Submitted to: Biochemica
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/8/2005
Publication Date: 4/15/2005
Citation: Elpidina, E.N., Tsybina, T.A., Dunaevsky, Y.E., Belozersky, M.A., Zhuzhikov, D.P., Oppert, B.S. 2005. Purification and characterization of a chymotrypsin-like proteinase from the midgut of Tenebrio molitor larvae. Biochemie 87: 771-779. Interpretive Summary: Very little is known about the detailed characteristics of digestive enzymes in storage pests. We purified and characterized a chymotrypsin enzyme from the yellow mealworm. Detailed studies were provided on the kinetic parameters and structural properties of the enzyme. Information on inhibitors may lead to the development of novel biopesticides for coleopteran storage pests.
Technical Abstract: A chymotrypsin-like proteinase was isolated from the posterior midgut of larvae of the yellow mealworm, Tenebrio molitor, by ion exchange and gel filtration chromatography. The enzyme, TmC1, was purified to homogeneity as determined by SDS-PAGE and postelectrophoretic activity detection. TmC1 had a molecular mass of 23.2 kDa, pI of 8.4, a pH optimum of 9.5, and the optimal temperature for activity was 51°C. The proteinase displayed high stability at temperatures below 43°C and in the pH range 6.5-11.2, which is inclusive of the pH of the posterior and middle midgut of the insect. The enzyme hydrolyzed long chymotrypsin peptide substrates SucAAPFpNA, SucAAPLpNA and GlpAALpNA and did not hydrolyze short chymotrypsin substrates. Kinetic parameters of the enzymatic reaction demonstrated that the best substrate was SucAAPFpNA, with kcat app 36.5 s-1 and Km 1.59 mM. However, the enzyme had a lower Km for SucAAPLpNA, 0.5 mM. PMSF was an effective inhibitor of TmC1, and the proteinase was not inhibited by either TPCK or TLCK. However, the activity of TmC1 was reduced with sulfhydryl reagents, suggesting the importance of disulfide bonds for activity. Several plant and insect proteinaceous proteinase inhibitors were active against the purified enzyme, the most effective being Kunitz soybean trypsin inhibitor. The N-terminal sequence of the enzyme was IISGSAASKGQFPWQ, which was up to 67% similar to other insect chymotrypsin-like proteinases and 47% similar to mammalian chymotrypsin A. The amino acid composition of TmC1 differed significantly from previously isolated T. molitor enzymes.