The Cloning and Expression of Trypsins from Yellow Mealworm (Tenebrio molitor)

Seven non-redundant trypsin genes including two sequences without signal peptide (TMT2 and TMT5) and five sequences with signal peptide (pTMT2-pTMT6) were screened from yellow mealworms survived from stress of Bt toxin. Three amino acid variations were unique to TMT/pTMT, which were the substitution of I87T from pTMT6, and the substitutions of S29P and N205D from TMT2. The substitution of S29P caused the terminal of a beta-sheet to transform into coil. The recombinant TMT2 and TMT5 fused with 6-His tag were both expressed in M15 and refolded by immobilized metal affinity chromatography (IMAC). At 25(degrees)C, the K-m values of TMT2 and TMT5 showed no significant difference, as indicated that there was little difference in the binding capacity and specificity for the substrate BAEE between TMT2 and TMT5. However, the k(cat) and k(cat)/K-m value of TMT2 were 25.3% and 29.4% higher than TMT5, respectively, as indicated that the k(cat) value contributed to the higher catalytic efficiency of TMT2. The effects of pH, temperature, metal ions and inhibitors on the activity of TMT2 and TMT5 showed no significant differences. However, pH and temperature showed greater influence on the stability of TMT2 than TMT5. In general, TMT2 improved the catalytic efficiency at the cost of stability through the substitution of S29P, as contributed to the survive of yellow mealworm from stress of Bt toxin.