Purification and properties of trypsin-like enzyme from the midgut of Morimus funereus (coleoptera, cerambycidae) Larvae

摘要

Trypsin-like enzyme (TLE) from the anterior midgut of Morimus funereus larvae was purified by anion exchange chromatography and gel filtration chromatography and characterized. Specific TLE activity was increased 322-fold by purification of the crude midgut extract. The purified enzyme had a pH optimum of 9.0 (optimum pH range 8.5–9.5) and temperature optimum of 45°C with the K_M ratio of 0.065 mM for benzoyl-arginine-p-nitroanilide (BApNA). Among a number of inhibitors tested, the most efficient was benzamidine (K_I value of 0.012 mM, Ic₅₀ value of 0.204 mM) while inhibition of TLE activity by SBTI, TLCK, and PMSF was partial. Almost all divalent cations tested enhanced the enzyme activity, amongst them Co2+ and Mn2+ stimulated TLE activity for 2.5 times. The purified TLE (after gel-filtration on Superose 12 column) had a molecular mass of 37.5 kDa with an isoelectric point over 9.3. Sodium dodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed one band of 38 kDa, suggesting that the enzyme is a monomer. © 2010 Wiley Periodicals, Inc.