Biochemical and kinetic characterization of the digestive trypsin-like activity of the lesser grain borer Rhyzopertha dominica (F.) (Coleoptera: Bostrichidae)

Abstract The digestive trypsin-like activity of the lesser grain borer Rhyzopertha dominica was characterized in some of its biochemical and kinetic properties. The enzyme activity from insect midguts was isolated using hydrophobic interaction chromatography with phenyl-sepharose CL-4B. Eight bands (identified from A through H) with caseinolytic activity and molecular weights in the range of 22–51.3 kDa were detected by zymography in casein-polyacrylamide gels. The strongest bands were D, G, and H, and showed estimated molecular weights of 33.6, 25.4, and 22 kDa, respectively. In-gel inhibition of caseinolytic activity showed that the serine protease inhibitors TLCK and SBTI inhibited all the proteases, except E. In-vitro inhibitory assays showed that SBTI and TLCK suppressed the BA p NAase activity by 92.3% and 79.2%, respectively, indicating the presence of serine proteases. Wheat hexaploid albumin extracts were highly effective in inhibiting all the proteolytic activity. The chymotrypsin inhibitor TPCK did not affect the BA p NAase activity, indicating that the proteolytic activity in R. dominica belongs to the trypsin-like type. With BA p NA as the substrate, the proteolytic activity was high across a broad pH range of 6–11 with two peaks of maximum activity at pH 8 and 10 with an optimum temperature of 50 °C. SBTI inhibited the BA p NAase activity with IC 50 and K i values of 0.02 μM and 1.17 × 10 −8  M, respectively. The kinetic constants K m and V max were 0.07 mM and 2.8 mM/min, respectively. The activation energy ( E a ) for BA p NA hydrolysis was 33.5 kJ/mol. The results of this study confirm that R. dominica rely on serine protease activity for food digestion.