Characterisation of midgut digestive proteases from the maize stem borer Busseola fusca

BACKGROUND: Insect damage is a major constraint on maize production. Control of Busseola fusca (Fuller) insub-Saharan Africa is relatively ineffective; the major larval digestive enzymes were characterised with a view todeveloping future control strategies.RESULTS: Using BODIPY-FL Casein, maximal activity was at pH 9.5, with six protease forms visualised bygelatin-PAGE. Synthetic substrates and diagnostic inhibitors demonstrated the presence of serine proteases.Chymostatin was a potent inhibitor of general proteolysis (90%), providing strong evidence for the presenceof chymotrypsin; it also caused significant inhibition (>95%) with SA2PFpNA as substrate. The I50 values forchymostatin with casein and SA2PFpNA were 0.0075 μM and 0.06 μM respectively. Z-Phe-Arg-pNA activity wasinhibited by chymostatin and TLCK (50 and 30% respectively), suggesting the presence of trypsin-like activity.BApNA hydrolysis was also strongly inhibited by chymostatin and TLCK (92 and 75%), suggesting trypsin activity,while SBBI, PMSF, pepstatin and E-64 had no significant effect. Interestingly, SBBI (I50 = 0.39 μM) and SBTIboth inhibited general proteolysis by approximately 70%, suggesting that SBBI’s dual inhibitory role makes thisinhibitor a potentially useful candidate for expression in maize for control of B. fusca.CONCLUSION: These results provide a basis for the rational design of insect-resistant transgenic maize expressingprotease inhibitors