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dc.creatorBlažić, Marija
dc.creatorKovačević, Gordana
dc.creatorProdanović, Olivera
dc.creatorOstafe, Raluca
dc.creatorGavrović-Jankulović, Marija
dc.creatorFischer, Rainer
dc.creatorProdanović, Radivoje
dc.date.accessioned2019-01-30T17:37:23Z
dc.date.available2019-01-30T17:37:23Z
dc.date.issued2013
dc.identifier.issn1046-5928
dc.identifier.urihttp://cer.ihtm.bg.ac.rs/handle/123456789/1331
dc.description.abstractGlucose oxidase (GOx) catalyzes the oxidation of glucose to form gluconic acid and hydrogen peroxide, a reaction with important applications in food preservation, the manufacture of cosmetics and pharmaceuticals, and the development of glucose monitoring devices and biofuel cells. We expressed Aspergillus niger wild type GOx and the B11 mutant, which has twice the activity of the wild type enzyme at pH 5.5, as C-terminal fusions with the Saccharomyces cerevisiae Aga2 protein, allowing the fusion proteins to be displayed on the surface of yeast EBY100 cells. After expression, we extracted the proteins from the yeast cell wall and purified them by ion-exchange chromatography and ultrafiltration. This produced a broad 100-140 kDa band by denaturing SDS-PAGE and a high-molecular-weight band by native PAGE corresponding to the activity band revealed by zymography. The wild type and B11 fusion proteins had k(cat) values of 33.3 and 61.3 s(-1) and K-m values for glucose of 33.4 and 27.9 mM, respectively. The pH optimum for both enzymes was 5.0. The kinetic properties of the fusion proteins displayed the same ratio as their native counterparts, confirming that yeast surface display is suitable for the high-throughput directed evolution of GOx using flow cytometry for selection. Aga2-GOx fusion proteins in the yeast cell wall could also be used as immobilized catalysts for the production of gluconic acid.en
dc.publisherAcademic Press Inc Elsevier Science, San Diego
dc.relationinfo:eu-repo/grantAgreement/MESTD/Basic Research (BR or ON)/172049/RS//
dc.relationinfo:eu-repo/grantAgreement/MESTD/Basic Research (BR or ON)/173017/RS//
dc.rightsrestrictedAccess
dc.sourceProtein Expression and Purification
dc.subjectSaccharomyces cerevisiaeen
dc.subjectGlucose oxidaseen
dc.subjectDirected evolutionen
dc.subjectHigh throughput screeningen
dc.subjectChimeraen
dc.titleYeast surface display for the expression, purification and characterization of wild-type and B11 mutant glucose oxidasesen
dc.typearticle
dc.rights.licenseARR
dcterms.abstractБлажић, Марија; Продановиц, Оливера; Остафе, Ралуца; Продановиц, Радивоје; Фисцхер, Раинер; Ковацевиц, Гордана; Гавровиц-Јанкуловиц, Марија;
dc.citation.volume89
dc.citation.issue2
dc.citation.spage175
dc.citation.epage180
dc.citation.other89(2): 175-180
dc.citation.rankM23
dc.identifier.pmid23562736
dc.identifier.doi10.1016/j.pep.2013.03.014
dc.identifier.rcubConv_2981
dc.identifier.scopus2-s2.0-84876377170
dc.identifier.wos000319373300009
dc.type.versionpublishedVersion


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