Purification of Leptinotarsa decemlineata (Say) Gut Specific Cysteine Protease Inhibitor(s) From Rapeseed

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dc.contributor.authorAshouri, Sh.
dc.contributor.authorZihnioglu, F.
dc.contributor.authorPourabad, R. Farshbaf
dc.contributor.authorKocadag, E.
dc.date.accessioned2019-10-27T11:08:11Z
dc.date.available2019-10-27T11:08:11Z
dc.date.issued2017
dc.departmentEge Üniversitesien_US
dc.description.abstractThe aim of the present work was to purify cysteine protease inhibitors from rapeseed (Brassica napus L.), with potential activity on digestive protease of Colorado Potato Beetle (CPB), Leptinotarsa decemlineata (Say). Ammonium sulfate precipitated proteinaceous fractions; 30, 50, 70, and 100% showed 39.07, 57.03, 51.47, and 22.44% inhibition on the fourth instar larval gut general protease activity, respectively. Fraction 50% showed the highest inhibitory effect on digestive general protease activity of all developmental stages. Gel assays approved the inhibition of the enzyme activity. Fraction 50% was purified by using various chromatography techniques; ion-exchange using DEAE, gel filtration and affinity using SiO2-CPB larval gut homogenate. Three peaks of protein were eluted from ion exchange chromatography using NaCl step gradient, also from gel filtration chromatography. When Z-Ala-Arg-Arg-4mBNA was used as cysteine protease substrate, the purification fold of second fraction of ion exchange chromatography was obtained 24.80, also the yield was 59.09%, the third fraction of gel permeation resulted in a 25.60 fold purification with 28.53% of recovery, and the fraction of affinity chromatography obtained a 22.72 fold purification and yielded 36.35%. In the SDS-PAGE, apparent molecular mass of purified proteins were 34 and 32 kDa by ion-exchange and 24 and 22 kDa by affinity. However, gel filtration was not an appropriate method in this study, because the purified protein band(s) were not observed on the gel. Consequently, these chromatography methods were appropriate methods to purification of inhibitor cystatins, specially affinity which was prepared by using CPB gut enzyme as ligand and obtained specific inhibitor proteins of CPB gut protease activity.en_US
dc.description.sponsorshipUniversity of Tabrizen_US
dc.description.sponsorshipThis project was funded by a grant from University of Tabriz. The authors are thankful to the Biochemistry Department, Ege University, Izmir, Turkey, where a big part of the study was conducted and to Dr. Serap Evran. Thanks to the Seed and Plant Improvement Institute of Karaj (Iran) for supplementation of seeds.en_US
dc.identifier.endpage667en_US
dc.identifier.issn1680-7073
dc.identifier.issn1680-7073en_US
dc.identifier.issue3en_US
dc.identifier.startpage653en_US
dc.identifier.urihttps://hdl.handle.net/11454/32133
dc.identifier.volume19en_US
dc.identifier.wosWOS:000403203600013en_US
dc.identifier.wosqualityQ2en_US
dc.indekslendigikaynakWeb of Scienceen_US
dc.language.isoenen_US
dc.publisherTarbiat Modares Univen_US
dc.relation.ispartofJournal of Agricultural Science and Technologyen_US
dc.relation.publicationcategoryMakale - Uluslararası Hakemli Dergi - Kurum Öğretim Elemanıen_US
dc.rightsinfo:eu-repo/semantics/closedAccessen_US
dc.subjectChromatographyen_US
dc.subjectColorado potato beetleen_US
dc.subjectEnzyme inhibitionen_US
dc.subjectProteinen_US
dc.subjectRapeseeden_US
dc.titlePurification of Leptinotarsa decemlineata (Say) Gut Specific Cysteine Protease Inhibitor(s) From Rapeseeden_US
dc.typeArticleen_US

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