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dc.creatorProdanović, Radivoje
dc.creatorOstafe, Raluca
dc.creatorBlanusa, Milan
dc.creatorSchwaneberg, Ulrich
dc.date.accessioned2018-11-22T00:21:52Z
dc.date.available2018-11-22T00:21:52Z
dc.date.issued2012
dc.identifier.issn1618-2642
dc.identifier.urihttp://cherry.chem.bg.ac.rs/handle/123456789/1525
dc.description.abstractA Vanadium bromoPeroxidase-coupled fluorescent assay (ViPer) for ultrahigh-throughput screening of glucose oxidase (GOx) gene libraries employing double emulsions and flow cytometry was developed. The assay is based on detection of the product of a GOx reaction, hydrogen peroxide, that is first converted to a hypobromide by vanadium bromoperoxidase in the presence of sodium bromide. The hypobromide is afterwards detected in a reaction with a fluorogenic probe, 3-carboxy-7-(4'-aminophenoxy)-coumarine, where fluorescent 3-carboxy-coumarine is released. The ViPer screening system is three times more sensitive than a horseradish peroxidase coupled detection system and more resistant to bleaching of fluorescence in excess of peroxide. Using the ViPer screening system a high epPCR gene library containing 100,000 different GOx variants was screened for active clones in less than 1 h by flow cytometry. A library containing 0.15 % of yeast cells expressing active enzyme variants and with an average GOx activity in the liquid culture of 0.47 U/mL, after one round of sorting, had 28.12 % of the yeast cells expressing the active GOx (an enrichment factor of 200) and 26.8 U/mL of the GOx activity in the liquid culture (an enrichment factor of 57). The developed screening system could be adapted and used in a directed evolution of GOx and other hydrogen peroxide-producing enzymes (oxidases) and glycosidases if coupled with a carbohydrate oxidase.en
dc.publisherSpringer Heidelberg, Heidelberg
dc.relationAlexander von Humboldt foundation
dc.relationBRAIN AG company
dc.relationBMBF BiochancePlus program
dc.rightsrestrictedAccess
dc.sourceAnalytical and Bioanalytical Chemistry
dc.subjectIn vitro compartmentalizationen
dc.subjectDirected evolutionen
dc.subjectHydrogen peroxideen
dc.subjectFACSen
dc.titleVanadium bromoperoxidase-coupled fluorescent assay for flow cytometry sorting of glucose oxidase gene libraries in double emulsionsen
dc.typearticle
dc.rights.licenseARR
dcterms.abstractБлануса, Милан; Продановић, Радивоје; Остафе, Ралуца; Сцхwанеберг, Улрицх;
dc.citation.volume404
dc.citation.issue5
dc.citation.spage1439
dc.citation.epage1447
dc.identifier.wos000307957800016
dc.identifier.doi10.1007/s00216-012-6234-x
dc.citation.other404(5): 1439-1447
dc.citation.rankM21
dc.identifier.pmid22811062
dc.type.versionpublishedVersionen
dc.identifier.scopus2-s2.0-84865466722
dc.identifier.rcubKon_2356


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