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

dc.creatorOliveira, M. G. A.
dc.creatorDe Simone, Salvatore Gianini
dc.creatorXavier, L. P.
dc.creatorGuedes, R. N. C.
dc.date2019-12-17T15:15:01Z
dc.date2019-12-17T15:15:01Z
dc.date2005
dc.date.accessioned2023-09-26T20:13:39Z
dc.date.available2023-09-26T20:13:39Z
dc.identifierOLIVEIRA, M. G. A. et al. Partial purification and characterization of digestive trypsin-like proteases from the velvet bean caterpillar, Anticarsia gemmatalis. Comparative Biochemistry and Physiology, Part B, v. 140, p. 369-380, 2005.
dc.identifier1096-4959
dc.identifierhttps://www.arca.fiocruz.br/handle/icict/38656
dc.identifier10.1016/j.cbpc.2004.10.018
dc.identifier.urihttps://repositorioslatinoamericanos.uchile.cl/handle/2250/8851890
dc.descriptionTrypsin-like proteases from the midgut of Anticarsia gemmatalis Hqbner (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-a-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, Wor 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 Na- p-tosyl-l-Arg methyl ester (l-TAME). Higher activity was observed at pH 8.5 and 35 8C when using l-BApNA as substrate and at pH 8.0 and 30 8C 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-a-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 AM for l-TAME.
dc.description2025-01-01
dc.formatapplication/pdf
dc.languageeng
dc.publisherElsevier
dc.rightsrestricted access
dc.subjectTripsina
dc.subjectProteases serinas
dc.subjectProteases digestivas
dc.subjectEspecificidade do substrato
dc.subjectInibição da protease
dc.subjectCinética da protease
dc.subjectHidrólise de peptídeos sintéticos
dc.subjectTrypsin
dc.subjectSerine proteases
dc.subjectDigestive proteases
dc.subjectLepidoptera midgut
dc.subjectSubstrate specificity
dc.subjectCleavage site
dc.subjectNoctuidae
dc.subjectProtease inhibition
dc.subjectProtease kinetics
dc.subjectSynthetic peptide hydrolysis
dc.titlePartial purification and characterization of digestive trypsin-like proteases from the velvet bean caterpillar, Anticarsia gemmatalis
dc.typeArticle


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