Purification and characterization of midgut α-amylases of Eurygaster integriceps

Abstract

In the current study, midgut α-amylase from Sunn pest (Eurygaster integriceps Puton) (Hemiptera: Scutelleridae), one of the most serious pests of wheat and barley in the wide area of the Near and Middle East, West Asia, and many of the new independent states of central Asia, were purified and characterized. Amylase activity was detected in the midgut of the insects which were collected from both over-wintering sites during winter and feeding insects during spring. Amylase activities in the midgut of over-wintering and feeding insects were 5.71 and 3.43 U/mg protein, respectively. Initially, a native electrophoretic analysis of E. integriceps crude midgut extract showed that there are two major amylase forms in the midgut. Through the sequence of ammonium sulfate precipitation, first by gel filtration chromatography (Sephadex G-75), anion exchange chromatography (diethylaminoethylcellulose) and second by gel filtration chromatography, specific activity of α-amylase of E. integriceps increased 44-fold from approximately 3 to 133 U/mg protein. Analysis of purified amylases by sodium dodecylsulfate polyacrylamide gel electrophoresis showed that these proteins had estimated molecular masses of 49 and 52 kDa. Optimum temperature was determined to be 30–40°C. The optimum pH value was 6.5 and the K_mapp for soluble starch was 0.54%.