Vanadium bromoperoxidase-coupled fluorescent assay for flow cytometry sorting of glucose oxidase gene libraries in double emulsions
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 ave...rage 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.
Keywords:
In vitro compartmentalization / Directed evolution / Hydrogen peroxide / FACSSource:
Analytical and Bioanalytical Chemistry, 2012, 404, 5, 1439-1447Publisher:
- Springer Heidelberg, Heidelberg
Funding / projects:
- Alexander von Humboldt foundation
- BRAIN AG company
- BMBF BiochancePlus program
DOI: 10.1007/s00216-012-6234-x
ISSN: 1618-2642
PubMed: 22811062
WoS: 000307957800016
Scopus: 2-s2.0-84865466722
Collections
Institution/Community
Hemijski fakultet / Faculty of ChemistryTY - JOUR AU - Prodanović, Radivoje AU - Ostafe, Raluca AU - Blanusa, Milan AU - Schwaneberg, Ulrich PY - 2012 UR - https://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", 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. in Analytical and Bioanalytical Chemistry Springer 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. in Analytical and Bioanalytical Chemistry. 2012;404(5):1439-1447. doi:10.1007/s00216-012-6234-x .
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" in Analytical and Bioanalytical Chemistry, 404, no. 5 (2012):1439-1447, https://doi.org/10.1007/s00216-012-6234-x . .