Isolation and partial characterization of two trypsins from the larval midgut of Spodoptera littoralis (Boisduval)

Two serine-proteases (P I and P II) were isolated from the larval midgut of Spodoptera littoralis by affinity chromatography. These hydrolysed casein and BApNA and were strongly inhibited by TLCK. On SDS-PAGE, an apparent molecular weight of 27 kDa was determined for both proteases. When mass spectrometric analysis was performed directly on the eluted fractions, one main component per peak was observed, having a molecular mass of 24,359±2 Da (P I) and 24,448±2 Da (P II). The enzymes had their maximum activity and stability at pH 10 and 4, respectively. P I was significantly more active than P II at 20°C or less; it was also more resistant to denaturation by heat (55 versus 50°C). A breakpoint between activity and stability was recorded at 30°C for both proteases. P II was strongly inhibited by aprotinin, leupeptin and KI, and partly inactivated by LBTI and chymostatin; P I was unaffected by LBTI and chymostatin and was also less susceptible to aprotinin and KI. On the basis of substrate degradation and inactivation rates, it was concluded that P I and P II are very different from the two proteases found in another strain of S. littoralis. Though P I and P II were found to belong to the same mechanistic class of bovine trypsin, they were not activated by Ca 2+ over the concentration range 0–20 mM. It is suggested that transgenic plants harbouring genes coding for effective P I inhibitors should have a high level of resistance towards this insect pest.