Development of GFP-based high-throughput screening system for directed evolution of glucose oxidase
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 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. © 2018 The Society for Biotechnology, Japan
Keywords:
Directed evolution / Glucose oxidase / High-throughput screening / Yeast enhanced green-fluorescent protein / Yeast surface displaySource:
Journal of Bioscience and Bioengineering, 2018Publisher:
- Elsevier
Funding / projects:
- Allergens, antibodies, enzymes and small physiologically important molecules: design, structure, function and relevance (RS-MESTD-Basic Research (BR or ON)-172049)
- Study of structure-function relationships in the plant cell wall and modifications of the wall structure by enzyme engineering (RS-MESTD-Basic Research (BR or ON)-173017)
- DAAD bilateral project 451-03-01038/2015-09/21
Note:
- Supplementary material: http://cherry.chem.bg.ac.rs/handle/123456789/3013
DOI: 10.1016/j.jbiosc.2018.07.002
ISSN: 1389-1723
WoS: 000462808700005
Scopus: 2-s2.0-85050129752
Collections
Institution/Community
Hemijski fakultet / Faculty of ChemistryTY - JOUR AU - Kovačević, Gordana AU - Ostafe, Raluca AU - Balaž, Ana Marija AU - Fischer, Rainer AU - Prodanović, Radivoje PY - 2018 UR - https://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", 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. in 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. in Journal of Bioscience and Bioengineering. 2018;. 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 (2018), https://doi.org/10.1016/j.jbiosc.2018.07.002 . .