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Digestive Proteinases of Yellow Mealworm (Tenebrio molitor) Larvae: Purification and Characterization of a Trypsin-Like Proteinase

T. A. Tsybina1,2, Y. E. Dunaevsky2, M. A. Belozersky2, D. P. Zhuzhikov3, B. Oppert4, and E. N. Elpidina2*

1Bach Institute of Biochemistry, Russian Academy of Sciences, Leninsky pr. 33, 119071 Moscow, Russia

2Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, 119992 Moscow, Russia; fax: (7-095) 939-3181; E-mail: elp@belozersky.msu.ru

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