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Chem Biol. 2014 Mar 20;21(3):414-21. doi: 10.1016/j.chembiol.2014.01.010. Epub 2014 Mar 6.

Ultra-high-throughput screening method for the directed evolution of glucose oxidase.

Author information

1
Fraunhofer Institute for Molecular Biology and Applied Ecology (IME), Forckenbeckstrasse 6, 52074 Aachen, Germany; Institute of Molecular Biotechnology, RWTH Aachen University, Worringerweg 1, 52074 Aachen, Germany.
2
Institute of Molecular Biotechnology, RWTH Aachen University, Worringerweg 1, 52074 Aachen, Germany; Faculty of Chemistry, University of Belgrade, Studentski trg 12, 11000, Belgrade, Serbia.
3
Codexis, 200 Penobscot Drive, Redwood City, CA 94063, USA.
4
Fraunhofer Institute for Molecular Biology and Applied Ecology (IME), Forckenbeckstrasse 6, 52074 Aachen, Germany; Institute of Molecular Biotechnology, RWTH Aachen University, Worringerweg 1, 52074 Aachen, Germany. Electronic address: rainer.fischer@ime.fraunhofer.de.

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.

PMID:
24613019
DOI:
10.1016/j.chembiol.2014.01.010
[Indexed for MEDLINE]
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