2Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, 119992 Moscow, Russia; fax: (7-095) 939-3181; E-mail: email@example.com
3Department of Entomology, Faculty of Biology, Lomonosov Moscow State University, 119992 Moscow, Russia
4Grain Marketing and Production Research Center, USDA Agricultural Research Service, 1515 College Ave, Manhattan KS, 66502, USA
* To whom correspondence should be addressed.
Received October 14, 2004; Revision received November 1, 2004
A new trypsin-like proteinase was purified to homogeneity from the posterior midgut of Tenebrio molitor larvae by ion-exchange chromatography on DEAE-Sephadex A-50 and gel filtration on Superdex-75. The isolated enzyme had molecular mass of 25.5 kD and pI 7.4. The enzyme was also characterized by temperature optimum at 55°C, pH optimum at 8.5, and Km value of 0.04 mM (for hydrolysis of Bz-Arg-pNA). According to inhibitor analysis the enzyme is a trypsin-like serine proteinase stable within the pH range of 5.0-9.5. The enzyme hydrolyzes peptide bonds formed by Arg or Lys residues in the P1 position with a preference for relatively long peptide substrates. The N-terminal amino acid sequence, IVGGSSISISSVPXQIXLQY, shares 50-72% identity with other insect trypsin-like proteinases, and 44-50% identity to mammalian trypsins. The isolated enzyme is sensitive to inhibition by plant proteinase inhibitors and it can serve as a suitable target for control of digestion in this stored product pest.
KEY WORDS: digestive proteinase, Tenebrio molitor, trypsin