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) in sub‐Saharan Africa is relatively ineffective; the major larval digestive enzymes were characterised with a view to developing future control strategies. RESULTS: Using BODIPY‐FL Casein, maximal activity was at pH 9.5, with six protease forms visualised by gelatin‐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 presence of chymotrypsin; it also caused significant inhibition (>95%) with SA₂PFpNA as substrate. The I₅₀ values for chymostatin with casein and SA₂PFpNA were 0.0075 µM and 0.06 µM respectively. Z‐Phe‐Arg‐pNA activity was inhibited 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 SBTI both inhibited general proteolysis by approximately 70%, suggesting that SBBI's dual inhibitory role makes this inhibitor 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 expressing protease inhibitors. Copyright © 2008 Society of Chemical Industry