Schwaneberg, Ulrich

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Authority KeyName Variants
orcid::0000-0003-4026-701X
  • Schwaneberg, Ulrich (7)
  • Schwaneberg, U. (3)

Author's Bibliography

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 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. 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" Analytical and Bioanalytical Chemistry, 404, no. 5 (2012):1439-1447,
https://doi.org/10.1007/s00216-012-6234-x .
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Fluorescent Assay for Directed Evolution of Perhydrolases

Despotovic, Dragana; Vojcic, Ljubica; Prodanović, Radivoje; Martinez, Ronny; Maurer, Karl-Heinz; Schwaneberg, Ulrich

(Sage Publications Inc, Thousand Oaks, 2012)

TY  - JOUR
AU  - Despotovic, Dragana
AU  - Vojcic, Ljubica
AU  - Prodanović, Radivoje
AU  - Martinez, Ronny
AU  - Maurer, Karl-Heinz
AU  - Schwaneberg, Ulrich
PY  - 2012
UR  - http://cherry.chem.bg.ac.rs/handle/123456789/1308
AB  - Directed evolution offers opportunities to improve promiscuous activities of hydrolases in rounds of diversity generation and high-throughput screening. In this article, we developed and validated a screening platform to improve the perhydrolytic activity of proteases and likely other hydrolases (e.g., lipases or esterases). Key was the development of a highly sensitive fluorescent assay (sensitivity in the mu M range) based on 3-carboxy-7-hydroxycoumarin (HCC) formation. HCC is released through an hypobromite-mediated oxidation of 7-(4'-aminophenoxy)-3-carboxycoumarin (APCC), which enables for the first time a continuous measurement of peroxycarboxylic acid formation with a standard deviation of 11% in microtiter plates with a wide pH range window (5-9). As example, subtilisin Carlsberg was subjected to site saturation mutagenesis at position G165, yielding a variant T58A/G165L/L216W with 5.4-fold increased k(cat) for perhydrolytic activity compared with wild type.
PB  - Sage Publications Inc, Thousand Oaks
T2  - Journal of Biomolecular Screening
T1  - Fluorescent Assay for Directed Evolution of Perhydrolases
VL  - 17
IS  - 6
SP  - 796
EP  - 805
DO  - 10.1177/1087057112438464
ER  - 
@article{
author = "Despotovic, Dragana and Vojcic, Ljubica and Prodanović, Radivoje and Martinez, Ronny and Maurer, Karl-Heinz and Schwaneberg, Ulrich",
year = "2012",
url = "http://cherry.chem.bg.ac.rs/handle/123456789/1308",
abstract = "Directed evolution offers opportunities to improve promiscuous activities of hydrolases in rounds of diversity generation and high-throughput screening. In this article, we developed and validated a screening platform to improve the perhydrolytic activity of proteases and likely other hydrolases (e.g., lipases or esterases). Key was the development of a highly sensitive fluorescent assay (sensitivity in the mu M range) based on 3-carboxy-7-hydroxycoumarin (HCC) formation. HCC is released through an hypobromite-mediated oxidation of 7-(4'-aminophenoxy)-3-carboxycoumarin (APCC), which enables for the first time a continuous measurement of peroxycarboxylic acid formation with a standard deviation of 11% in microtiter plates with a wide pH range window (5-9). As example, subtilisin Carlsberg was subjected to site saturation mutagenesis at position G165, yielding a variant T58A/G165L/L216W with 5.4-fold increased k(cat) for perhydrolytic activity compared with wild type.",
publisher = "Sage Publications Inc, Thousand Oaks",
journal = "Journal of Biomolecular Screening",
title = "Fluorescent Assay for Directed Evolution of Perhydrolases",
volume = "17",
number = "6",
pages = "796-805",
doi = "10.1177/1087057112438464"
}
Despotovic, D., Vojcic, L., Prodanović, R., Martinez, R., Maurer, K.,& Schwaneberg, U. (2012). Fluorescent Assay for Directed Evolution of Perhydrolases.
Journal of Biomolecular Screening
Sage Publications Inc, Thousand Oaks., 17(6), 796-805.
https://doi.org/10.1177/1087057112438464
Despotovic D, Vojcic L, Prodanović R, Martinez R, Maurer K, Schwaneberg U. Fluorescent Assay for Directed Evolution of Perhydrolases. Journal of Biomolecular Screening. 2012;17(6):796-805
Despotovic Dragana, Vojcic Ljubica, Prodanović Radivoje, Martinez Ronny, Maurer Karl-Heinz, Schwaneberg Ulrich, "Fluorescent Assay for Directed Evolution of Perhydrolases" Journal of Biomolecular Screening, 17, no. 6 (2012):796-805,
https://doi.org/10.1177/1087057112438464 .
12
10
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11

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 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. 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" Progress in Colloid and Polymer Science, 139 (2012):51,
https://doi.org/10.1007/978-3-642-28974-3_10 .
1
1
1

Supplementary data for article: Yu, E. H.; Prodanović, R.; Gueven, G.; Ostafe, R.; Schwaneberg, U. Electrochemical Oxidation of Glucose Using Mutant Glucose Oxidase from Directed Protein Evolution for Biosensor and Biofuel Cell Applications. Applied Biochemistry and Biotechnology 2011, 165 (7–8), 1448–1457. https://doi.org/10.1007/s12010-011-9366-0

Yu, Eileen Hao; Prodanović, Radivoje; Gueven, Gueray; Ostafe, Raluca; Schwaneberg, U.

(Humana Press Inc, Totowa, 2011)

TY  - BOOK
AU  - Yu, Eileen Hao
AU  - Prodanović, Radivoje
AU  - Gueven, Gueray
AU  - Ostafe, Raluca
AU  - Schwaneberg, U.
PY  - 2011
UR  - http://cherry.chem.bg.ac.rs/handle/123456789/3611
PB  - Humana Press Inc, Totowa
T2  - Applied Biochemistry and Biotechnology
T1  - Supplementary data for article: Yu, E. H.; Prodanović, R.; Gueven, G.; Ostafe, R.; Schwaneberg, U. Electrochemical Oxidation of Glucose Using Mutant Glucose Oxidase from Directed Protein Evolution for Biosensor and Biofuel Cell Applications. Applied Biochemistry and Biotechnology 2011, 165 (7–8), 1448–1457. https://doi.org/10.1007/s12010-011-9366-0
ER  - 
@book{
author = "Yu, Eileen Hao and Prodanović, Radivoje and Gueven, Gueray and Ostafe, Raluca and Schwaneberg, U.",
year = "2011",
url = "http://cherry.chem.bg.ac.rs/handle/123456789/3611",
publisher = "Humana Press Inc, Totowa",
journal = "Applied Biochemistry and Biotechnology",
title = "Supplementary data for article: Yu, E. H.; Prodanović, R.; Gueven, G.; Ostafe, R.; Schwaneberg, U. Electrochemical Oxidation of Glucose Using Mutant Glucose Oxidase from Directed Protein Evolution for Biosensor and Biofuel Cell Applications. Applied Biochemistry and Biotechnology 2011, 165 (7–8), 1448–1457. https://doi.org/10.1007/s12010-011-9366-0"
}
Yu, E. H., Prodanović, R., Gueven, G., Ostafe, R.,& Schwaneberg, U. (2011). Supplementary data for article: Yu, E. H.; Prodanović, R.; Gueven, G.; Ostafe, R.; Schwaneberg, U. Electrochemical Oxidation of Glucose Using Mutant Glucose Oxidase from Directed Protein Evolution for Biosensor and Biofuel Cell Applications. Applied Biochemistry and Biotechnology 2011, 165 (7–8), 1448–1457. https://doi.org/10.1007/s12010-011-9366-0.
Applied Biochemistry and Biotechnology
Humana Press Inc, Totowa..
Yu EH, Prodanović R, Gueven G, Ostafe R, Schwaneberg U. Supplementary data for article: Yu, E. H.; Prodanović, R.; Gueven, G.; Ostafe, R.; Schwaneberg, U. Electrochemical Oxidation of Glucose Using Mutant Glucose Oxidase from Directed Protein Evolution for Biosensor and Biofuel Cell Applications. Applied Biochemistry and Biotechnology 2011, 165 (7–8), 1448–1457. https://doi.org/10.1007/s12010-011-9366-0. Applied Biochemistry and Biotechnology. 2011;
Yu Eileen Hao, Prodanović Radivoje, Gueven Gueray, Ostafe Raluca, Schwaneberg U., "Supplementary data for article: Yu, E. H.; Prodanović, R.; Gueven, G.; Ostafe, R.; Schwaneberg, U. Electrochemical Oxidation of Glucose Using Mutant Glucose Oxidase from Directed Protein Evolution for Biosensor and Biofuel Cell Applications. Applied Biochemistry and Biotechnology 2011, 165 (7–8), 1448–1457. https://doi.org/10.1007/s12010-011-9366-0" Applied Biochemistry and Biotechnology (2011)

Electrochemical Oxidation of Glucose Using Mutant Glucose Oxidase from Directed Protein Evolution for Biosensor and Biofuel Cell Applications

Yu, Eileen Hao; Prodanović, Radivoje; Gueven, Gueray; Ostafe, Raluca; Schwaneberg, U.

(Humana Press Inc, Totowa, 2011)

TY  - JOUR
AU  - Yu, Eileen Hao
AU  - Prodanović, Radivoje
AU  - Gueven, Gueray
AU  - Ostafe, Raluca
AU  - Schwaneberg, U.
PY  - 2011
UR  - http://cherry.chem.bg.ac.rs/handle/123456789/1221
AB  - In this study, electrochemical characterisation of glucose oxidation has been carried out in solution and using enzyme polymer electrodes prepared by mutant glucose oxidase (B11-GOx) obtained from directed protein evolution and wild-type enzymes. Higher glucose oxidation currents were obtained from B11-GOx both in solution and polymer electrodes compared to wt-GOx. This demonstrates an improved electrocatalytic activity towards electrochemical oxidation of glucose from the mutant enzyme. The enzyme electrode with B11-GOx also showed a faster electron transfer indicating a better electronic interaction with the polymer mediator. These encouraging results have shown a promising application of enzymes developed by directed evolution tailored for the applications of biosensors and biofuel cells.
PB  - Humana Press Inc, Totowa
T2  - Applied Biochemistry and Biotechnology
T1  - Electrochemical Oxidation of Glucose Using Mutant Glucose Oxidase from Directed Protein Evolution for Biosensor and Biofuel Cell Applications
VL  - 165
IS  - 7-8
SP  - 1448
EP  - 1457
DO  - 10.1007/s12010-011-9366-0
ER  - 
@article{
author = "Yu, Eileen Hao and Prodanović, Radivoje and Gueven, Gueray and Ostafe, Raluca and Schwaneberg, U.",
year = "2011",
url = "http://cherry.chem.bg.ac.rs/handle/123456789/1221",
abstract = "In this study, electrochemical characterisation of glucose oxidation has been carried out in solution and using enzyme polymer electrodes prepared by mutant glucose oxidase (B11-GOx) obtained from directed protein evolution and wild-type enzymes. Higher glucose oxidation currents were obtained from B11-GOx both in solution and polymer electrodes compared to wt-GOx. This demonstrates an improved electrocatalytic activity towards electrochemical oxidation of glucose from the mutant enzyme. The enzyme electrode with B11-GOx also showed a faster electron transfer indicating a better electronic interaction with the polymer mediator. These encouraging results have shown a promising application of enzymes developed by directed evolution tailored for the applications of biosensors and biofuel cells.",
publisher = "Humana Press Inc, Totowa",
journal = "Applied Biochemistry and Biotechnology",
title = "Electrochemical Oxidation of Glucose Using Mutant Glucose Oxidase from Directed Protein Evolution for Biosensor and Biofuel Cell Applications",
volume = "165",
number = "7-8",
pages = "1448-1457",
doi = "10.1007/s12010-011-9366-0"
}
Yu, E. H., Prodanović, R., Gueven, G., Ostafe, R.,& Schwaneberg, U. (2011). Electrochemical Oxidation of Glucose Using Mutant Glucose Oxidase from Directed Protein Evolution for Biosensor and Biofuel Cell Applications.
Applied Biochemistry and Biotechnology
Humana Press Inc, Totowa., 165(7-8), 1448-1457.
https://doi.org/10.1007/s12010-011-9366-0
Yu EH, Prodanović R, Gueven G, Ostafe R, Schwaneberg U. Electrochemical Oxidation of Glucose Using Mutant Glucose Oxidase from Directed Protein Evolution for Biosensor and Biofuel Cell Applications. Applied Biochemistry and Biotechnology. 2011;165(7-8):1448-1457
Yu Eileen Hao, Prodanović Radivoje, Gueven Gueray, Ostafe Raluca, Schwaneberg U., "Electrochemical Oxidation of Glucose Using Mutant Glucose Oxidase from Directed Protein Evolution for Biosensor and Biofuel Cell Applications" Applied Biochemistry and Biotechnology, 165, no. 7-8 (2011):1448-1457,
https://doi.org/10.1007/s12010-011-9366-0 .
15
14
14

A Flow Cytometry-Based Screening System for Directed Evolution of Proteases

Tu, Ran; Martinez, Ronny; Prodanović, Radivoje; Klein, Mathias; Schwaneberg, Ulrich

(Sage Publications Inc, Thousand Oaks, 2011)

TY  - JOUR
AU  - Tu, Ran
AU  - Martinez, Ronny
AU  - Prodanović, Radivoje
AU  - Klein, Mathias
AU  - Schwaneberg, Ulrich
PY  - 2011
UR  - http://cherry.chem.bg.ac.rs/handle/123456789/1158
AB  - Proteases are industrially important enzymes but often have to be improved for their catalytic efficiency and stabilities to suit applications. Flow cytometry screening technology based on in vitro compartmentalization in double emulsion had been developed and applied on directed evolution of paraoxonase and beta-galactosidase. Further advancements of flow cytometry-based screening technologies will enable an ultra-high throughput of variants offering novel opportunities in directed enzyme evolution under high mutational loads. For the industrially important enzyme class of proteases, a first flow cytometry-based screening system for directed protease evolution has been developed based on an extracellular protease-deficient Bacillus subtilis strain (WB800N), a model protease (subtilisin Carlsberg), and a water-in-oil-in-water double-emulsion technology. B. subtilis WB800N cells are encapsulated in double emulsion with a fluorogenic substrate (rhodamine 110-containing peptide), allowing the screening of protease variants in femtoliter compartments at high throughput. The protease screening technology was validated by employing an epPCR mutant library with a high mutational load and screened for increased resistance toward the inhibitor antipain dihydrochloride. A variant (K127R, T237P, M239I, I269V, Y310F, I372V) with an improved relative resistance was isolated from a small population of active variants, validating the reported protease flow cytometry screening technology for increased inhibitor resistance. (Journal of Biomolecular Screening 2011;16:285-294)
PB  - Sage Publications Inc, Thousand Oaks
T2  - Journal of Biomolecular Screening
T1  - A Flow Cytometry-Based Screening System for Directed Evolution of Proteases
VL  - 16
IS  - 3
SP  - 285
EP  - 294
DO  - 10.1177/1087057110396361
ER  - 
@article{
author = "Tu, Ran and Martinez, Ronny and Prodanović, Radivoje and Klein, Mathias and Schwaneberg, Ulrich",
year = "2011",
url = "http://cherry.chem.bg.ac.rs/handle/123456789/1158",
abstract = "Proteases are industrially important enzymes but often have to be improved for their catalytic efficiency and stabilities to suit applications. Flow cytometry screening technology based on in vitro compartmentalization in double emulsion had been developed and applied on directed evolution of paraoxonase and beta-galactosidase. Further advancements of flow cytometry-based screening technologies will enable an ultra-high throughput of variants offering novel opportunities in directed enzyme evolution under high mutational loads. For the industrially important enzyme class of proteases, a first flow cytometry-based screening system for directed protease evolution has been developed based on an extracellular protease-deficient Bacillus subtilis strain (WB800N), a model protease (subtilisin Carlsberg), and a water-in-oil-in-water double-emulsion technology. B. subtilis WB800N cells are encapsulated in double emulsion with a fluorogenic substrate (rhodamine 110-containing peptide), allowing the screening of protease variants in femtoliter compartments at high throughput. The protease screening technology was validated by employing an epPCR mutant library with a high mutational load and screened for increased resistance toward the inhibitor antipain dihydrochloride. A variant (K127R, T237P, M239I, I269V, Y310F, I372V) with an improved relative resistance was isolated from a small population of active variants, validating the reported protease flow cytometry screening technology for increased inhibitor resistance. (Journal of Biomolecular Screening 2011;16:285-294)",
publisher = "Sage Publications Inc, Thousand Oaks",
journal = "Journal of Biomolecular Screening",
title = "A Flow Cytometry-Based Screening System for Directed Evolution of Proteases",
volume = "16",
number = "3",
pages = "285-294",
doi = "10.1177/1087057110396361"
}
Tu, R., Martinez, R., Prodanović, R., Klein, M.,& Schwaneberg, U. (2011). A Flow Cytometry-Based Screening System for Directed Evolution of Proteases.
Journal of Biomolecular Screening
Sage Publications Inc, Thousand Oaks., 16(3), 285-294.
https://doi.org/10.1177/1087057110396361
Tu R, Martinez R, Prodanović R, Klein M, Schwaneberg U. A Flow Cytometry-Based Screening System for Directed Evolution of Proteases. Journal of Biomolecular Screening. 2011;16(3):285-294
Tu Ran, Martinez Ronny, Prodanović Radivoje, Klein Mathias, Schwaneberg Ulrich, "A Flow Cytometry-Based Screening System for Directed Evolution of Proteases" Journal of Biomolecular Screening, 16, no. 3 (2011):285-294,
https://doi.org/10.1177/1087057110396361 .
6
38
31
32

Ultrahigh Throughput Screening System for Directed Glucose Oxidase Evolution in Yeast Cells

Prodanović, Radivoje; Ostafe, Raluca; Scacioc, Andreea; Schwaneberg, Ulrich

(Bentham Science Publ Ltd, Sharjah, 2011)

TY  - JOUR
AU  - Prodanović, Radivoje
AU  - Ostafe, Raluca
AU  - Scacioc, Andreea
AU  - Schwaneberg, Ulrich
PY  - 2011
UR  - http://cherry.chem.bg.ac.rs/handle/123456789/1142
AB  - A compartmentalized tyramide labeling system (CoaTi) employing flow cytometry for sorting of yeast cells was developed as ultrahigh throughput screening for Glucose oxidase (GOx) from Aspergillus niger. CoaTi combines in vitro compartmentalization technology with the CARD reporter system which uses fluorescein tyramide labels for detection of peroxidase activity. Physical connection between cells and fluorescein tyramide radicals was achieved by compartmentalization of yeast cells inside microdroplets of single water-in-oil emulsions. After reaction cells were recovered from single emulsions and sorted by flow cytometry, an error prone PCR mutant library of Glucose oxidase (GOx) containing 10(7) cells and similar to 10(5) of different GOx variants was screened. Mutagenic conditions of GOx mutant library were selected to generate  lt 1% of active GOx population in order to explore influence of high mutation frequency on GOx activity. GOx variant Mut12 that contains 5 mutations (N2Y, K13E, T30V, I94V, K152R) showed a 1.2 times decreased K-m (22.0 vs 18.1 mM) and a 2.7 fold increased k(cat) (150 s(-1) vs 54.8 s(-1)) compared to wt GOx. Compared to the employed parent B11 GOx (16 mM, 80 s(-1)) it has a slightly increased K-m and 1.8 times increased k(cat).
PB  - Bentham Science Publ Ltd, Sharjah
T2  - Combinatorial Chemistry and High Throughput Screening
T1  - Ultrahigh Throughput Screening System for Directed Glucose Oxidase Evolution in Yeast Cells
VL  - 14
IS  - 1
SP  - 55
EP  - 60
DO  - 10.2174/1386207311107010055
ER  - 
@article{
author = "Prodanović, Radivoje and Ostafe, Raluca and Scacioc, Andreea and Schwaneberg, Ulrich",
year = "2011",
url = "http://cherry.chem.bg.ac.rs/handle/123456789/1142",
abstract = "A compartmentalized tyramide labeling system (CoaTi) employing flow cytometry for sorting of yeast cells was developed as ultrahigh throughput screening for Glucose oxidase (GOx) from Aspergillus niger. CoaTi combines in vitro compartmentalization technology with the CARD reporter system which uses fluorescein tyramide labels for detection of peroxidase activity. Physical connection between cells and fluorescein tyramide radicals was achieved by compartmentalization of yeast cells inside microdroplets of single water-in-oil emulsions. After reaction cells were recovered from single emulsions and sorted by flow cytometry, an error prone PCR mutant library of Glucose oxidase (GOx) containing 10(7) cells and similar to 10(5) of different GOx variants was screened. Mutagenic conditions of GOx mutant library were selected to generate  lt 1% of active GOx population in order to explore influence of high mutation frequency on GOx activity. GOx variant Mut12 that contains 5 mutations (N2Y, K13E, T30V, I94V, K152R) showed a 1.2 times decreased K-m (22.0 vs 18.1 mM) and a 2.7 fold increased k(cat) (150 s(-1) vs 54.8 s(-1)) compared to wt GOx. Compared to the employed parent B11 GOx (16 mM, 80 s(-1)) it has a slightly increased K-m and 1.8 times increased k(cat).",
publisher = "Bentham Science Publ Ltd, Sharjah",
journal = "Combinatorial Chemistry and High Throughput Screening",
title = "Ultrahigh Throughput Screening System for Directed Glucose Oxidase Evolution in Yeast Cells",
volume = "14",
number = "1",
pages = "55-60",
doi = "10.2174/1386207311107010055"
}
Prodanović, R., Ostafe, R., Scacioc, A.,& Schwaneberg, U. (2011). Ultrahigh Throughput Screening System for Directed Glucose Oxidase Evolution in Yeast Cells.
Combinatorial Chemistry and High Throughput Screening
Bentham Science Publ Ltd, Sharjah., 14(1), 55-60.
https://doi.org/10.2174/1386207311107010055
Prodanović R, Ostafe R, Scacioc A, Schwaneberg U. Ultrahigh Throughput Screening System for Directed Glucose Oxidase Evolution in Yeast Cells. Combinatorial Chemistry and High Throughput Screening. 2011;14(1):55-60
Prodanović Radivoje, Ostafe Raluca, Scacioc Andreea, Schwaneberg Ulrich, "Ultrahigh Throughput Screening System for Directed Glucose Oxidase Evolution in Yeast Cells" Combinatorial Chemistry and High Throughput Screening, 14, no. 1 (2011):55-60,
https://doi.org/10.2174/1386207311107010055 .
9
26
23
25

Rationalizing perhydrolase activity of aryl-esterase and subtilisin Carlsberg mutants by molecular dynamics simulations of the second tetrahedral intermediate state

Lee, Wook; Vojcic, Ljubica; Despotovic, Dragana; Prodanović, Radivoje; Maurer, Karl-Heinz; Schwaneberg, Ulrich; Zacharias, Martin

(Springer, New York, 2010)

TY  - JOUR
AU  - Lee, Wook
AU  - Vojcic, Ljubica
AU  - Despotovic, Dragana
AU  - Prodanović, Radivoje
AU  - Maurer, Karl-Heinz
AU  - Schwaneberg, Ulrich
AU  - Zacharias, Martin
PY  - 2010
UR  - http://cherry.chem.bg.ac.rs/handle/123456789/1039
AB  - The perhydrolysis reaction in hydrolases is an important example of catalytic promiscuity and has many potential industrial applications. The mechanisms of perhydrolase activity of a subtilisin Carlsberg mutant and of an aryl-esterase mutant have been investigated using classical molecular dynamics simulations of the second tetrahedral intermediate (TI) state. The simulations demonstrated that hydrogen bonding between the second TI of the perhydrolysis reaction is possible in the mutants but not wild type. The stabilization by hydrogen bonds was specific for the perhydrolysis intermediate and either no hydrogen bonding or only weakened hydrogen bonding to the second TI state of the hydrolysis reaction was observed. Furthermore, a significant hindrance to the formation of the catalytically important hydrogen bond between His64 and Ser221 in the catalytic triad by competing hydrogen bonds was found for the subtilisin mutant but not wild type enzyme in case of the hydrolysis intermediate. The opposite was observed in case of the perhydrolysis intermediate. The result offers a qualitative explanation for the overall reduced hydrolysis activity of the subtilisin mutant. In addition, the simulations also explain qualitatively the perhydrolysis activity of the enzyme variants and may be helpful for designing enzyme mutants with further improved perhydrolysis activity.
PB  - Springer, New York
T2  - Theoretical Chemistry Accounts
T1  - Rationalizing perhydrolase activity of aryl-esterase and subtilisin Carlsberg mutants by molecular dynamics simulations of the second tetrahedral intermediate state
VL  - 125
IS  - 3-6
SP  - 375
EP  - 386
DO  - 10.1007/s00214-009-0611-3
ER  - 
@article{
author = "Lee, Wook and Vojcic, Ljubica and Despotovic, Dragana and Prodanović, Radivoje and Maurer, Karl-Heinz and Schwaneberg, Ulrich and Zacharias, Martin",
year = "2010",
url = "http://cherry.chem.bg.ac.rs/handle/123456789/1039",
abstract = "The perhydrolysis reaction in hydrolases is an important example of catalytic promiscuity and has many potential industrial applications. The mechanisms of perhydrolase activity of a subtilisin Carlsberg mutant and of an aryl-esterase mutant have been investigated using classical molecular dynamics simulations of the second tetrahedral intermediate (TI) state. The simulations demonstrated that hydrogen bonding between the second TI of the perhydrolysis reaction is possible in the mutants but not wild type. The stabilization by hydrogen bonds was specific for the perhydrolysis intermediate and either no hydrogen bonding or only weakened hydrogen bonding to the second TI state of the hydrolysis reaction was observed. Furthermore, a significant hindrance to the formation of the catalytically important hydrogen bond between His64 and Ser221 in the catalytic triad by competing hydrogen bonds was found for the subtilisin mutant but not wild type enzyme in case of the hydrolysis intermediate. The opposite was observed in case of the perhydrolysis intermediate. The result offers a qualitative explanation for the overall reduced hydrolysis activity of the subtilisin mutant. In addition, the simulations also explain qualitatively the perhydrolysis activity of the enzyme variants and may be helpful for designing enzyme mutants with further improved perhydrolysis activity.",
publisher = "Springer, New York",
journal = "Theoretical Chemistry Accounts",
title = "Rationalizing perhydrolase activity of aryl-esterase and subtilisin Carlsberg mutants by molecular dynamics simulations of the second tetrahedral intermediate state",
volume = "125",
number = "3-6",
pages = "375-386",
doi = "10.1007/s00214-009-0611-3"
}
Lee, W., Vojcic, L., Despotovic, D., Prodanović, R., Maurer, K., Schwaneberg, U.,& Zacharias, M. (2010). Rationalizing perhydrolase activity of aryl-esterase and subtilisin Carlsberg mutants by molecular dynamics simulations of the second tetrahedral intermediate state.
Theoretical Chemistry Accounts
Springer, New York., 125(3-6), 375-386.
https://doi.org/10.1007/s00214-009-0611-3
Lee W, Vojcic L, Despotovic D, Prodanović R, Maurer K, Schwaneberg U, Zacharias M. Rationalizing perhydrolase activity of aryl-esterase and subtilisin Carlsberg mutants by molecular dynamics simulations of the second tetrahedral intermediate state. Theoretical Chemistry Accounts. 2010;125(3-6):375-386
Lee Wook, Vojcic Ljubica, Despotovic Dragana, Prodanović Radivoje, Maurer Karl-Heinz, Schwaneberg Ulrich, Zacharias Martin, "Rationalizing perhydrolase activity of aryl-esterase and subtilisin Carlsberg mutants by molecular dynamics simulations of the second tetrahedral intermediate state" Theoretical Chemistry Accounts, 125, no. 3-6 (2010):375-386,
https://doi.org/10.1007/s00214-009-0611-3 .
9
8
11

Protein Engineering - An Option for Enzymatic Biofuel Cell Design

Gueven, Gueray; Prodanović, Radivoje; Schwaneberg, Ulrich

(Wiley-V C H Verlag Gmbh, Weinheim, 2010)

TY  - JOUR
AU  - Gueven, Gueray
AU  - Prodanović, Radivoje
AU  - Schwaneberg, Ulrich
PY  - 2010
UR  - http://cherry.chem.bg.ac.rs/handle/123456789/1076
AB  - This review summarizes and discusses from a protein engineering point of view strategies to improve performances of biocatalysts in enzymatic biofuel cells. Emphasis will be given on biocatalysts employed in biofuel cells and protein engineering principles for achieving an efficient electrical communication between electrode and biocatalyst(s). Biocatalyst engineering by Rational Design and Directed Evolution offers opportunities to redesign and to improve biocatalysts for biofuel cell applications instead of accepting insufficient biocatalyst properties as unalterable limitations in the development of biofuel cells.
PB  - Wiley-V C H Verlag Gmbh, Weinheim
T2  - Electroanalysis
T1  - Protein Engineering - An Option for Enzymatic Biofuel Cell Design
VL  - 22
IS  - 7-8
SP  - 765
DO  - 10.1002/elan.200980017
ER  - 
@article{
author = "Gueven, Gueray and Prodanović, Radivoje and Schwaneberg, Ulrich",
year = "2010",
url = "http://cherry.chem.bg.ac.rs/handle/123456789/1076",
abstract = "This review summarizes and discusses from a protein engineering point of view strategies to improve performances of biocatalysts in enzymatic biofuel cells. Emphasis will be given on biocatalysts employed in biofuel cells and protein engineering principles for achieving an efficient electrical communication between electrode and biocatalyst(s). Biocatalyst engineering by Rational Design and Directed Evolution offers opportunities to redesign and to improve biocatalysts for biofuel cell applications instead of accepting insufficient biocatalyst properties as unalterable limitations in the development of biofuel cells.",
publisher = "Wiley-V C H Verlag Gmbh, Weinheim",
journal = "Electroanalysis",
title = "Protein Engineering - An Option for Enzymatic Biofuel Cell Design",
volume = "22",
number = "7-8",
pages = "765",
doi = "10.1002/elan.200980017"
}
Gueven, G., Prodanović, R.,& Schwaneberg, U. (2010). Protein Engineering - An Option for Enzymatic Biofuel Cell Design.
Electroanalysis
Wiley-V C H Verlag Gmbh, Weinheim., 22(7-8), 765.
https://doi.org/10.1002/elan.200980017
Gueven G, Prodanović R, Schwaneberg U. Protein Engineering - An Option for Enzymatic Biofuel Cell Design. Electroanalysis. 2010;22(7-8):765
Gueven Gueray, Prodanović Radivoje, Schwaneberg Ulrich, "Protein Engineering - An Option for Enzymatic Biofuel Cell Design" Electroanalysis, 22, no. 7-8 (2010):765,
https://doi.org/10.1002/elan.200980017 .
48
46
48

Flow cytometry based high throughput screening system for screening and improving industrially important enzymes

Tu, R.; Prodanović, Radivoje; Blanusa, M.; Ostafe, Raluca; Niehaus, F.; Eck, J.; Schwaneberg, U.

(Elsevier Science Bv, Amsterdam, 2009)

TY  - CONF
AU  - Tu, R.
AU  - Prodanović, Radivoje
AU  - Blanusa, M.
AU  - Ostafe, Raluca
AU  - Niehaus, F.
AU  - Eck, J.
AU  - Schwaneberg, U.
PY  - 2009
UR  - http://cherry.chem.bg.ac.rs/handle/123456789/1118
PB  - Elsevier Science Bv, Amsterdam
C3  - New Biotechnology
T1  - Flow cytometry based high throughput screening system for screening and improving industrially important enzymes
VL  - 25
DO  - 10.1016/j.nbt.2009.06.474
ER  - 
@conference{
author = "Tu, R. and Prodanović, Radivoje and Blanusa, M. and Ostafe, Raluca and Niehaus, F. and Eck, J. and Schwaneberg, U.",
year = "2009",
url = "http://cherry.chem.bg.ac.rs/handle/123456789/1118",
publisher = "Elsevier Science Bv, Amsterdam",
journal = "New Biotechnology",
title = "Flow cytometry based high throughput screening system for screening and improving industrially important enzymes",
volume = "25",
doi = "10.1016/j.nbt.2009.06.474"
}
Tu, R., Prodanović, R., Blanusa, M., Ostafe, R., Niehaus, F., Eck, J.,& Schwaneberg, U. (2009). Flow cytometry based high throughput screening system for screening and improving industrially important enzymes.
New Biotechnology
Elsevier Science Bv, Amsterdam., 25.
https://doi.org/10.1016/j.nbt.2009.06.474
Tu R, Prodanović R, Blanusa M, Ostafe R, Niehaus F, Eck J, Schwaneberg U. Flow cytometry based high throughput screening system for screening and improving industrially important enzymes. New Biotechnology. 2009;25
Tu R., Prodanović Radivoje, Blanusa M., Ostafe Raluca, Niehaus F., Eck J., Schwaneberg U., "Flow cytometry based high throughput screening system for screening and improving industrially important enzymes" New Biotechnology, 25 (2009),
https://doi.org/10.1016/j.nbt.2009.06.474 .