title: Ultra-high-throughput screening method for the directed evolution of glucose oxidase.
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.
authors.affiliation: 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.
authors.forename: Raluca
authors.initials: R
authors.lastname: Ostafe
authors.affiliation: Institute of Molecular Biotechnology, RWTH Aachen University, Worringerweg 1, 52074 Aachen, Germany; Faculty of Chemistry, University of Belgrade, Studentski trg 12, 11000, Belgrade, Serbia.
authors.forename: Radivoje
authors.initials: R
authors.lastname: Prodanovic
authors.affiliation: Codexis, 200 Penobscot Drive, Redwood City, CA 94063, USA.
authors.forename: Jovana
authors.initials: J
authors.lastname: Nazor
authors.affiliation: 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.
authors.forename: Rainer
authors.initials: R
authors.lastname: Fischer
mesh_terms: D000595:Amino Acid Sequence; D001234:Aspergillus niger / Q000201:enzymology; D019020:Directed Molecular Evolution; D005434:Flow Cytometry; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015723:Gene Library; D005947:Glucose / Q000378:metabolism; D005949:Glucose Oxidase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006207:Half-Life; D057166:High-Throughput Screening Assays; D006735:Horseradish Peroxidase / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D055550:Protein Stability; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D013696:Temperature
chemical_list: D005656:Fungal Proteins; D005949:Glucose Oxidase; D006735:Horseradish Peroxidase; D005947:Glucose