A fluorescent hydrogel-based flow cytometry screening platform for hydrolytic enzymes
September 24-28, 2017
In directed evolution experiments and enzyme discovery, screening throughput plays a key role. In this study, for the first time a high-throughput screening platform based on a coupled reaction of glucose oxidase and a hydrolase (Yersinia mollaretii phytase [YmPh]) is described. The coupled reaction produces hydroxyl radicals through Fenton’s reaction, which initiate a poly(ethylene- glycol)-acrylate-based polymerization incorporating a fluorescent monomer. Consequently, a fluorescent hydrogel is formed around Escherichia coli cells expressing active YmPh. We validate the performance of the fluorescent polymer shell (fur-shell) technology by directed phytase evolution that yielded variant M1 with 97 U/mg increased specific activity compared to YmPh wild type (315 U/mg). Thus, fur-shell technology represents a rapid and nonlaborious way of identifying the most active variants from vast populations, as well as a platform for generation of polymer-hybrid cells for biobased interactive materials.
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Volkan Besirlioglu, Christian Pitzler, Ljubica Vojcic, Ronny Martinez, Ulrich Schwaneberg, Georgette Wirtz, Stephanie Hiltl, and Alexander Böker, "A fluorescent hydrogel-based flow cytometry screening platform for hydrolytic enzymes" in "Enzyme Engineering XXIV", Pierre Monsan, Toulouse White Biotechnology, France Magali Remaud-Simeon, LISBP-INSA, University of Toulouse, France Eds, ECI Symposium Series, (2017). http://dc.engconfintl.org/enzyme_xxiv/26