Ostafe, Vasile

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  • Ostafe, Vasile (2)
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A High-Throughput Screening System Based on Fluorescence-Activated Cell Sorting for the Directed Evolution of Chitinase A

Menghiu, Gheorghita; Ostafe, Vasile; Prodanović, Radivoje; Fischer, Rainer; Ostafe, Raluca

(MDPI, 2021)

TY  - 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  - http://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",
url = "https://www.mdpi.com/1422-0067/22/6/3041, http://cherry.chem.bg.ac.rs/handle/123456789/4393",
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.
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. International Journal of Molecular Sciences. 2021;22(6):3041
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" International Journal of Molecular Sciences, 22, no. 6 (2021):3041,
https://doi.org/10.3390/ijms22063041 .

Supplementary data for the article: 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.

Menghiu, Gheorghita; Ostafe, Vasile; Prodanović, Radivoje; Fischer, Rainer; Ostafe, Raluca

(MDPI, 2021)


                                            

                                            
Menghiu, G., Ostafe, V., Prodanović, R., Fischer, R.,& Ostafe, R. (2021). Supplementary data for the article: 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..
International Journal of Molecular Sciences
MDPI..
Menghiu G, Ostafe V, Prodanović R, Fischer R, Ostafe R. Supplementary data for the article: 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.. International Journal of Molecular Sciences. 2021;
Menghiu Gheorghita, Ostafe Vasile, Prodanović Radivoje, Fischer Rainer, Ostafe Raluca, "Supplementary data for the article: 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." International Journal of Molecular Sciences (2021)