A High-Throughput Screening System Based on Fluorescence-Activated Cell Sorting for the Directed Evolution of Chitinase A
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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 hydr...olytic 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.
Keywords:
bactericidal effect / error-prone PCR / FACS / fluorescence assay / improved enzymes / mutants / protein engineeringSource:
International Journal of Molecular Sciences, 2021, 22, 6, 3041-Publisher:
- MDPI
Funding / projects:
- G. Menghiu acknowledges support from the strategic grant POSDRU/159/1.5/S/137750: Project “Doctoral and postdoctoral programs support for increased competitiveness in exact sciences research” co-financed by the European Social Fund within the Sectorial Operational Program Human Resources Development 2007–2013. This research was funded by the GRANT PNIII-P3- 284, ChitoWound—Biotechnological tools implementation for new wound healing applications of byproducts from the crustacean seafood processing industry.
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- Supplementary material: https://cherry.chem.bg.ac.rs/handle/123456789/4394
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- Version of
https://doi.org/10.3390/ijms22063041 - Referenced by
https://cherry.chem.bg.ac.rs/handle/123456789/4394
DOI: 10.3390/ijms22063041
ISSN: 1422-0067
WoS: 000645728100001
Scopus: 2-s2.0-85102521701
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Hemijski fakultet / Faculty of ChemistryTY - JOUR AU - Menghiu, Gheorghita AU - Ostafe, Vasile AU - Prodanović, Radivoje AU - Fischer, Rainer AU - Ostafe, Raluca PY - 2021 UR - https://www.mdpi.com/1422-0067/22/6/3041 UR - https://cherry.chem.bg.ac.rs/handle/123456789/4393 AB - 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. PB - MDPI T2 - International Journal of Molecular Sciences T1 - A High-Throughput Screening System Based on Fluorescence-Activated Cell Sorting for the Directed Evolution of Chitinase A VL - 22 IS - 6 SP - 3041 DO - 10.3390/ijms22063041 ER -
@article{ author = "Menghiu, Gheorghita and Ostafe, Vasile and Prodanović, Radivoje and Fischer, Rainer and Ostafe, Raluca", year = "2021", abstract = "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.", publisher = "MDPI", journal = "International Journal of Molecular Sciences", title = "A High-Throughput Screening System Based on Fluorescence-Activated Cell Sorting for the Directed Evolution of Chitinase A", volume = "22", number = "6", pages = "3041", doi = "10.3390/ijms22063041" }
Menghiu, G., Ostafe, V., Prodanović, R., Fischer, R.,& Ostafe, R.. (2021). A High-Throughput Screening System Based on Fluorescence-Activated Cell Sorting for the Directed Evolution of Chitinase A. in International Journal of Molecular Sciences MDPI., 22(6), 3041. https://doi.org/10.3390/ijms22063041
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. in International Journal of Molecular Sciences. 2021;22(6):3041. doi:10.3390/ijms22063041 .
Menghiu, Gheorghita, Ostafe, Vasile, Prodanović, Radivoje, Fischer, Rainer, Ostafe, Raluca, "A High-Throughput Screening System Based on Fluorescence-Activated Cell Sorting for the Directed Evolution of Chitinase A" in International Journal of Molecular Sciences, 22, no. 6 (2021):3041, https://doi.org/10.3390/ijms22063041 . .