A high.throughput screening system based on droplet microfluidics for glucose oxidase gene libraries
Lloyd Ung, W.
Ilić Đurđić, Karla
Weitz, David A.
Article (Published version)
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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 combinati...on 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. © 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
Keywords:Enzyme optimization / Fluorescent label / Protein engineering / Sorting
Source:Molecules, 2020, 25, 10
- Fulbright Foundation.
- Ministry of Education, Science and Technological Development, Republic of Serbia, Grant no. 451-03-68/2020-14/200168 (University of Belgrade, Faculty of Chemistry) (RS-200168)
- National Science Foundation (NSF) (DMR-1310266) and Harvard MRSEC (DMR-0820484).
- RWTH Aachen University.
- Cluster of Excellence “Tailor-made Fuels from Biomass”.
- National Science Foundation under NSF Award No. ECS-0335765.