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Flow cytometry-based system for screening of lignin peroxidase mutants with higher oxidative stability
dc.creator | Ilić Đurđić, Karla | |
dc.creator | Ece, Selin | |
dc.creator | Ostafe, Raluca | |
dc.creator | Vogel, Simon | |
dc.creator | Balaž, Ana Marija | |
dc.creator | Schillberg, Stefan | |
dc.creator | Fischer, Rainer | |
dc.creator | Prodanović, Radivoje | |
dc.date.accessioned | 2020-06-26T09:48:35Z | |
dc.date.available | 2020-06-26T09:48:35Z | |
dc.date.issued | 2020 | |
dc.identifier.issn | 1389-1723 | |
dc.identifier.uri | http://cherry.chem.bg.ac.rs/handle/123456789/3974 | |
dc.identifier.uri | https://cer.ihtm.bg.ac.rs/handle/123456789/3580 | |
dc.description.abstract | Lignin 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.publisher | Elsevier | |
dc.relation | info:eu-repo/grantAgreement/MESTD/Integrated and Interdisciplinary Research (IIR or III)/46010/RS// | |
dc.relation | info:eu-repo/grantAgreement/MESTD/Basic Research (BR or ON)/172049/RS// | |
dc.relation | info:eu-repo/grantAgreement/MESTD/Basic Research (BR or ON)/173017/RS// | |
dc.rights | restrictedAccess | |
dc.source | Journal of Bioscience and Bioengineering | |
dc.subject | Chimera | |
dc.subject | Directed evolution | |
dc.subject | Fluorescence activated cell sorting | |
dc.subject | Hydrogen-peroxide stability | |
dc.subject | Yeast surface display | |
dc.title | Flow cytometry-based system for screening of lignin peroxidase mutants with higher oxidative stability | en |
dc.type | article | |
dc.rights.license | ARR | |
dcterms.abstract | Илић Ђурђић, Карла; Остафе, Ралуца; Балаж, Aна Марија; Фисцхер, Раинер; Продановић, Радивоје; Еце, Селин; Вогел, Симон; Сцхиллберг, Стефан; | |
dc.citation.volume | 129 | |
dc.citation.issue | 6 | |
dc.citation.spage | 664 | |
dc.citation.epage | 671 | |
dc.citation.rank | M22~ | |
dc.identifier.doi | 10.1016/j.jbiosc.2019.12.009 | |
dc.identifier.scopus | 2-s2.0-85079014891 | |
dc.identifier.wos | 000614233200003 | |
dc.type.version | publishedVersion |