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dc.creatorIlić Đurđić, Karla
dc.creatorEce, Selin
dc.creatorOstafe, Raluca
dc.creatorVogel, Simon
dc.creatorSchillberg, Stefan
dc.creatorFischer, Rainer
dc.creatorProdanović, Radivoje
dc.date.accessioned2020-03-27T17:47:22Z
dc.date.available2020-03-27T17:47:22Z
dc.date.issued2020
dc.identifier.issn1369-703X
dc.identifier.urihttp://cherry.chem.bg.ac.rs/handle/123456789/3888
dc.description.abstractPleurotus eryngii wild-type versatile peroxidase (wtVP) oxidizes structurally diverse substrates in an H2O2-dependent manner, but its ability to oxidize many pollutants is limited by suicidal enzyme inactivation in the presence of excess H2O2. To address this drawback, we generated random mutagenesis libraries containing 3 × 106 mutated VP genes and screened for enzymes with higher oxidative stability expressed on the surface of yeast cells. This was achieved by flow cytometry using the substrate fluorescein tyramide. After two rounds of sorting, the percentage of cells expressing variants with improved oxidative stability had increased from 1 % to 56 %. The most stable variants featured 3–5 amino acid substitutions and retained up to 70 % of their initial activity after incubation for 1 h in 30 mM H2O2 (conditions that completely inactivate wtVP). Selected variants were extracted from yeast cell walls and purified for kinetic characterization. We also prepared yeast cell walls with wtVP and the three most stable VP variants for multiple cycles of azo dye (Reactive black 5) degradation. After 10 cycles of 12 h, two of the variants retained more than 97 % of their initial activity, whereas the activity of wtVP declined by ∼30 %. These results confirm that our high-throughput screening system can improve the oxidative stability of versatile peroxidase, providing a source of novel enzymes for remediation applications.
dc.publisherElsevier
dc.relationinfo:eu-repo/grantAgreement/MESTD/Basic Research (BR or ON)/172049/RS//
dc.relationinfo:eu-repo/grantAgreement/MESTD/Integrated and Interdisciplinary Research (IIR or III)/46010/RS//
dc.rightsrestrictedAccess
dc.sourceBiochemical Engineering Journal
dc.subjectYeast surface display
dc.subjectDirected evolution
dc.subjectHydrogen-peroxide stability
dc.subjectFACS
dc.titleImprovement in oxidative stability of versatile peroxidase by flow cytometry-based high-throughput screening system
dc.typearticle
dc.rights.licenseARR
dcterms.abstractВогел, Симон; Илић Ђурђић, Карла; Остафе, Ралуца; Сцхиллберг, Стефан; Фисцхер, Раинер; Продановић, Радивоје; Еце, Селин;
dc.citation.volume157
dc.identifier.wos000527325100023
dc.identifier.doi10.1016/j.bej.2020.107555
dc.citation.rankM21~
dc.type.versionpublishedVersion
dc.identifier.scopus2-s2.0-85081216986


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