Digestive proteinases of yellow mealworm (Tenebrio molitor) larvae: purification and characterization of a trypsin-like proteinase

• Published in: Biochemistry (Moscow) 2005-03, Vol.70 (3), p.300-305
• Main Authors: Tsybina, T A, Dunaevsky, Y E, Belozersky, M A, Zhuzhikov, D P, Oppert, B, Elpidina, E N
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Amino acids; Animals; Digestive enzymes; Electrophoresis, Polyacrylamide Gel; Enzyme Stability; Enzymes; Hydrogen-Ion Concentration; Intestines - enzymology; Larva - enzymology; Larvae; Molecular Sequence Data; Proteases; Proteinase inhibitors; Sequence Alignment; Serine Endopeptidases - isolation & purification; Serine Endopeptidases - metabolism; Serine Proteinase Inhibitors - pharmacology; Temperature; Tenebrio - enzymology; Tenebrio molitor; Trypsin
• ISSN: 0006-2979; 1608-3040; 0320-9725
• DOI: 10.1007/s10541-005-0115-2

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 degrees C, pH optimum at 8.5, and K(m) 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.