Partial purification and characterization of digestive trypsin-like proteases from the velvet bean caterpillar, Anticarsia gemmatalis

• Autor(a) principal: Oliveira, M.G.A.
• Data de Publicação: 2004
• Outros Autores: Simone, S.G. De, Xavier, L.P., Guedes, R.N.C.
• Tipo de documento: Artigo
• Idioma: eng
• Título da fonte: LOCUS Repositório Institucional da UFV
• Texto Completo: https://doi.org/10.1016/j.cbpc.2004.10.018; http://www.locus.ufv.br/handle/123456789/19221
• topic: Trypsin; Serine proteases; Digestive proteases; Lepidoptera midgut; Substrate specificity; Cleavage site; Noctuidae; Protease inhibition; Protease kinetics; Synthetic peptide hydrolysis

Trypsin-like proteases from the midgut of Anticarsia gemmatalis Hübner (Lepidoptera: Noctuidae) were purified on an aprotinin-agarose column equilibrated with 0.01 M Tris–HCl containing 5 mM CaCl2 (pH 7.5). The yield was 66.7% with a purification factor of 107 and a final specific activity of 6.88 mM/min/mg protein with the substrate N-α-benzoyl-l-Arg-p-nitroanilide (l-BApNA). The purified fraction showed three bands with proteolytic activity and molecular weights of 66,000, 71,000 and 91,000 (sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis (PAGE)). Enzyme specificity assays were carried out using seven synthetic peptides containing 13 amino acid residues, but differing only on the 5th residue (K, R, Y, L, W or P). Peptide cleavage takes place only with amino acids K or R at the 5th position, which is typical of trypsin. The partially purified enzymes hydrolyzed casein and the synthetic trypsin substrates l-BApNA and N-α-p-tosyl-l-Arg methyl ester (l-TAME). Higher activity was observed at pH 8.5 and 35 °C when using l-BApNA as substrate and at pH 8.0 and 30 °C when using l-TAME. Maximum enzyme activity against l-BApNA was obtained with 20 mM CaCl2 in the reaction mixture. The partially purified enzymes showing trypsin activity were sensitive to inhibition by ethylenediaminetetraacetic acid (EDTA), phenylmethyl sulphonyl fluoride (PMSF), N-α-tosyl-l-lysine chloromethyl ketone (TLCK), benzamidine and aprotinin. Highest inhibition was obtained with TLCK and benzamidine. KM values obtained were 0.32 mM for l-BApNA and 52.5 μM for l-TAME.