Ultra-High-Throughput Screening Method for the Directed Evolution of Glucose Oxidase

• HTS platform for GOx libraries based on tyramide-fluorescein and flow cytometry
• Internal delivery of substrate by glucosidase after compartmentalization
• A library containing 107 clones was screened using the developed system
• Mutants with 5.8-fold higher activity and double thermal stability were selected

SummaryGlucose 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.

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