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A paper-based whole-cell screening assay for directed evolution-driven enzyme engineering
- Source :
- Applied Microbiology and Biotechnology. 104:6013-6022
- Publication Year :
- 2020
- Publisher :
- Springer Science and Business Media LLC, 2020.
-
Abstract
- Directed evolution has become an important method to unleash the latent potential of enzymes to make them uniquely suited for human purposes. However, the need for a large reagent volume and sophisticated instrumentation hampers its broad implementation. In an attempt to address this problem, here we report a paper-based high-throughput screening approach that should find broad application in generating desired enzymes. As an example case, the dehalogenation reaction of the halohydrin dehalogenase was adopted for assay development. In addition to visual detection, quantitative measurements were performed by measuring the color intensity of an image that was photographed by a smartphone and processed using ImageJ free software. The proposed method was first validated using a gold standard method and then applied to mutagenesis library screening with reduced consumption of reagents (i.e., ≤ 10 μl per assay) and a shorter assay time. We identified two active mutants (P135A and G137A) with improved activities toward four tested substrates. The assay not only consumes less reagents but also eliminates the need for expensive instrumentation. The proposed method demonstrates the potential of paper-based whole-cell screening coupled with digital image colorimetry as a promising approach for the discovery of industrially important enzymes.Key Points• A frugal method was developed for directed enzyme evolution.• Mutagenesis libraries were successfully screened on a paper platform.• Smartphone imaging was efficiently used to measure enzyme activities.
- Subjects :
- Paper
Hydrolases
Computer science
Halohydrin dehalogenase
Applied Microbiology and Biotechnology
Catalysis
03 medical and health sciences
Digital image
Software
Escherichia coli
Instrumentation (computer programming)
Gene Library
030304 developmental biology
0303 health sciences
030306 microbiology
business.industry
Reproducibility of Results
Screening assay
General Medicine
Protein engineering
Paper based
Directed evolution
High-Throughput Screening Assays
Mutagenesis
Mutation
Colorimetry
Smartphone
Biochemical engineering
Directed Molecular Evolution
business
Biotechnology
Subjects
Details
- ISSN :
- 14320614 and 01757598
- Volume :
- 104
- Database :
- OpenAIRE
- Journal :
- Applied Microbiology and Biotechnology
- Accession number :
- edsair.doi.dedup.....d18e904f8e9feb38b09ba88f4bfb3d72