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.
Please click Additional Files below to see the full abstract.
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). https://dc.engconfintl.org/enzyme_xxiv/26