Show simple item record

dc.creatorKovačević, Gordana
dc.creatorOstafe, Raluca
dc.creatorBalaž, Ana Marija
dc.creatorFischer, Rainer
dc.creatorProdanović, Radivoje
dc.date.accessioned2018-11-22T00:42:38Z
dc.date.available2018-11-22T00:42:38Z
dc.date.issued2018
dc.identifier.issn1389-1723
dc.identifier.urihttp://cherry.chem.bg.ac.rs/handle/123456789/336
dc.description.abstractGlucose oxidase (GOx) mutants with higher activity or stability have important role in industry and in the development of biosensors and biofuel cells. Discovering these mutants can be time-consuming if appropriate high-throughput screening (HTS) systems are not available. GOx gene libraries were successfully screened and sorted using a HTS system based on GOx activity dependent fluorescent labeling of yeast cells with tyramids and quantification of the amount of expressed enzyme by yeast enhanced green fluorescent protein (yGFP) tagging and flow cytometry. For this purpose, we expressed wild type and a mutant GOx as a chimera with the yGFP to confirm differences in catalytic activity between wild-type and mutant GOx. Fluorescence of yGFP is preserved during expression of chimera, and also after the oxidative enzymatic reaction. We have obtained a 2.5-fold enrichment in population of cells expressing active enzyme, and percentage of enzyme variants with enzymatic mean activity higher than wild type activity was increased to 44% after a single round of GOx gene library sorting. We have found two mutants with 1.3 and 2.3-fold increase in Vmax values compared to the wtGOx. By simultaneous detection of protein expression level and enzyme activity we have increased the likelihood of finding GOx variants with increased activity in a single round of flow cytometry sorting. © 2018 The Society for Biotechnology, Japanen
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.relationDAAD bilateral project 451-03-01038/2015-09/21
dc.rightsrestrictedAccess
dc.sourceJournal of Bioscience and Bioengineering
dc.subjectDirected evolutionen
dc.subjectGlucose oxidaseen
dc.subjectHigh-throughput screeningen
dc.subjectYeast enhanced green-fluorescent proteinen
dc.subjectYeast surface displayen
dc.titleDevelopment of GFP-based high-throughput screening system for directed evolution of glucose oxidaseen
dc.typearticle
dc.rights.licenseARR
dcterms.abstractБалаж, A.М.; Продановић, Радивоје; Фисцхер, Р.; Остафе, Р.; Ковачевић, Гордана;
dc.identifier.doi10.1016/j.jbiosc.2018.07.002
dc.citation.rankM22~
dc.type.versionpublishedVersionen
dc.identifier.scopus2-s2.0-85050129752
dc.identifier.rcubKon_1307


Files in this item

Thumbnail

This item appears in the following Collection(s)

Show simple item record