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.
TY - JOUR
AU - Ostafe, Raluca
AU - Prodanović, Radivoje
AU - Nazor, Jovana
AU - Fischer, Rainer
PY - 2014
UR - http://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",
url = "http://cherry.chem.bg.ac.rs/handle/123456789/1519",
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.
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. Chemistry and Biology. 2014;21(3):414-421
Ostafe Raluca, Prodanović Radivoje, Nazor Jovana, Fischer Rainer, "Ultra-High-Throughput Screening Method for the Directed Evolution of Glucose Oxidase" Chemistry and Biology, 21, no. 3 (2014):414-421,
https://doi.org/10.1016/j.chembiol.2014.01.010 .
