A High-Throughput Screening System Based on Droplet Microfluidics for Glucose Oxidase Gene Libraries
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...
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| Format: | Article |
| Language: | English |
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MDPI AG
2020-05-01
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| Series: | Molecules |
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| Online Access: | https://www.mdpi.com/1420-3049/25/10/2418 |
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| author | Radivoje Prodanović W. Lloyd Ung Karla Ilić Đurđić Rainer Fischer David A. Weitz Raluca Ostafe |
| author_facet | Radivoje Prodanović W. Lloyd Ung Karla Ilić Đurđić Rainer Fischer David A. Weitz Raluca Ostafe |
| author_sort | Radivoje Prodanović |
| collection | DOAJ |
| description | 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 k<sub>cat</sub> 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. |
| first_indexed | 2024-03-10T19:39:55Z |
| format | Article |
| id | doaj.art-9f364736ea3f41698edabf6a16dcf4cd |
| institution | Directory Open Access Journal |
| issn | 1420-3049 |
| language | English |
| last_indexed | 2024-03-10T19:39:55Z |
| publishDate | 2020-05-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Molecules |
| spelling | doaj.art-9f364736ea3f41698edabf6a16dcf4cd2023-11-20T01:22:10ZengMDPI AGMolecules1420-30492020-05-012510241810.3390/molecules25102418A High-Throughput Screening System Based on Droplet Microfluidics for Glucose Oxidase Gene LibrariesRadivoje Prodanović0W. Lloyd Ung1Karla Ilić Đurđić2Rainer Fischer3David A. Weitz4Raluca Ostafe5Faculty of Chemistry, University of Belgrade, Studentski trg 12, 11000 Belgrade, SerbiaDepartment of Physics, School of Engineering and Applied Sciences, Harvard University, Cambridge, MA 02138, USAFaculty of Chemistry, University of Belgrade, Studentski trg 12, 11000 Belgrade, SerbiaDepartments of Biological Sciences and Chemistry, Purdue University, 207 S. Martin Jischke Dr., West Lafayette, IN 47907, USADepartment of Physics, School of Engineering and Applied Sciences, Harvard University, Cambridge, MA 02138, USAPurdue Institute of Inflammation, Immunology and Infectious Disease, Molecular Evolution, Protein Engineering and Production, Purdue University, 207 S. Martin Jischke Dr., West Lafayette, IN 47907, USAGlucose 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 k<sub>cat</sub> 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.https://www.mdpi.com/1420-3049/25/10/2418fluorescent labelsortingprotein engineeringenzyme optimization |
| spellingShingle | Radivoje Prodanović 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 Molecules fluorescent label sorting protein engineering enzyme optimization |
| title | A High-Throughput Screening System Based on Droplet Microfluidics for Glucose Oxidase Gene Libraries |
| title_full | A High-Throughput Screening System Based on Droplet Microfluidics for Glucose Oxidase Gene Libraries |
| title_fullStr | A High-Throughput Screening System Based on Droplet Microfluidics for Glucose Oxidase Gene Libraries |
| title_full_unstemmed | A High-Throughput Screening System Based on Droplet Microfluidics for Glucose Oxidase Gene Libraries |
| title_short | A High-Throughput Screening System Based on Droplet Microfluidics for Glucose Oxidase Gene Libraries |
| title_sort | high throughput screening system based on droplet microfluidics for glucose oxidase gene libraries |
| topic | fluorescent label sorting protein engineering enzyme optimization |
| url | https://www.mdpi.com/1420-3049/25/10/2418 |
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