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Article

A High-Throughput Screening System Based on Fluorescence-Activated Cell Sorting for the Directed Evolution of Chitinase A

1
Institute for Biology VII, Molecular Biotechnology, RWTH Aachen University, Worringerweg 1, 52074 Aachen, Germany
2
Advanced Environmental Research Laboratories, Department of Biology–Chemistry, West University of Timisoara, Oituz 4, 300086 Timisoara, Romania
3
Faculty of Chemistry, University of Belgrade, Studentski trg 12-16, 11000 Belgrade, Serbia
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Departments of Biological Sciences and Chemistry, Purdue University, 207 S. Martin Jischke Dr., West Lafayette, IN 47907, USA
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Purdue Institute of Inflammation, Immunology and Infectious Disease, Molecular Evolution, Protein Engineering and Production, Purdue University, 207 S. Martin Jischke Dr., West Lafayette, IN 47907, USA
*
Author to whom correspondence should be addressed.
Academic Editor: Iolanda Francolini
Int. J. Mol. Sci. 2021, 22(6), 3041; https://doi.org/10.3390/ijms22063041
Received: 3 February 2021 / Revised: 10 March 2021 / Accepted: 12 March 2021 / Published: 16 March 2021
(This article belongs to the Special Issue Chitosan Functionalizations, Formulations and Composites 2.0)
Chitinases catalyze the degradation of chitin, a polymer of N-acetylglucosamine found in crustacean shells, insect cuticles, and fungal cell walls. There is great interest in the development of improved chitinases to address the environmental burden of chitin waste from the food processing industry as well as the potential medical, agricultural, and industrial uses of partially deacetylated chitin (chitosan) and its products (chito-oligosaccharides). The depolymerization of chitin can be achieved using chemical and physical treatments, but an enzymatic process would be more environmentally friendly and more sustainable. However, chitinases are slow-acting enzymes, limiting their biotechnological exploitation, although this can be overcome by molecular evolution approaches to enhance the features required for specific applications. The two main goals of this study were the development of a high-throughput screening system for chitinase activity (which could be extrapolated to other hydrolytic enzymes), and the deployment of this new method to select improved chitinase variants. We therefore cloned and expressed the Bacillus licheniformis DSM8785 chitinase A (chiA) gene in Escherichia coli BL21 (DE3) cells and generated a mutant library by error-prone PCR. We then developed a screening method based on fluorescence-activated cell sorting (FACS) using the model substrate 4-methylumbelliferyl β-d-N,N′,N″-triacetyl chitotrioside to identify improved enzymes. We prevented cross-talk between emulsion compartments caused by the hydrophobicity of 4-methylumbelliferone, the fluorescent product of the enzymatic reaction, by incorporating cyclodextrins into the aqueous phases. We also addressed the toxicity of long-term chiA expression in E. coli by limiting the reaction time. We identified 12 mutants containing 2–8 mutations per gene resulting in up to twofold higher activity than wild-type ChiA. View Full-Text
Keywords: FACS; protein engineering; error-prone PCR; mutants; bactericidal effect; improved enzymes; fluorescence assay FACS; protein engineering; error-prone PCR; mutants; bactericidal effect; improved enzymes; fluorescence assay
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MDPI and ACS Style

Menghiu, G.; Ostafe, V.; Prodanović, R.; Fischer, R.; Ostafe, R. A High-Throughput Screening System Based on Fluorescence-Activated Cell Sorting for the Directed Evolution of Chitinase A. Int. J. Mol. Sci. 2021, 22, 3041. https://doi.org/10.3390/ijms22063041

AMA Style

Menghiu G, Ostafe V, Prodanović R, Fischer R, Ostafe R. A High-Throughput Screening System Based on Fluorescence-Activated Cell Sorting for the Directed Evolution of Chitinase A. International Journal of Molecular Sciences. 2021; 22(6):3041. https://doi.org/10.3390/ijms22063041

Chicago/Turabian Style

Menghiu, Gheorghita, Vasile Ostafe, Radivoje Prodanović, Rainer Fischer, and Raluca Ostafe. 2021. "A High-Throughput Screening System Based on Fluorescence-Activated Cell Sorting for the Directed Evolution of Chitinase A" International Journal of Molecular Sciences 22, no. 6: 3041. https://doi.org/10.3390/ijms22063041

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