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dc.creatorIlić Đurđić, Karla
dc.creatorEce, Selin
dc.creatorOstafe, Raluca
dc.creatorVogel, Simon
dc.creatorBalaž, Ana Marija
dc.creatorSchillberg, Stefan
dc.creatorFischer, Rainer
dc.creatorProdanović, Radivoje
dc.date.accessioned2020-06-26T09:48:35Z
dc.date.available2020-06-26T09:48:35Z
dc.date.issued2020
dc.identifier.issn1389-1723
dc.identifier.urihttp://cherry.chem.bg.ac.rs/handle/123456789/3974
dc.identifier.urihttp://cer.ihtm.bg.ac.rs/handle/123456789/3580
dc.description.abstractLignin peroxidase (LiP) is a heme-containing oxidoreductase that oxidizes structurally diverse substrates in an H2O2-dependent manner. Its ability to oxidize many pollutants makes it suitable for bioremediation applications and an ideal candidate for optimization by mutagenesis and selection. In order to increase oxidative stability of LiP we generated a random mutagenesis library comprising 106 mutated LiP genes and screened for expressed enzymes with higher than wild-type activity after incubation in 30 mM H2O2 by flow cytometry with fluorescein-tyramide as a substrate. To preserve the genotype-phenotype connection, the LiP mutants were displayed on the yeast cell surface. Two rounds of sorting were performed, recovered colonies were then screened in microtiter plates, and activity analysis revealed a significant increase in the percentage of cells expressing LiP variants with higher oxidative stability than wtLiP. Two rounds of sorting increased the proportion of more-stable variants from 1.4% in the original library to 52.3%. The most stable variants after two rounds of sorting featured between two and four mutations and retained up to 80% of initial activity after 1 h incubation in 30 mM H2O2. We for the first-time applied flow cytometry for screening of any ligninolytic peroxidase library. Obtained results suggest that developed system may be applied for improvement of industrially important characteristics of lignin peroxidase.en
dc.publisherElsevier
dc.relationinfo:eu-repo/grantAgreement/MESTD/Integrated and Interdisciplinary Research (IIR or III)/46010/RS//
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.sourceJournal of Bioscience and Bioengineering
dc.subjectChimera
dc.subjectDirected evolution
dc.subjectFluorescence activated cell sorting
dc.subjectHydrogen-peroxide stability
dc.subjectYeast surface display
dc.titleFlow cytometry-based system for screening of lignin peroxidase mutants with higher oxidative stabilityen
dc.typearticle
dc.rights.licenseARR
dcterms.abstractИлић Ђурђић, Карла; Остафе, Ралуца; Балаж, Aна Марија; Фисцхер, Раинер; Продановић, Радивоје; Еце, Селин; Вогел, Симон; Сцхиллберг, Стефан;
dc.citation.volume129
dc.citation.issue6
dc.citation.spage664
dc.citation.epage671
dc.citation.rankM22~
dc.identifier.doi10.1016/j.jbiosc.2019.12.009
dc.identifier.scopus2-s2.0-85079014891
dc.identifier.wos000614233200003
dc.type.versionpublishedVersion


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