Development of GFP-based high-throughput screening system for directed evolution of glucose oxidase
Samo za registrovane korisnike
2019
Članak u časopisu (Objavljena verzija)
Metapodaci
Prikaz svih podataka o dokumentuApstrakt
Glucose 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 t...han 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.
Ključne reči:
Directed evolution / Glucose oxidase / High-throughput screening / Yeast enhanced green-fluorescent protein / Yeast surface displayIzvor:
Journal of Bioscience and Bioengineering, 2019, 127, 1, 30-37Izdavač:
- Elsevier
Finansiranje / projekti:
- Alergeni, antitela, enzimi i mali fiziološki značajni molekuli: dizajn, struktura, funkcija i značaj (RS-MESTD-Basic Research (BR or ON)-172049)
- Ispitivanja odnosa struktura-funkcija u ćelijskom zidu biljaka i izmene strukture zida enzimskim inženjeringom (RS-MESTD-Basic Research (BR or ON)-173017)
- DAAD bilateral project 451-03-01038/2015-09/21
DOI: 10.1016/j.jbiosc.2018.07.002
ISSN: 1389-1723
PubMed: 30033354
WoS: 000462808700005
Scopus: 2-s2.0-85050129752
Institucija/grupa
IHTMTY - JOUR AU - Kovačević, Gordana AU - Ostafe, Raluca AU - Balaž, Ana Marija AU - Fischer, Rainer AU - Prodanović, Radivoje PY - 2019 UR - https://cer.ihtm.bg.ac.rs/handle/123456789/2497 AB - Glucose 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. PB - Elsevier T2 - Journal of Bioscience and Bioengineering T1 - Development of GFP-based high-throughput screening system for directed evolution of glucose oxidase VL - 127 IS - 1 SP - 30 EP - 37 DO - 10.1016/j.jbiosc.2018.07.002 ER -
@article{ author = "Kovačević, Gordana and Ostafe, Raluca and Balaž, Ana Marija and Fischer, Rainer and Prodanović, Radivoje", year = "2019", abstract = "Glucose 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.", publisher = "Elsevier", journal = "Journal of Bioscience and Bioengineering", title = "Development of GFP-based high-throughput screening system for directed evolution of glucose oxidase", volume = "127", number = "1", pages = "30-37", doi = "10.1016/j.jbiosc.2018.07.002" }
Kovačević, G., Ostafe, R., Balaž, A. M., Fischer, R.,& Prodanović, R.. (2019). Development of GFP-based high-throughput screening system for directed evolution of glucose oxidase. in Journal of Bioscience and Bioengineering Elsevier., 127(1), 30-37. https://doi.org/10.1016/j.jbiosc.2018.07.002
Kovačević G, Ostafe R, Balaž AM, Fischer R, Prodanović R. Development of GFP-based high-throughput screening system for directed evolution of glucose oxidase. in Journal of Bioscience and Bioengineering. 2019;127(1):30-37. doi:10.1016/j.jbiosc.2018.07.002 .
Kovačević, Gordana, Ostafe, Raluca, Balaž, Ana Marija, Fischer, Rainer, Prodanović, Radivoje, "Development of GFP-based high-throughput screening system for directed evolution of glucose oxidase" in Journal of Bioscience and Bioengineering, 127, no. 1 (2019):30-37, https://doi.org/10.1016/j.jbiosc.2018.07.002 . .