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Open AccessArticle

A High-Throughput Screening System Based on Droplet Microfluidics for Glucose Oxidase Gene Libraries

1
Faculty of Chemistry, University of Belgrade, Studentski trg 12, 11000 Belgrade, Serbia
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Department of Physics, School of Engineering and Applied Sciences, Harvard University, Cambridge, MA 02138, USA
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Departments of Biological Sciences and Chemistry, Purdue University, 207 S. Martin Jischke Dr., West Lafayette, IN 47907, USA
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Purdue Institute of Inflammation, Immunology and Infectious Disease, Molecular Evolution, Protein Engineering and Production, Purdue University, 207 S. Martin Jischke Dr., West Lafayette, IN 47907, USA
*
Author to whom correspondence should be addressed.
Academic Editors: Goran T. Vladisavljević and Guido Bolognesi
Molecules 2020, 25(10), 2418; https://doi.org/10.3390/molecules25102418
Received: 20 April 2020 / Revised: 13 May 2020 / Accepted: 15 May 2020 / Published: 22 May 2020
Glucose oxidase (GOx) is an important industrial enzyme that can be optimized for specific applications by mutagenesis and activity-based screening. To increase the efficiency of this approach, we have developed a new ultrahigh-throughput screening platform based on a microfluidic lab-on-chip device that allows the sorting of GOx mutants from a saturation mutagenesis library expressed on the surface of yeast cells. GOx activity was measured by monitoring the fluorescence of water microdroplets dispersed in perfluorinated oil. The signal was generated via a series of coupled enzyme reactions leading to the formation of fluorescein. Using this new method, we were able to enrich the yeast cell population by more than 35-fold for GOx mutants with higher than wild-type activity after two rounds of sorting, almost double the efficiency of our previously described flow cytometry platform. We identified and characterized novel GOx mutants, the most promising of which (M6) contained a combination of six point mutations that increased the catalytic constant kcat by 2.1-fold compared to wild-type GOx and by 1.4-fold compared to a parental GOx variant. The new microfluidic platform for GOx was therefore more sensitive than flow cytometry and supports comprehensive screens of gene libraries containing multiple mutations per gene. View Full-Text
Keywords: fluorescent label; sorting; protein engineering; enzyme optimization fluorescent label; sorting; protein engineering; enzyme optimization
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Prodanović, R.; Ung, W.L.; Ilić Đurđić, K.; Fischer, R.; Weitz, D.A.; Ostafe, R. A High-Throughput Screening System Based on Droplet Microfluidics for Glucose Oxidase Gene Libraries. Molecules 2020, 25, 2418.

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