A trypsin- like protease was purified and characterized from the mid guts of Helicoverpa armigera using ion exchange chromatography and gel filtration on QAE- sephadex column. The protease activity was found to reside in two protein peaks. High BAPNAase activity was associated with a second peak eluted with 0.3M NaCl. The enzyme was found to be homogeneous by the criteria of native PAGE. SDS-PAGE analysis in the presence of 2-mercaptoethaonol gave a single band corresponding to a molecular weight of about 30.4 kDa. Trypsin like protease from the mid gut was alkali-stable and its pH optimum for activity was about pH 11. The specific activity of the purified enzyme was 1426.92 TU per mg protein. The final yield was 52.81% with a fold purification of 3.81. The enzyme followed Michaelis-Menten kinetics when the protease activity was measured at different concentrations (0.9-4.0 mM) of BAPNA. Km and Vmax values for the protease were found to be 3.84 x 10-3 M and 45 nmol/10min/mg protein with BAPNA as the substrate respectively. Strong inhibition of protease activity by TLCK not by TPCK suggests that the isolated mid gut protease is trypsin like but not chymotrypsin like specificity of serine protease.
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