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.
Highlights
Enzymes are widely used for industrial processes, but natural enzymes tend to be incompatible with harsh process conditions and must be optimized using molecular evolution methods to improve properties such as stability or substrate preference [1,2]
The high-throughput screening of enzyme libraries by droplet microfluidics should allow the top 5% most active enzyme variants to be sorted from a library containing only active mutants
After two rounds of sorting, the proportion of cells in the population expressing glucose oxidase (GOx) variants with activity exceeded 60% which compares favorably to the 36% obtained after sorting the same library higher than wild-type activity exceeded 60% which compares favorably to the 36% obtained after by flow cytometry [6]
Summary
Enzymes are widely used for industrial processes, but natural enzymes tend to be incompatible with harsh process conditions and must be optimized using molecular evolution methods to improve properties such as stability or substrate preference [1,2]. Once the assay has plateaued, any correlation between enzyme activity and fluorescence intensity is lost and there will be no measurable difference between droplets containing enzyme variants with high and low activity This problem is exacerbated by FACS because the timeframe between emulsion preparation and screening/sorting is difficult to control [17]. The smaller reaction volumes and use of disposable microchips significantly reduce the cost of the process [9] Given these combined advantages, the high-throughput screening of enzyme libraries by droplet microfluidics should allow the top 5% most active enzyme variants to be sorted from a library containing only active mutants.
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