TY  - 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 . .

TY  - CONF
AU  - Prodanović, Radivoje
AU  - Ostafe, Raluca
AU  - Blanusa, Milan
AU  - Schwaneberg, Ulrich
PY  - 2012
UR  - https://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",
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. in Progress in Colloid and Polymer Science
Springer-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. in Progress in Colloid and Polymer Science. 2012;139:51.
doi:10.1007/978-3-642-28974-3_10 .

Prodanović, Radivoje, Ostafe, Raluca, Blanusa, Milan, Schwaneberg, Ulrich, "FACS Based High Throughput Screening Systems for Gene Libraries in Double Emulsions" in Progress in Colloid and Polymer Science, 139 (2012):51,
https://doi.org/10.1007/978-3-642-28974-3_10 . .

TY  - JOUR
AU  - Radosavljević, Jelena
AU  - Dobrijevic, Dragana
AU  - Jadranin, Milka
AU  - Blanusa, Milan
AU  - Vukmirica, Jelena
AU  - Ćirković-Veličković, Tanja
PY  - 2010
UR  - https://cherry.chem.bg.ac.rs/handle/123456789/1095
AB  - BACKGROUND: The major peanut allergens are Ara h 1, Ara h 2 and Ara h 6. Proteolytic processing has been shown to be required for the maturation process of Ara h 6. The aim of this study was to examine whether Ara h 2 undergoes proteolytic processing and, if so, whether proteolytic processing influences its ability to bind human immunoglobulin E (IgE). RESULTS: Ara h 2 isolated from peanut extract under conditions of protease inhibition revealed a single additional peak for its two known isoforms (Ara h 2.01 and Ara h 2.02), corresponding to a C-terminally truncated form lacking a dipeptide (RY). Ara h 2 isolated in the absence of protease inhibition, however, yielded two additional peaks, identified as C-terminally truncated forms lacking either a dipeptide (RY) or a single tyrosine residue. The IgE-binding capacity of the Ara h 2 truncated forms was not altered. CONCLUSION: Ara h 2 undergoes proteolytic processing by peanut proteases that involves C-terminal removal of a dipeptide. Hence Ara h 2 isolated from peanut extract is a complex mixture of two isoforms expressed by different genes, Ara h 2.01 and Ara h 2.02, as well as truncated forms generated by the proteolytic processing of these isoforms. (C) 2010 Society of Chemical Industry
PB  - John Wiley & Sons Ltd, Chichester
T2  - Journal of the Science of Food and Agriculture
T1  - Insights into proteolytic processing of the major peanut allergen Ara h 2 by endogenous peanut proteases
VL  - 90
IS  - 10
SP  - 1702
EP  - 1708
DO  - 10.1002/jsfa.4005
ER  - 

@article{
author = "Radosavljević, Jelena and Dobrijevic, Dragana and Jadranin, Milka and Blanusa, Milan and Vukmirica, Jelena and Ćirković-Veličković, Tanja",
year = "2010",
abstract = "BACKGROUND: The major peanut allergens are Ara h 1, Ara h 2 and Ara h 6. Proteolytic processing has been shown to be required for the maturation process of Ara h 6. The aim of this study was to examine whether Ara h 2 undergoes proteolytic processing and, if so, whether proteolytic processing influences its ability to bind human immunoglobulin E (IgE). RESULTS: Ara h 2 isolated from peanut extract under conditions of protease inhibition revealed a single additional peak for its two known isoforms (Ara h 2.01 and Ara h 2.02), corresponding to a C-terminally truncated form lacking a dipeptide (RY). Ara h 2 isolated in the absence of protease inhibition, however, yielded two additional peaks, identified as C-terminally truncated forms lacking either a dipeptide (RY) or a single tyrosine residue. The IgE-binding capacity of the Ara h 2 truncated forms was not altered. CONCLUSION: Ara h 2 undergoes proteolytic processing by peanut proteases that involves C-terminal removal of a dipeptide. Hence Ara h 2 isolated from peanut extract is a complex mixture of two isoforms expressed by different genes, Ara h 2.01 and Ara h 2.02, as well as truncated forms generated by the proteolytic processing of these isoforms. (C) 2010 Society of Chemical Industry",
publisher = "John Wiley & Sons Ltd, Chichester",
journal = "Journal of the Science of Food and Agriculture",
title = "Insights into proteolytic processing of the major peanut allergen Ara h 2 by endogenous peanut proteases",
volume = "90",
number = "10",
pages = "1702-1708",
doi = "10.1002/jsfa.4005"
}

Radosavljević, J., Dobrijevic, D., Jadranin, M., Blanusa, M., Vukmirica, J.,& Ćirković-Veličković, T.. (2010). Insights into proteolytic processing of the major peanut allergen Ara h 2 by endogenous peanut proteases. in Journal of the Science of Food and Agriculture
John Wiley & Sons Ltd, Chichester., 90(10), 1702-1708.
https://doi.org/10.1002/jsfa.4005

Radosavljević J, Dobrijevic D, Jadranin M, Blanusa M, Vukmirica J, Ćirković-Veličković T. Insights into proteolytic processing of the major peanut allergen Ara h 2 by endogenous peanut proteases. in Journal of the Science of Food and Agriculture. 2010;90(10):1702-1708.
doi:10.1002/jsfa.4005 .

Radosavljević, Jelena, Dobrijevic, Dragana, Jadranin, Milka, Blanusa, Milan, Vukmirica, Jelena, Ćirković-Veličković, Tanja, "Insights into proteolytic processing of the major peanut allergen Ara h 2 by endogenous peanut proteases" in Journal of the Science of Food and Agriculture, 90, no. 10 (2010):1702-1708,
https://doi.org/10.1002/jsfa.4005 . .

TY  - JOUR
AU  - Blanusa, Milan
AU  - Perovic, Iva
AU  - Popović, Milica
AU  - Polović, Natalija
AU  - Burazer, Lidija M.
AU  - Milovanovic, Mina
AU  - Gavrović-Jankulović, Marija
AU  - Jankov, Ratko M.
AU  - Ćirković-Veličković, Tanja
PY  - 2007
UR  - https://cherry.chem.bg.ac.rs/handle/123456789/885
AB  - A simple ion-exchange HPLC-UV method was developed for determination of major allergens from mugwort pollen and kiwi fruit extracts in mass-units. The separation of Art v 1 and Act c 1 from other components in the extracts was achieved in one step. The extinction coefficients used in the study here theoretically determined and compared to the extinction coefficients determined by gravimetry. We also reported a close correlation of the major allergen contents with the overall allergenic potency of the extracts determined by inhibition ELISA. This method could be a useful tool for standardization of allergenic extracts for clinical use. (c) 2007 Elsevier B.V. All rights reserved.
PB  - Elsevier Science Bv, Amsterdam
T2  - Journal of Chromatography B: Analytical Technologies in the Biomedical and L
T1  - Quantification of Art v 1 and Act c 1 being major allergens of mugwort pollen and kiwi fruit extracts in mass-units by ion-exchange HPLC-UV method
VL  - 857
IS  - 2
SP  - 188
EP  - 194
DO  - 10.1016/j.jchromb.2007.07.015
ER  - 

@article{
author = "Blanusa, Milan and Perovic, Iva and Popović, Milica and Polović, Natalija and Burazer, Lidija M. and Milovanovic, Mina and Gavrović-Jankulović, Marija and Jankov, Ratko M. and Ćirković-Veličković, Tanja",
year = "2007",
abstract = "A simple ion-exchange HPLC-UV method was developed for determination of major allergens from mugwort pollen and kiwi fruit extracts in mass-units. The separation of Art v 1 and Act c 1 from other components in the extracts was achieved in one step. The extinction coefficients used in the study here theoretically determined and compared to the extinction coefficients determined by gravimetry. We also reported a close correlation of the major allergen contents with the overall allergenic potency of the extracts determined by inhibition ELISA. This method could be a useful tool for standardization of allergenic extracts for clinical use. (c) 2007 Elsevier B.V. All rights reserved.",
publisher = "Elsevier Science Bv, Amsterdam",
journal = "Journal of Chromatography B: Analytical Technologies in the Biomedical and L",
title = "Quantification of Art v 1 and Act c 1 being major allergens of mugwort pollen and kiwi fruit extracts in mass-units by ion-exchange HPLC-UV method",
volume = "857",
number = "2",
pages = "188-194",
doi = "10.1016/j.jchromb.2007.07.015"
}

Blanusa, M., Perovic, I., Popović, M., Polović, N., Burazer, L. M., Milovanovic, M., Gavrović-Jankulović, M., Jankov, R. M.,& Ćirković-Veličković, T.. (2007). Quantification of Art v 1 and Act c 1 being major allergens of mugwort pollen and kiwi fruit extracts in mass-units by ion-exchange HPLC-UV method. in Journal of Chromatography B: Analytical Technologies in the Biomedical and L
Elsevier Science Bv, Amsterdam., 857(2), 188-194.
https://doi.org/10.1016/j.jchromb.2007.07.015

Blanusa M, Perovic I, Popović M, Polović N, Burazer LM, Milovanovic M, Gavrović-Jankulović M, Jankov RM, Ćirković-Veličković T. Quantification of Art v 1 and Act c 1 being major allergens of mugwort pollen and kiwi fruit extracts in mass-units by ion-exchange HPLC-UV method. in Journal of Chromatography B: Analytical Technologies in the Biomedical and L. 2007;857(2):188-194.
doi:10.1016/j.jchromb.2007.07.015 .

Blanusa, Milan, Perovic, Iva, Popović, Milica, Polović, Natalija, Burazer, Lidija M., Milovanovic, Mina, Gavrović-Jankulović, Marija, Jankov, Ratko M., Ćirković-Veličković, Tanja, "Quantification of Art v 1 and Act c 1 being major allergens of mugwort pollen and kiwi fruit extracts in mass-units by ion-exchange HPLC-UV method" in Journal of Chromatography B: Analytical Technologies in the Biomedical and L, 857, no. 2 (2007):188-194,
https://doi.org/10.1016/j.jchromb.2007.07.015 . .