Flow cytometry-based system for screening of lignin peroxidase mutants with higher oxidative stability
Samo za registrovane korisnike
2020
Autori
Ilić Đurđić, KarlaEce, Selin
Ostafe, Raluca
Vogel, Simon
Balaž, Ana Marija
Schillberg, Stefan
Fischer, Rainer
Prodanović, Radivoje
Članak u časopisu (Objavljena verzija)
Metapodaci
Prikaz svih podataka o dokumentuApstrakt
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 variant...s 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.
Ključne reči:
Chimera / Directed evolution / Fluorescence activated cell sorting / Hydrogen-peroxide stability / Yeast surface displayIzvor:
Journal of Bioscience and Bioengineering, 2020, 129, 6, 664-671Izdavač:
- Elsevier
Finansiranje / projekti:
- Razvoj novih inkapsulacionih i enzimskih tehnologija za proizvodnju biokatalizatora i biološki aktivnih komponenata hrane u cilju povećanja njene konkurentnosti, kvaliteta i bezbednosti (RS-46010)
- Alergeni, antitela, enzimi i mali fiziološki značajni molekuli: dizajn, struktura, funkcija i značaj (RS-172049)
- Ispitivanja odnosa struktura-funkcija u ćelijskom zidu biljaka i izmene strukture zida enzimskim inženjeringom (RS-173017)
DOI: 10.1016/j.jbiosc.2019.12.009
ISSN: 1389-1723
WoS: 000614233200003
Scopus: 2-s2.0-85079014891
URI
http://cherry.chem.bg.ac.rs/handle/123456789/3974https://cer.ihtm.bg.ac.rs/handle/123456789/3580
Institucija/grupa
IHTMTY - JOUR AU - Ilić Đurđić, Karla AU - Ece, Selin AU - Ostafe, Raluca AU - Vogel, Simon AU - Balaž, Ana Marija AU - Schillberg, Stefan AU - Fischer, Rainer AU - Prodanović, Radivoje PY - 2020 UR - http://cherry.chem.bg.ac.rs/handle/123456789/3974 UR - https://cer.ihtm.bg.ac.rs/handle/123456789/3580 AB - 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. PB - Elsevier T2 - Journal of Bioscience and Bioengineering T1 - Flow cytometry-based system for screening of lignin peroxidase mutants with higher oxidative stability VL - 129 IS - 6 SP - 664 EP - 671 DO - 10.1016/j.jbiosc.2019.12.009 ER -
@article{ author = "Ilić Đurđić, Karla and Ece, Selin and Ostafe, Raluca and Vogel, Simon and Balaž, Ana Marija and Schillberg, Stefan and Fischer, Rainer and Prodanović, Radivoje", year = "2020", 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.", publisher = "Elsevier", journal = "Journal of Bioscience and Bioengineering", title = "Flow cytometry-based system for screening of lignin peroxidase mutants with higher oxidative stability", volume = "129", number = "6", pages = "664-671", doi = "10.1016/j.jbiosc.2019.12.009" }
Ilić Đurđić, K., Ece, S., Ostafe, R., Vogel, S., Balaž, A. M., Schillberg, S., Fischer, R.,& Prodanović, R.. (2020). Flow cytometry-based system for screening of lignin peroxidase mutants with higher oxidative stability. in Journal of Bioscience and Bioengineering Elsevier., 129(6), 664-671. https://doi.org/10.1016/j.jbiosc.2019.12.009
Ilić Đurđić K, Ece S, Ostafe R, Vogel S, Balaž AM, Schillberg S, Fischer R, Prodanović R. Flow cytometry-based system for screening of lignin peroxidase mutants with higher oxidative stability. in Journal of Bioscience and Bioengineering. 2020;129(6):664-671. doi:10.1016/j.jbiosc.2019.12.009 .
Ilić Đurđić, Karla, Ece, Selin, Ostafe, Raluca, Vogel, Simon, Balaž, Ana Marija, Schillberg, Stefan, Fischer, Rainer, Prodanović, Radivoje, "Flow cytometry-based system for screening of lignin peroxidase mutants with higher oxidative stability" in Journal of Bioscience and Bioengineering, 129, no. 6 (2020):664-671, https://doi.org/10.1016/j.jbiosc.2019.12.009 . .