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

Authors: Radivoje Prodanovic; W. Lloyd Ung; Karla Ilić Đurđić; Rainer Fischer; David A. Weitz; Raluca Ostafe;

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

Abstract

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. © 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/).

Country
Serbia
Subjects by Vocabulary

Microsoft Academic Graph classification: Mutant Population Mutagenesis (molecular biology technique) Glucose oxidase Enzyme kinetics education Saturated mutagenesis education.field_of_study biology Chemistry Protein engineering Yeast Biochemistry biology.protein

Library of Congress Subject Headings: lcsh:QD241-441 lcsh:Organic chemistry

Keywords

Protein Conformation, Pharmaceutical Science, Saccharomyces cerevisiae, Article, Analytical Chemistry, Glucose Oxidase, Structure-Activity Relationship, QD241-441, Lab-On-A-Chip Devices, Drug Discovery, enzyme optimization, Physical and Theoretical Chemistry, Gene Library, Organic Chemistry, fluorescent label, protein engineering, Flow Cytometry, High-Throughput Screening Assays, Mutagenesis, Chemistry (miscellaneous), Molecular Medicine, Mutant Proteins, Directed Molecular Evolution, sorting

18 references, page 1 of 2

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PNAS 2008, 105, 17694-17699. [CrossRef] 19. Agresti, J.J.; Antipov, E.; Abate, A.R.; Ahn, K.; Rowat, A.C.; Baret, J.C.; Marquez, M.; Klibanov, A.M.; Gri ths, A.D.; Weitz, D.A. Ultrahigh-throughput screening in drop-based microfluidics for directed evolution. Proc. Natl. Acad. Sci. USA 2010, 107, 4004-4009. [CrossRef] 20. Swoboda, B.E.; Massey, V. Purification and proporties of the glucose oxidase from Aspergillus niger. J. Biol. Chem.

21. Dubey, M.K.; Zehra, A.; Aamir, M.; Meena, M.; Ahirwal, L.; Singh, S.; Shukla, S.; Upadhyay, R.S.; Bueno-Mari, R.; Bajpai, V.K. Improvement strategies, cost e ective production, and potential applications of fungal glucose oxidase (GOD): Current updates. Front. Microbiol. 2017, 8, 1032. [CrossRef] [PubMed] 22. Ste olani, M.E.; Ribotta, P.D.; Pérez, G.T.; León, A.E. E ect of glucose oxidase, transglutaminase, and pentosanase on wheat proteins: Relationship with dough properties and bread-making quality. J. Cereal Sci.

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This is an alternative to the "Influence" indicator, which also reflects the overall/total impact of an article in the research community at large, based on the underlying citation network (diachronically).
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popularity
This indicator reflects the "current" impact/attention (the "hype") of an article in the research community at large, based on the underlying citation network.
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impulse
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