2+ and Ba 2+ and were inhibited by Ethylenediaminetetraacetic acid (EDTA) and SDS. Concerning the sulfhydryl reagent as 5,5-dithio-bis (2-nitrobenzoate) (DTNB) and p-chloromercurybenzoate (pCMB), whilst these compounds acted as inhibitors for Amy A2, they did not affect Amy A1. The analysis of hydrolytic products after soluble starch hydrolysis by each amylase revealed that maltose, maltotriose and maltotetraose were the major products. This indicated that Amy A1 and Amy A2 can be classified as the �-amylases (the endoamylases). From a physiological point of view, �-amylases Amy A1 and Amy A2 play a fundamental role in energy production for this insect. Consequently, the digestive tract of this insect could be a good source of �-amylases for starch saccharification.