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Characterization of a prolyl endoprotease from Eurygaster integriceps puton (Sunn pest) infested wheat
Author(s) -
Darkoh Charles,
ElBouhssini Mustapha,
Baum Michael,
Clack Beatrice
Publication year - 2010
Publication title -
archives of insect biochemistry and physiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.576
H-Index - 66
eISSN - 1520-6327
pISSN - 0739-4462
DOI - 10.1002/arch.20370
Subject(s) - gluten , enzyme , proline , biology , enzyme assay , digestive enzyme , biochemistry , size exclusion chromatography , food science , amino acid , amylase
Sunn pest, Eurygaster integriceps , Puton, infested and uninfested wheat seeds were obtained from the International Center for Agriculture Research in the Dry Areas (ICARDA), Aleppo, Syria, with the primary objective to identify the type of enzyme deposited by the Sunn pest on the wheat responsible for the gluten degradation. Enzyme levels were extremely low due to the enzyme being secreted by the insect in localized areas on the seed. Only extract from the infested wheat contained glutenase activity. Anion exchange, Cu 2+ sepharose, and gel filtration chromatography were used to partially purify and enrich protein samples from both infested wheat and uninfested wheat. An SDS‐gluten assay was used to show gluten specificity while a commercially available chromogenic proline peptide, benzyloxycarbonyl‐Gly‐Pro‐p‐nitroanalide (ZGPpNA), was utilized to identify fractions containing the active proline specific enzyme activity and to determine Michaelis‐Menten kinetics. Despite low levels of enzyme on the infested wheat, the enzyme was partially purified and enriched exhibiting a specific activity of 4.5 U/mg of total protein for gluten in a SDS gluten assay (1 U of enzyme activity was defined as the decrease in gel height in millimeters in 1 h) and exhibited a high‐affinity Km of 65 µM for ZGPpNA, cleaving at the carboxy terminus of the proline residue. The enzyme exhibited optimal activity between pH 8 and 10.0 at temperatures between 20° and 35°C. The enzyme was identified to be a prolyl endoprotease. © 2010 Wiley Periodicals, Inc.