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Vanadium bromoperoxidase-coupled fluorescent assay for flow cytometry sorting of glucose oxidase gene libraries in double emulsions
dc.creator | Prodanović, Radivoje | |
dc.creator | Ostafe, Raluca | |
dc.creator | Blanusa, Milan | |
dc.creator | Schwaneberg, Ulrich | |
dc.date.accessioned | 2018-11-22T00:21:52Z | |
dc.date.available | 2018-11-22T00:21:52Z | |
dc.date.issued | 2012 | |
dc.identifier.issn | 1618-2642 | |
dc.identifier.uri | https://cherry.chem.bg.ac.rs/handle/123456789/1525 | |
dc.description.abstract | A 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.publisher | Springer Heidelberg, Heidelberg | |
dc.relation | Alexander von Humboldt foundation | |
dc.relation | BRAIN AG company | |
dc.relation | BMBF BiochancePlus program | |
dc.rights | restrictedAccess | |
dc.source | Analytical and Bioanalytical Chemistry | |
dc.subject | In vitro compartmentalization | en |
dc.subject | Directed evolution | en |
dc.subject | Hydrogen peroxide | en |
dc.subject | FACS | en |
dc.title | Vanadium bromoperoxidase-coupled fluorescent assay for flow cytometry sorting of glucose oxidase gene libraries in double emulsions | en |
dc.type | article | |
dc.rights.license | ARR | |
dcterms.abstract | Блануса, Милан; Продановић, Радивоје; Остафе, Ралуца; Сцхwанеберг, Улрицх; | |
dc.citation.volume | 404 | |
dc.citation.issue | 5 | |
dc.citation.spage | 1439 | |
dc.citation.epage | 1447 | |
dc.identifier.wos | 000307957800016 | |
dc.identifier.doi | 10.1007/s00216-012-6234-x | |
dc.citation.other | 404(5): 1439-1447 | |
dc.citation.rank | M21 | |
dc.identifier.pmid | 22811062 | |
dc.type.version | publishedVersion | en |
dc.identifier.scopus | 2-s2.0-84865466722 |