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Vanadium bromoperoxidase-coupled fluorescent assay for flow cytometry sorting of glucose oxidase gene libraries in double emulsions

Prodanović, Radivoje; Ostafe, Raluca; Blanusa, Milan; Schwaneberg, Ulrich

(Springer Heidelberg, Heidelberg, 2012)

TY  - JOUR
AU  - Prodanović, Radivoje
AU  - Ostafe, Raluca
AU  - Blanusa, Milan
AU  - Schwaneberg, Ulrich
PY  - 2012
UR  - http://cherry.chem.bg.ac.rs/handle/123456789/1525
AB  - A Vanadium bromoPeroxidase-coupled fluorescent assay (ViPer) for ultrahigh-throughput screening of glucose oxidase (GOx) gene libraries employing double emulsions and flow cytometry was developed. The assay is based on detection of the product of a GOx reaction, hydrogen peroxide, that is first converted to a hypobromide by vanadium bromoperoxidase in the presence of sodium bromide. The hypobromide is afterwards detected in a reaction with a fluorogenic probe, 3-carboxy-7-(4'-aminophenoxy)-coumarine, where fluorescent 3-carboxy-coumarine is released. The ViPer screening system is three times more sensitive than a horseradish peroxidase coupled detection system and more resistant to bleaching of fluorescence in excess of peroxide. Using the ViPer screening system a high epPCR gene library containing 100,000 different GOx variants was screened for active clones in less than 1 h by flow cytometry. A library containing 0.15 % of yeast cells expressing active enzyme variants and with an average GOx activity in the liquid culture of 0.47 U/mL, after one round of sorting, had 28.12 % of the yeast cells expressing the active GOx (an enrichment factor of 200) and 26.8 U/mL of the GOx activity in the liquid culture (an enrichment factor of 57). The developed screening system could be adapted and used in a directed evolution of GOx and other hydrogen peroxide-producing enzymes (oxidases) and glycosidases if coupled with a carbohydrate oxidase.
PB  - Springer Heidelberg, Heidelberg
T2  - Analytical and Bioanalytical Chemistry
T1  - Vanadium bromoperoxidase-coupled fluorescent assay for flow cytometry sorting of glucose oxidase gene libraries in double emulsions
VL  - 404
IS  - 5
SP  - 1439
EP  - 1447
DO  - 10.1007/s00216-012-6234-x
ER  - 
@article{
author = "Prodanović, Radivoje and Ostafe, Raluca and Blanusa, Milan and Schwaneberg, Ulrich",
year = "2012",
url = "http://cherry.chem.bg.ac.rs/handle/123456789/1525",
abstract = "A Vanadium bromoPeroxidase-coupled fluorescent assay (ViPer) for ultrahigh-throughput screening of glucose oxidase (GOx) gene libraries employing double emulsions and flow cytometry was developed. The assay is based on detection of the product of a GOx reaction, hydrogen peroxide, that is first converted to a hypobromide by vanadium bromoperoxidase in the presence of sodium bromide. The hypobromide is afterwards detected in a reaction with a fluorogenic probe, 3-carboxy-7-(4'-aminophenoxy)-coumarine, where fluorescent 3-carboxy-coumarine is released. The ViPer screening system is three times more sensitive than a horseradish peroxidase coupled detection system and more resistant to bleaching of fluorescence in excess of peroxide. Using the ViPer screening system a high epPCR gene library containing 100,000 different GOx variants was screened for active clones in less than 1 h by flow cytometry. A library containing 0.15 % of yeast cells expressing active enzyme variants and with an average GOx activity in the liquid culture of 0.47 U/mL, after one round of sorting, had 28.12 % of the yeast cells expressing the active GOx (an enrichment factor of 200) and 26.8 U/mL of the GOx activity in the liquid culture (an enrichment factor of 57). The developed screening system could be adapted and used in a directed evolution of GOx and other hydrogen peroxide-producing enzymes (oxidases) and glycosidases if coupled with a carbohydrate oxidase.",
publisher = "Springer Heidelberg, Heidelberg",
journal = "Analytical and Bioanalytical Chemistry",
title = "Vanadium bromoperoxidase-coupled fluorescent assay for flow cytometry sorting of glucose oxidase gene libraries in double emulsions",
volume = "404",
number = "5",
pages = "1439-1447",
doi = "10.1007/s00216-012-6234-x"
}
Prodanović, R., Ostafe, R., Blanusa, M.,& Schwaneberg, U. (2012). Vanadium bromoperoxidase-coupled fluorescent assay for flow cytometry sorting of glucose oxidase gene libraries in double emulsions.
Analytical and Bioanalytical ChemistrySpringer Heidelberg, Heidelberg., 404(5), 1439-1447.
https://doi.org/10.1007/s00216-012-6234-x
Prodanović R, Ostafe R, Blanusa M, Schwaneberg U. Vanadium bromoperoxidase-coupled fluorescent assay for flow cytometry sorting of glucose oxidase gene libraries in double emulsions. Analytical and Bioanalytical Chemistry. 2012;404(5):1439-1447
Prodanović Radivoje, Ostafe Raluca, Blanusa Milan, Schwaneberg Ulrich, "Vanadium bromoperoxidase-coupled fluorescent assay for flow cytometry sorting of glucose oxidase gene libraries in double emulsions" 404, no. 5 (2012):1439-1447,
https://doi.org/10.1007/s00216-012-6234-x .
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FACS Based High Throughput Screening Systems for Gene Libraries in Double Emulsions

Prodanović, Radivoje; Ostafe, Raluca; Blanusa, Milan; Schwaneberg, Ulrich

(Springer-Verlag Berlin, Berlin, 2012)

TY  - CONF
AU  - Prodanović, Radivoje
AU  - Ostafe, Raluca
AU  - Blanusa, Milan
AU  - Schwaneberg, Ulrich
PY  - 2012
UR  - http://cherry.chem.bg.ac.rs/handle/123456789/1542
AB  - A flow cytometry based high throughput screening system for glucose oxidase (GOx) gene libraries in double emulsions was developed. Firstly, encapsulation of yeast cells in double emulsion was optimized by changing the ABIL EM90 concentration in light mineral oil from 2.9% to 1.5%. This enabled formation of larger water droplets and more efficient yeast cell encapsulation. Several fluorescent assays for hydrogen peroxide were tested and the 3-carboxy-7-(4'-aminophenoxy)-coumarine (APCC) oxidation by horseradish peroxidase based assay best fit the requirements of the double emulsion technology. Using an optimized substrate solution consisting of 0.5 mM APCC, 40 mM glucose and 10 U/mL of horse radish peroxidase, a referent gene library containing 10(7) yeast cells was sorted in 30 min and enriched from 1% to 15% of yeast cells expressing wt GOx.
PB  - Springer-Verlag Berlin, Berlin
C3  - Progress in Colloid and Polymer Science
T1  - FACS Based High Throughput Screening Systems for Gene Libraries in Double Emulsions
VL  - 139
SP  - 51
DO  - 10.1007/978-3-642-28974-3_10
ER  - 
@conference{
author = "Prodanović, Radivoje and Ostafe, Raluca and Blanusa, Milan and Schwaneberg, Ulrich",
year = "2012",
url = "http://cherry.chem.bg.ac.rs/handle/123456789/1542",
abstract = "A flow cytometry based high throughput screening system for glucose oxidase (GOx) gene libraries in double emulsions was developed. Firstly, encapsulation of yeast cells in double emulsion was optimized by changing the ABIL EM90 concentration in light mineral oil from 2.9% to 1.5%. This enabled formation of larger water droplets and more efficient yeast cell encapsulation. Several fluorescent assays for hydrogen peroxide were tested and the 3-carboxy-7-(4'-aminophenoxy)-coumarine (APCC) oxidation by horseradish peroxidase based assay best fit the requirements of the double emulsion technology. Using an optimized substrate solution consisting of 0.5 mM APCC, 40 mM glucose and 10 U/mL of horse radish peroxidase, a referent gene library containing 10(7) yeast cells was sorted in 30 min and enriched from 1% to 15% of yeast cells expressing wt GOx.",
publisher = "Springer-Verlag Berlin, Berlin",
journal = "Progress in Colloid and Polymer Science",
title = "FACS Based High Throughput Screening Systems for Gene Libraries in Double Emulsions",
volume = "139",
pages = "51",
doi = "10.1007/978-3-642-28974-3_10"
}
Prodanović, R., Ostafe, R., Blanusa, M.,& Schwaneberg, U. (2012). FACS Based High Throughput Screening Systems for Gene Libraries in Double Emulsions.
Progress in Colloid and Polymer ScienceSpringer-Verlag Berlin, Berlin., 139, 51.
https://doi.org/10.1007/978-3-642-28974-3_10
Prodanović R, Ostafe R, Blanusa M, Schwaneberg U. FACS Based High Throughput Screening Systems for Gene Libraries in Double Emulsions. Progress in Colloid and Polymer Science. 2012;139:51
Prodanović Radivoje, Ostafe Raluca, Blanusa Milan, Schwaneberg Ulrich, "FACS Based High Throughput Screening Systems for Gene Libraries in Double Emulsions" 139 (2012):51,
https://doi.org/10.1007/978-3-642-28974-3_10 .
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