Ultra-High-Throughput Screening Method for the Directed Evolution of Glucose Oxidase
Apstrakt
Glucose oxidase (GOx) is used in many industrial processes that could benefit from improved versions of the enzyme. Some improvements like higher activity under physiological conditions and thermal stability could be useful for GOx applications in biosensors and biofuel cells. Directed evolution is one of the currently available methods to engineer improved GOx variants. Here, we describe an ultra-high-throughput screening system for sorting the best enzyme variants generated by directed evolution that incorporates several methodological refinements: flow cytometry, in vitro compartmentalization, yeast surface display, fluorescent labeling of the expressed enzyme, delivery of glucose substrate to the reaction mixture through the oil phase, and covalent labeling of the cells with fluorescein-tyramide. The method enables quantitative screening of gene libraries to identify clones with improved activity and it also allows cells to be selected based not only on the overall activity but als...o on the specific activity of the enzyme.
Izvor:
Chemistry and Biology, 2014, 21, 3, 414-421Izdavač:
- Cell Press, Cambridge
DOI: 10.1016/j.chembiol.2014.01.010
ISSN: 1074-5521
PubMed: 24613019
WoS: 000333407000014
Scopus: 2-s2.0-84897065790
Kolekcije
Institucija/grupa
Hemijski fakultet / Faculty of ChemistryTY - JOUR AU - Ostafe, Raluca AU - Prodanović, Radivoje AU - Nazor, Jovana AU - Fischer, Rainer PY - 2014 UR - https://cherry.chem.bg.ac.rs/handle/123456789/1519 AB - Glucose oxidase (GOx) is used in many industrial processes that could benefit from improved versions of the enzyme. Some improvements like higher activity under physiological conditions and thermal stability could be useful for GOx applications in biosensors and biofuel cells. Directed evolution is one of the currently available methods to engineer improved GOx variants. Here, we describe an ultra-high-throughput screening system for sorting the best enzyme variants generated by directed evolution that incorporates several methodological refinements: flow cytometry, in vitro compartmentalization, yeast surface display, fluorescent labeling of the expressed enzyme, delivery of glucose substrate to the reaction mixture through the oil phase, and covalent labeling of the cells with fluorescein-tyramide. The method enables quantitative screening of gene libraries to identify clones with improved activity and it also allows cells to be selected based not only on the overall activity but also on the specific activity of the enzyme. PB - Cell Press, Cambridge T2 - Chemistry and Biology T1 - Ultra-High-Throughput Screening Method for the Directed Evolution of Glucose Oxidase VL - 21 IS - 3 SP - 414 EP - 421 DO - 10.1016/j.chembiol.2014.01.010 ER -
@article{ author = "Ostafe, Raluca and Prodanović, Radivoje and Nazor, Jovana and Fischer, Rainer", year = "2014", abstract = "Glucose oxidase (GOx) is used in many industrial processes that could benefit from improved versions of the enzyme. Some improvements like higher activity under physiological conditions and thermal stability could be useful for GOx applications in biosensors and biofuel cells. Directed evolution is one of the currently available methods to engineer improved GOx variants. Here, we describe an ultra-high-throughput screening system for sorting the best enzyme variants generated by directed evolution that incorporates several methodological refinements: flow cytometry, in vitro compartmentalization, yeast surface display, fluorescent labeling of the expressed enzyme, delivery of glucose substrate to the reaction mixture through the oil phase, and covalent labeling of the cells with fluorescein-tyramide. The method enables quantitative screening of gene libraries to identify clones with improved activity and it also allows cells to be selected based not only on the overall activity but also on the specific activity of the enzyme.", publisher = "Cell Press, Cambridge", journal = "Chemistry and Biology", title = "Ultra-High-Throughput Screening Method for the Directed Evolution of Glucose Oxidase", volume = "21", number = "3", pages = "414-421", doi = "10.1016/j.chembiol.2014.01.010" }
Ostafe, R., Prodanović, R., Nazor, J.,& Fischer, R.. (2014). Ultra-High-Throughput Screening Method for the Directed Evolution of Glucose Oxidase. in Chemistry and Biology Cell Press, Cambridge., 21(3), 414-421. https://doi.org/10.1016/j.chembiol.2014.01.010
Ostafe R, Prodanović R, Nazor J, Fischer R. Ultra-High-Throughput Screening Method for the Directed Evolution of Glucose Oxidase. in Chemistry and Biology. 2014;21(3):414-421. doi:10.1016/j.chembiol.2014.01.010 .
Ostafe, Raluca, Prodanović, Radivoje, Nazor, Jovana, Fischer, Rainer, "Ultra-High-Throughput Screening Method for the Directed Evolution of Glucose Oxidase" in Chemistry and Biology, 21, no. 3 (2014):414-421, https://doi.org/10.1016/j.chembiol.2014.01.010 . .