DAAD bilateral project 451-03-01038/2015-09/21

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DAAD bilateral project 451-03-01038/2015-09/21

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Publications

Development of GFP-based high-throughput screening system for directed evolution of glucose oxidase

Kovačević, Gordana; Ostafe, Raluca; Balaž, Ana Marija; Fischer, Rainer; Prodanović, Radivoje

(Elsevier, 2018)

TY  - JOUR
AU  - Kovačević, Gordana
AU  - Ostafe, Raluca
AU  - Balaž, Ana Marija
AU  - Fischer, Rainer
AU  - Prodanović, Radivoje
PY  - 2018
UR  - http://cherry.chem.bg.ac.rs/handle/123456789/336
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. © 2018 The Society for Biotechnology, Japan
PB  - Elsevier
T2  - Journal of Bioscience and Bioengineering
T1  - Development of GFP-based high-throughput screening system for directed evolution of glucose oxidase
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 = "2018",
url = "http://cherry.chem.bg.ac.rs/handle/123456789/336",
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. © 2018 The Society for Biotechnology, Japan",
publisher = "Elsevier",
journal = "Journal of Bioscience and Bioengineering",
title = "Development of GFP-based high-throughput screening system for directed evolution of glucose oxidase",
doi = "10.1016/j.jbiosc.2018.07.002"
}
Kovačević, G., Ostafe, R., Balaž, A. M., Fischer, R.,& Prodanović, R. (2018). Development of GFP-based high-throughput screening system for directed evolution of glucose oxidase.
Journal of Bioscience and Bioengineering
Elsevier..
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. Journal of Bioscience and Bioengineering. 2018;
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" Journal of Bioscience and Bioengineering (2018),
https://doi.org/10.1016/j.jbiosc.2018.07.002 .
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Supplementary data for the article: Kovačević, G.; Ostafe, R.; Balaž, A. M.; Fischer, R.; Prodanović, R. Development of GFP-Based High-Throughput Screening System for Directed Evolution of Glucose Oxidase. Journal of Bioscience and Bioengineering 2019, 127 (1), 30–37. https://doi.org/10.1016/j.jbiosc.2018.07.002

Kovačević, Gordana; Ostafe, Raluca; Balaž, Ana Marija; Fischer, Rainer; Prodanović, Radivoje

(Elsevier, 2018)

TY  - BOOK
AU  - Kovačević, Gordana
AU  - Ostafe, Raluca
AU  - Balaž, Ana Marija
AU  - Fischer, Rainer
AU  - Prodanović, Radivoje
PY  - 2018
UR  - http://cherry.chem.bg.ac.rs/handle/123456789/3013
PB  - Elsevier
T2  - Journal of Bioscience and Bioengineering
T1  - Supplementary data for the article: Kovačević, G.; Ostafe, R.; Balaž, A. M.; Fischer, R.; Prodanović, R. Development of GFP-Based High-Throughput Screening System for Directed Evolution of Glucose Oxidase. Journal of Bioscience and Bioengineering 2019, 127 (1), 30–37. https://doi.org/10.1016/j.jbiosc.2018.07.002
ER  - 
@book{
author = "Kovačević, Gordana and Ostafe, Raluca and Balaž, Ana Marija and Fischer, Rainer and Prodanović, Radivoje",
year = "2018",
url = "http://cherry.chem.bg.ac.rs/handle/123456789/3013",
publisher = "Elsevier",
journal = "Journal of Bioscience and Bioengineering",
title = "Supplementary data for the article: Kovačević, G.; Ostafe, R.; Balaž, A. M.; Fischer, R.; Prodanović, R. Development of GFP-Based High-Throughput Screening System for Directed Evolution of Glucose Oxidase. Journal of Bioscience and Bioengineering 2019, 127 (1), 30–37. https://doi.org/10.1016/j.jbiosc.2018.07.002"
}
Kovačević, G., Ostafe, R., Balaž, A. M., Fischer, R.,& Prodanović, R. (2018). Supplementary data for the article: Kovačević, G.; Ostafe, R.; Balaž, A. M.; Fischer, R.; Prodanović, R. Development of GFP-Based High-Throughput Screening System for Directed Evolution of Glucose Oxidase. Journal of Bioscience and Bioengineering 2019, 127 (1), 30–37. https://doi.org/10.1016/j.jbiosc.2018.07.002.
Journal of Bioscience and Bioengineering
Elsevier..
Kovačević G, Ostafe R, Balaž AM, Fischer R, Prodanović R. Supplementary data for the article: Kovačević, G.; Ostafe, R.; Balaž, A. M.; Fischer, R.; Prodanović, R. Development of GFP-Based High-Throughput Screening System for Directed Evolution of Glucose Oxidase. Journal of Bioscience and Bioengineering 2019, 127 (1), 30–37. https://doi.org/10.1016/j.jbiosc.2018.07.002. Journal of Bioscience and Bioengineering. 2018;
Kovačević Gordana, Ostafe Raluca, Balaž Ana Marija, Fischer Rainer, Prodanović Radivoje, "Supplementary data for the article: Kovačević, G.; Ostafe, R.; Balaž, A. M.; Fischer, R.; Prodanović, R. Development of GFP-Based High-Throughput Screening System for Directed Evolution of Glucose Oxidase. Journal of Bioscience and Bioengineering 2019, 127 (1), 30–37. https://doi.org/10.1016/j.jbiosc.2018.07.002" Journal of Bioscience and Bioengineering (2018)