Partial characterization of amylasic activity present in the intestine of Pachymerus nucleorum larvae (Fabricius, 1792)

Pachymerus nucleorum (Fabricius, 1792) is a beetle specie of the Bruchinae sub-family, family Chysomelidae and principal host is coconut of the palm babassu (Orbinya spp), typical of the varzea zones and river valleys. The objective in this work was identify and to characterize the amylolytic activity in P. nucleorum larvae guts. The guts were immediately homogenized in phosphatate buffer 50 mM pH 7,5 containing protease inhibitor (Aprotinine 0,2 ug/mL and Benzamidine 1 mM), sucrose 250 mM and EDTA 1 mM, after that the samples were centrifugated at 15000xg for 30 minutes, 4 ºC. The supernatant fraction (S1) presented higher amylolytic activity in relation to the precipitated fraction (P1). Analyses in SDS-PAGE revealed polypeptides of 62 kDa, in fraction P1 and in fraction S1 polypeptides of 12 and 40 kDa. The fraction S1 presented maximum amylolytic activity in 50°C, and stimulation of the activity in 20 % and 45 % in presence of calcium 0,5 mM or sodium chloride 14 mM, respectively. The preparation containing sucrose and EDTA presented higher amylolytic activity in relation to the preparation without sucrose and EDTA. An increase in the amylolytic activity was observed in both preparations when stored in refrigerator, however the preparation without sucrose and EDTA presented higher increase in this activity.