Ostafe, Raluca

Link to this page

Authority KeyName Variants
orcid::0000-0003-0479-1527
  • Ostafe, Raluca (31)
Projects
Allergens, antibodies, enzymes and small physiologically important molecules: design, structure, function and relevance Study of structure-function relationships in the plant cell wall and modifications of the wall structure by enzyme engineering
Novel encapsulation and enzyme technologies for designing of new biocatalysts and biologically active compounds targeting enhancement of food quality, safety and competitiveness Alexander von Humboldt foundation
Harvard MRSEC [DMR-0820484] BMBF BiochancePlus program
Deutsche Forschungsgemeinschaft (DFG) Excellence Initiative by the German federal government
Fulbright Foundation [N0009552407] National Institute of Health [R01 EB014703, P01GM096971]
National Science Foundation [DMR-1310266] BMBF
BRAIN AG BRAIN AG company
company BRAIN AG DAAD bilateral project 451-03-01038/2015-09/21
Engineering and Physical Sciences Research Council [EP/C535456/1] EPSRC [EP/C535456/1]
FEBS Collaborative and Experimental Scholarship for Central and Eastern Europe Fulbright Foundation
G. Menghiu acknowledges support from the strategic grant POSDRU/159/1.5/S/137750: Project “Doctoral and postdoctoral programs support for increased competitiveness in exact sciences research” co-financed by the European Social Fund within the Sectorial Operational Program Human Resources Development 2007–2013. This research was funded by the GRANT PNIII-P3- 284, ChitoWound—Biotechnological tools implementation for new wound healing applications of byproducts from the crustacean seafood processing industry. Ministry of Education, Science and Technological Development, Republic of Serbia, Grant no. 451-03-68/2020-14/200053 (University of Belgrade, Institute for Multidisciplinary Research)
Ministry of Education, Science and Technological Development, Republic of Serbia, Grant no. 451-03-68/2020-14/200168 (University of Belgrade, Faculty of Chemistry) internal program MEF from the Fraunhofer Gesellschaft [125-600156]
National Science Foundation (NSF) [DMR-1310266] National Science Foundation under NSF Award [ECS-0335765]
NSERC PGS D RWTH Aachen University
US Office of Naval Research (ONR) [N00014-03-1-0026]

Author's Bibliography

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)

TY  - DATA
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/123456789/4394
PB  - MDPI
T2  - International Journal of Molecular Sciences
T1  - 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.
ER  - 
@misc{
author = "Menghiu, Gheorghita and Ostafe, Vasile and Prodanović, Radivoje and Fischer, Rainer and Ostafe, Raluca",
year = "2021",
publisher = "MDPI",
journal = "International Journal of Molecular Sciences",
title = "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, 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.. in 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.. in 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." in International Journal of Molecular Sciences (2021).

Supplementary data for the article: Ilić Đurđić, K.; Ostafe, R.; Prodanović, O.; Đurđević Đelmaš, A.; Popović, N.; Fischer, R.; Schillberg, S.; Prodanović, R. Improved Degradation of Azo Dyes by Lignin Peroxidase Following Mutagenesis at Two Sites near the Catalytic Pocket and the Application of Peroxidase-Coated Yeast Cell Walls. Front. Environ. Sci. Eng. 2020, 15 (2), 19. https://doi.org/10.1007/s11783-020-1311-4

Ilić Đurđić, Karla; Ostafe, Raluca; Prodanović, Olivera; Đurđević Đelmaš, Aleksandra; Popović, Nikolina; Fischer, Rainer; Schillberg, Stefan; Prodanović, Radivoje

(Springer, 2021)

TY  - DATA
AU  - Ilić Đurđić, Karla
AU  - Ostafe, Raluca
AU  - Prodanović, Olivera
AU  - Đurđević Đelmaš, Aleksandra
AU  - Popović, Nikolina
AU  - Fischer, Rainer
AU  - Schillberg, Stefan
AU  - Prodanović, Radivoje
PY  - 2021
UR  - http://cherry.chem.bg.ac.rs/handle/123456789/4103
PB  - Springer
T2  - Frontiers of Environmental Science & EngineeringFront. Environ. Sci. Eng.
T1  - Supplementary data for the article: Ilić Đurđić, K.; Ostafe, R.; Prodanović, O.; Đurđević Đelmaš, A.; Popović, N.; Fischer, R.; Schillberg, S.; Prodanović, R. Improved Degradation of Azo Dyes by Lignin Peroxidase Following Mutagenesis at Two Sites near the Catalytic Pocket and the Application of Peroxidase-Coated Yeast Cell Walls. Front. Environ. Sci. Eng. 2020, 15 (2), 19. https://doi.org/10.1007/s11783-020-1311-4
ER  - 
@misc{
author = "Ilić Đurđić, Karla and Ostafe, Raluca and Prodanović, Olivera and Đurđević Đelmaš, Aleksandra and Popović, Nikolina and Fischer, Rainer and Schillberg, Stefan and Prodanović, Radivoje",
year = "2021",
publisher = "Springer",
journal = "Frontiers of Environmental Science & EngineeringFront. Environ. Sci. Eng.",
title = "Supplementary data for the article: Ilić Đurđić, K.; Ostafe, R.; Prodanović, O.; Đurđević Đelmaš, A.; Popović, N.; Fischer, R.; Schillberg, S.; Prodanović, R. Improved Degradation of Azo Dyes by Lignin Peroxidase Following Mutagenesis at Two Sites near the Catalytic Pocket and the Application of Peroxidase-Coated Yeast Cell Walls. Front. Environ. Sci. Eng. 2020, 15 (2), 19. https://doi.org/10.1007/s11783-020-1311-4"
}
Ilić Đurđić, K., Ostafe, R., Prodanović, O., Đurđević Đelmaš, A., Popović, N., Fischer, R., Schillberg, S.,& Prodanović, R.. (2021). Supplementary data for the article: Ilić Đurđić, K.; Ostafe, R.; Prodanović, O.; Đurđević Đelmaš, A.; Popović, N.; Fischer, R.; Schillberg, S.; Prodanović, R. Improved Degradation of Azo Dyes by Lignin Peroxidase Following Mutagenesis at Two Sites near the Catalytic Pocket and the Application of Peroxidase-Coated Yeast Cell Walls. Front. Environ. Sci. Eng. 2020, 15 (2), 19. https://doi.org/10.1007/s11783-020-1311-4. in Frontiers of Environmental Science & EngineeringFront. Environ. Sci. Eng.
Springer..
Ilić Đurđić K, Ostafe R, Prodanović O, Đurđević Đelmaš A, Popović N, Fischer R, Schillberg S, Prodanović R. Supplementary data for the article: Ilić Đurđić, K.; Ostafe, R.; Prodanović, O.; Đurđević Đelmaš, A.; Popović, N.; Fischer, R.; Schillberg, S.; Prodanović, R. Improved Degradation of Azo Dyes by Lignin Peroxidase Following Mutagenesis at Two Sites near the Catalytic Pocket and the Application of Peroxidase-Coated Yeast Cell Walls. Front. Environ. Sci. Eng. 2020, 15 (2), 19. https://doi.org/10.1007/s11783-020-1311-4. in Frontiers of Environmental Science & EngineeringFront. Environ. Sci. Eng.. 2021;..
Ilić Đurđić, Karla, Ostafe, Raluca, Prodanović, Olivera, Đurđević Đelmaš, Aleksandra, Popović, Nikolina, Fischer, Rainer, Schillberg, Stefan, Prodanović, Radivoje, "Supplementary data for the article: Ilić Đurđić, K.; Ostafe, R.; Prodanović, O.; Đurđević Đelmaš, A.; Popović, N.; Fischer, R.; Schillberg, S.; Prodanović, R. Improved Degradation of Azo Dyes by Lignin Peroxidase Following Mutagenesis at Two Sites near the Catalytic Pocket and the Application of Peroxidase-Coated Yeast Cell Walls. Front. Environ. Sci. Eng. 2020, 15 (2), 19. https://doi.org/10.1007/s11783-020-1311-4" in Frontiers of Environmental Science & EngineeringFront. Environ. Sci. Eng. (2021).

Supplementary data for the article: Popović, N.; Pržulj, D.; Mladenović, M.; Prodanović, O.; Ece, S.; Ilić Đurđić, K.; Ostafe, R.; Fischer, R.; Prodanović, R. Immobilization of Yeast Cell Walls with Surface Displayed Laccase from Streptomyces Cyaneus within Dopamine-Alginate Beads for Dye Decolorization. International Journal of Biological Macromolecules 2021, 181, 1072–1080. https://doi.org/10.1016/j.ijbiomac.2021.04.115.

Popović, Nikolina; Pržulj, Dunja; Mladenović, Maja; Prodanović, Olivera; Ece, Selin; Ilić Đurđić, Karla; Ostafe, Raluca; Fischer, Rainer; Prodanović, Radivoje

(2021)

TY  - DATA
AU  - Popović, Nikolina
AU  - Pržulj, Dunja
AU  - Mladenović, Maja
AU  - Prodanović, Olivera
AU  - Ece, Selin
AU  - Ilić Đurđić, Karla
AU  - Ostafe, Raluca
AU  - Fischer, Rainer
AU  - Prodanović, Radivoje
PY  - 2021
UR  - https://www.sciencedirect.com/science/article/pii/S0141813021008813
UR  - http://cherry.chem.bg.ac.rs/handle/123456789/4405
T2  - International Journal of Biological Macromolecules
T1  - Supplementary data for the article: Popović, N.; Pržulj, D.; Mladenović, M.; Prodanović, O.; Ece, S.; Ilić Đurđić, K.; Ostafe, R.; Fischer, R.; Prodanović, R. Immobilization of Yeast Cell Walls with Surface Displayed Laccase from Streptomyces Cyaneus within Dopamine-Alginate Beads for Dye Decolorization. International Journal of Biological Macromolecules 2021, 181, 1072–1080. https://doi.org/10.1016/j.ijbiomac.2021.04.115.
ER  - 
@misc{
author = "Popović, Nikolina and Pržulj, Dunja and Mladenović, Maja and Prodanović, Olivera and Ece, Selin and Ilić Đurđić, Karla and Ostafe, Raluca and Fischer, Rainer and Prodanović, Radivoje",
year = "2021",
journal = "International Journal of Biological Macromolecules",
title = "Supplementary data for the article: Popović, N.; Pržulj, D.; Mladenović, M.; Prodanović, O.; Ece, S.; Ilić Đurđić, K.; Ostafe, R.; Fischer, R.; Prodanović, R. Immobilization of Yeast Cell Walls with Surface Displayed Laccase from Streptomyces Cyaneus within Dopamine-Alginate Beads for Dye Decolorization. International Journal of Biological Macromolecules 2021, 181, 1072–1080. https://doi.org/10.1016/j.ijbiomac.2021.04.115."
}
Popović, N., Pržulj, D., Mladenović, M., Prodanović, O., Ece, S., Ilić Đurđić, K., Ostafe, R., Fischer, R.,& Prodanović, R.. (2021). Supplementary data for the article: Popović, N.; Pržulj, D.; Mladenović, M.; Prodanović, O.; Ece, S.; Ilić Đurđić, K.; Ostafe, R.; Fischer, R.; Prodanović, R. Immobilization of Yeast Cell Walls with Surface Displayed Laccase from Streptomyces Cyaneus within Dopamine-Alginate Beads for Dye Decolorization. International Journal of Biological Macromolecules 2021, 181, 1072–1080. https://doi.org/10.1016/j.ijbiomac.2021.04.115.. in International Journal of Biological Macromolecules.
Popović N, Pržulj D, Mladenović M, Prodanović O, Ece S, Ilić Đurđić K, Ostafe R, Fischer R, Prodanović R. Supplementary data for the article: Popović, N.; Pržulj, D.; Mladenović, M.; Prodanović, O.; Ece, S.; Ilić Đurđić, K.; Ostafe, R.; Fischer, R.; Prodanović, R. Immobilization of Yeast Cell Walls with Surface Displayed Laccase from Streptomyces Cyaneus within Dopamine-Alginate Beads for Dye Decolorization. International Journal of Biological Macromolecules 2021, 181, 1072–1080. https://doi.org/10.1016/j.ijbiomac.2021.04.115.. in International Journal of Biological Macromolecules. 2021;..
Popović, Nikolina, Pržulj, Dunja, Mladenović, Maja, Prodanović, Olivera, Ece, Selin, Ilić Đurđić, Karla, Ostafe, Raluca, Fischer, Rainer, Prodanović, Radivoje, "Supplementary data for the article: Popović, N.; Pržulj, D.; Mladenović, M.; Prodanović, O.; Ece, S.; Ilić Đurđić, K.; Ostafe, R.; Fischer, R.; Prodanović, R. Immobilization of Yeast Cell Walls with Surface Displayed Laccase from Streptomyces Cyaneus within Dopamine-Alginate Beads for Dye Decolorization. International Journal of Biological Macromolecules 2021, 181, 1072–1080. https://doi.org/10.1016/j.ijbiomac.2021.04.115." in International Journal of Biological Macromolecules (2021).

One-shot optimization of multiple enzyme parameters: Tailoring glucose oxidase for pH and electron mediators

Ostafe, Raluca; Fontaine, Nicolas; Frank, David; Ng Fuk Chong, Matthieu; Prodanović, Radivoje; Pandjaitan, Rudy; Offmann, Bernard; Cadet, Frederic; Fischer, Rainer

(Willey, 2020)

TY  - JOUR
AU  - Ostafe, Raluca
AU  - Fontaine, Nicolas
AU  - Frank, David
AU  - Ng Fuk Chong, Matthieu
AU  - Prodanović, Radivoje
AU  - Pandjaitan, Rudy
AU  - Offmann, Bernard
AU  - Cadet, Frederic
AU  - Fischer, Rainer
PY  - 2020
UR  - http://cherry.chem.bg.ac.rs/handle/123456789/3784
AB  - Enzymes are biological catalysts with many industrial applications, but natural enzymes are usually unsuitable for industrial processes because they are not optimized for the process conditions. The properties of enzymes can be improved by directed evolution, which involves multiple rounds of mutagenesis and screening. By using mathematical models to predict the structure–activity relationship of an enzyme, and by defining the optimal combination of mutations in silico, we can significantly reduce the number of bench experiments needed, and hence the time and investment required to develop an optimized product. Here, we applied our innovative sequence–activity relationship methodology (innov'SAR) to improve glucose oxidase activity in the presence of different mediators across a range of pH values. Using this machine learning approach, a predictive model was developed and the optimal combination of mutations was determined, leading to a glucose oxidase mutant (P1) with greater specificity for the mediators ferrocene–methanol (12-fold) and nitrosoaniline (8-fold), compared to the wild-type enzyme, and better performance in three pH-adjusted buffers. The kcat/KM ratio of P1 increased by up to 121 folds compared to the wild type enzyme at pH 5.5 in the presence of ferrocene methanol.
PB  - Willey
T2  - Biotechnology and Bioengineering
T1  - One-shot optimization of multiple enzyme parameters: Tailoring glucose oxidase for pH and electron mediators
VL  - 117
IS  - 1
SP  - 17
EP  - 29
DO  - 10.1002/bit.27169
ER  - 
@article{
author = "Ostafe, Raluca and Fontaine, Nicolas and Frank, David and Ng Fuk Chong, Matthieu and Prodanović, Radivoje and Pandjaitan, Rudy and Offmann, Bernard and Cadet, Frederic and Fischer, Rainer",
year = "2020",
abstract = "Enzymes are biological catalysts with many industrial applications, but natural enzymes are usually unsuitable for industrial processes because they are not optimized for the process conditions. The properties of enzymes can be improved by directed evolution, which involves multiple rounds of mutagenesis and screening. By using mathematical models to predict the structure–activity relationship of an enzyme, and by defining the optimal combination of mutations in silico, we can significantly reduce the number of bench experiments needed, and hence the time and investment required to develop an optimized product. Here, we applied our innovative sequence–activity relationship methodology (innov'SAR) to improve glucose oxidase activity in the presence of different mediators across a range of pH values. Using this machine learning approach, a predictive model was developed and the optimal combination of mutations was determined, leading to a glucose oxidase mutant (P1) with greater specificity for the mediators ferrocene–methanol (12-fold) and nitrosoaniline (8-fold), compared to the wild-type enzyme, and better performance in three pH-adjusted buffers. The kcat/KM ratio of P1 increased by up to 121 folds compared to the wild type enzyme at pH 5.5 in the presence of ferrocene methanol.",
publisher = "Willey",
journal = "Biotechnology and Bioengineering",
title = "One-shot optimization of multiple enzyme parameters: Tailoring glucose oxidase for pH and electron mediators",
volume = "117",
number = "1",
pages = "17-29",
doi = "10.1002/bit.27169"
}
Ostafe, R., Fontaine, N., Frank, D., Ng Fuk Chong, M., Prodanović, R., Pandjaitan, R., Offmann, B., Cadet, F.,& Fischer, R.. (2020). One-shot optimization of multiple enzyme parameters: Tailoring glucose oxidase for pH and electron mediators. in Biotechnology and Bioengineering
Willey., 117(1), 17-29.
https://doi.org/10.1002/bit.27169
Ostafe R, Fontaine N, Frank D, Ng Fuk Chong M, Prodanović R, Pandjaitan R, Offmann B, Cadet F, Fischer R. One-shot optimization of multiple enzyme parameters: Tailoring glucose oxidase for pH and electron mediators. in Biotechnology and Bioengineering. 2020;117(1):17-29.
doi:10.1002/bit.27169 .
Ostafe, Raluca, Fontaine, Nicolas, Frank, David, Ng Fuk Chong, Matthieu, Prodanović, Radivoje, Pandjaitan, Rudy, Offmann, Bernard, Cadet, Frederic, Fischer, Rainer, "One-shot optimization of multiple enzyme parameters: Tailoring glucose oxidase for pH and electron mediators" in Biotechnology and Bioengineering, 117, no. 1 (2020):17-29,
https://doi.org/10.1002/bit.27169 . .
2
8
7
8

Saturation mutagenesis to improve the degradation of azo dyes by versatile peroxidase and application in form of VP-coated yeast cell walls

Ilić Đurđić, Karla; Ostafe, Raluca; Đurđević Đelmaš, Aleksandra; Popović, Nikolina; Schillberg, Stefan; Fischer, Rainer; Prodanović, Radivoje

(Elsevier, 2020)

TY  - JOUR
AU  - Ilić Đurđić, Karla
AU  - Ostafe, Raluca
AU  - Đurđević Đelmaš, Aleksandra
AU  - Popović, Nikolina
AU  - Schillberg, Stefan
AU  - Fischer, Rainer
AU  - Prodanović, Radivoje
PY  - 2020
UR  - http://cherry.chem.bg.ac.rs/handle/123456789/3834
AB  - Azo dyes are toxic and carcinogenic synthetic pigments that accumulate as pollutants in aquatic bodies near textile factories. The pigments are structurally diverse, and bioremediation is mostly limited to single dye compounds or related groups. Versatile peroxidase (VP) from Pleurotus eryngii is a heme-containing peroxidase with a broad substrate spectrum that can break down many structurally distinct pollutants, including azo dyes. The utilization of this enzyme could be facilitated by engineering to modify its catalytic activity and substrate range. We used saturation mutagenesis to alter two amino acids in the catalytic tryptophan environment of VP (V160 and A260). Library screening with three azo dyes revealed that these two positions had a significant influence on substrate specificity. We were able to isolate and sequence VP variants with up to 16-fold higher catalytic efficiency for different azo dyes. The same approach could be used to select for VP variants that catalyze the degradation of many other types of pollutants. To allow multiple cycles of dye degradation, we immobilized VP on the surface of yeast cells and used washed cell wall fragments after lysis. VP embedded in the cell wall retained ∼70 % of its initial activity after 10 cycles of dye degradation each lasting 12 h, making this platform ideal for the bioremediation of environments contaminated with azo dyes.
PB  - Elsevier
T2  - Enzyme and Microbial Technology
T1  - Saturation mutagenesis to improve the degradation of azo dyes by versatile peroxidase and application in form of VP-coated yeast cell walls
VL  - 136
SP  - e109509
DO  - 10.1016/j.enzmictec.2020.109509
ER  - 
@article{
author = "Ilić Đurđić, Karla and Ostafe, Raluca and Đurđević Đelmaš, Aleksandra and Popović, Nikolina and Schillberg, Stefan and Fischer, Rainer and Prodanović, Radivoje",
year = "2020",
abstract = "Azo dyes are toxic and carcinogenic synthetic pigments that accumulate as pollutants in aquatic bodies near textile factories. The pigments are structurally diverse, and bioremediation is mostly limited to single dye compounds or related groups. Versatile peroxidase (VP) from Pleurotus eryngii is a heme-containing peroxidase with a broad substrate spectrum that can break down many structurally distinct pollutants, including azo dyes. The utilization of this enzyme could be facilitated by engineering to modify its catalytic activity and substrate range. We used saturation mutagenesis to alter two amino acids in the catalytic tryptophan environment of VP (V160 and A260). Library screening with three azo dyes revealed that these two positions had a significant influence on substrate specificity. We were able to isolate and sequence VP variants with up to 16-fold higher catalytic efficiency for different azo dyes. The same approach could be used to select for VP variants that catalyze the degradation of many other types of pollutants. To allow multiple cycles of dye degradation, we immobilized VP on the surface of yeast cells and used washed cell wall fragments after lysis. VP embedded in the cell wall retained ∼70 % of its initial activity after 10 cycles of dye degradation each lasting 12 h, making this platform ideal for the bioremediation of environments contaminated with azo dyes.",
publisher = "Elsevier",
journal = "Enzyme and Microbial Technology",
title = "Saturation mutagenesis to improve the degradation of azo dyes by versatile peroxidase and application in form of VP-coated yeast cell walls",
volume = "136",
pages = "e109509",
doi = "10.1016/j.enzmictec.2020.109509"
}
Ilić Đurđić, K., Ostafe, R., Đurđević Đelmaš, A., Popović, N., Schillberg, S., Fischer, R.,& Prodanović, R.. (2020). Saturation mutagenesis to improve the degradation of azo dyes by versatile peroxidase and application in form of VP-coated yeast cell walls. in Enzyme and Microbial Technology
Elsevier., 136, e109509.
https://doi.org/10.1016/j.enzmictec.2020.109509
Ilić Đurđić K, Ostafe R, Đurđević Đelmaš A, Popović N, Schillberg S, Fischer R, Prodanović R. Saturation mutagenesis to improve the degradation of azo dyes by versatile peroxidase and application in form of VP-coated yeast cell walls. in Enzyme and Microbial Technology. 2020;136:e109509.
doi:10.1016/j.enzmictec.2020.109509 .
Ilić Đurđić, Karla, Ostafe, Raluca, Đurđević Đelmaš, Aleksandra, Popović, Nikolina, Schillberg, Stefan, Fischer, Rainer, Prodanović, Radivoje, "Saturation mutagenesis to improve the degradation of azo dyes by versatile peroxidase and application in form of VP-coated yeast cell walls" in Enzyme and Microbial Technology, 136 (2020):e109509,
https://doi.org/10.1016/j.enzmictec.2020.109509 . .
10
5
7

Saturation mutagenesis to improve the degradation of azo dyes by versatile peroxidase and application in form of VP-coated yeast cell walls

Ilić Đurđić, Karla; Ostafe, Raluca; Đurđević Đelmaš, Aleksandra; Popović, Nikolina; Schillberg, Stefan; Fischer, Rainer; Prodanović, Radivoje

(Elsevier, 2020)

TY  - JOUR
AU  - Ilić Đurđić, Karla
AU  - Ostafe, Raluca
AU  - Đurđević Đelmaš, Aleksandra
AU  - Popović, Nikolina
AU  - Schillberg, Stefan
AU  - Fischer, Rainer
AU  - Prodanović, Radivoje
PY  - 2020
UR  - http://cherry.chem.bg.ac.rs/handle/123456789/3835
AB  - Azo dyes are toxic and carcinogenic synthetic pigments that accumulate as pollutants in aquatic bodies near textile factories. The pigments are structurally diverse, and bioremediation is mostly limited to single dye compounds or related groups. Versatile peroxidase (VP) from Pleurotus eryngii is a heme-containing peroxidase with a broad substrate spectrum that can break down many structurally distinct pollutants, including azo dyes. The utilization of this enzyme could be facilitated by engineering to modify its catalytic activity and substrate range. We used saturation mutagenesis to alter two amino acids in the catalytic tryptophan environment of VP (V160 and A260). Library screening with three azo dyes revealed that these two positions had a significant influence on substrate specificity. We were able to isolate and sequence VP variants with up to 16-fold higher catalytic efficiency for different azo dyes. The same approach could be used to select for VP variants that catalyze the degradation of many other types of pollutants. To allow multiple cycles of dye degradation, we immobilized VP on the surface of yeast cells and used washed cell wall fragments after lysis. VP embedded in the cell wall retained ∼70 % of its initial activity after 10 cycles of dye degradation each lasting 12 h, making this platform ideal for the bioremediation of environments contaminated with azo dyes.
PB  - Elsevier
T2  - Enzyme and Microbial Technology
T1  - Saturation mutagenesis to improve the degradation of azo dyes by versatile peroxidase and application in form of VP-coated yeast cell walls
VL  - 136
SP  - e109509
DO  - 10.1016/j.enzmictec.2020.109509
ER  - 
@article{
author = "Ilić Đurđić, Karla and Ostafe, Raluca and Đurđević Đelmaš, Aleksandra and Popović, Nikolina and Schillberg, Stefan and Fischer, Rainer and Prodanović, Radivoje",
year = "2020",
abstract = "Azo dyes are toxic and carcinogenic synthetic pigments that accumulate as pollutants in aquatic bodies near textile factories. The pigments are structurally diverse, and bioremediation is mostly limited to single dye compounds or related groups. Versatile peroxidase (VP) from Pleurotus eryngii is a heme-containing peroxidase with a broad substrate spectrum that can break down many structurally distinct pollutants, including azo dyes. The utilization of this enzyme could be facilitated by engineering to modify its catalytic activity and substrate range. We used saturation mutagenesis to alter two amino acids in the catalytic tryptophan environment of VP (V160 and A260). Library screening with three azo dyes revealed that these two positions had a significant influence on substrate specificity. We were able to isolate and sequence VP variants with up to 16-fold higher catalytic efficiency for different azo dyes. The same approach could be used to select for VP variants that catalyze the degradation of many other types of pollutants. To allow multiple cycles of dye degradation, we immobilized VP on the surface of yeast cells and used washed cell wall fragments after lysis. VP embedded in the cell wall retained ∼70 % of its initial activity after 10 cycles of dye degradation each lasting 12 h, making this platform ideal for the bioremediation of environments contaminated with azo dyes.",
publisher = "Elsevier",
journal = "Enzyme and Microbial Technology",
title = "Saturation mutagenesis to improve the degradation of azo dyes by versatile peroxidase and application in form of VP-coated yeast cell walls",
volume = "136",
pages = "e109509",
doi = "10.1016/j.enzmictec.2020.109509"
}
Ilić Đurđić, K., Ostafe, R., Đurđević Đelmaš, A., Popović, N., Schillberg, S., Fischer, R.,& Prodanović, R.. (2020). Saturation mutagenesis to improve the degradation of azo dyes by versatile peroxidase and application in form of VP-coated yeast cell walls. in Enzyme and Microbial Technology
Elsevier., 136, e109509.
https://doi.org/10.1016/j.enzmictec.2020.109509
Ilić Đurđić K, Ostafe R, Đurđević Đelmaš A, Popović N, Schillberg S, Fischer R, Prodanović R. Saturation mutagenesis to improve the degradation of azo dyes by versatile peroxidase and application in form of VP-coated yeast cell walls. in Enzyme and Microbial Technology. 2020;136:e109509.
doi:10.1016/j.enzmictec.2020.109509 .
Ilić Đurđić, Karla, Ostafe, Raluca, Đurđević Đelmaš, Aleksandra, Popović, Nikolina, Schillberg, Stefan, Fischer, Rainer, Prodanović, Radivoje, "Saturation mutagenesis to improve the degradation of azo dyes by versatile peroxidase and application in form of VP-coated yeast cell walls" in Enzyme and Microbial Technology, 136 (2020):e109509,
https://doi.org/10.1016/j.enzmictec.2020.109509 . .
10
5
7

Supplementary data for article: Ilić Đurđić, K.; Ostafe, R.; Đurđević Đelmaš, A.; Popović, N.; Schillberg, S.; Fischer, R.; Prodanović, R. Saturation Mutagenesis to Improve the Degradation of Azo Dyes by Versatile Peroxidase and Application in Form of VP-Coated Yeast Cell Walls. Enzyme and Microbial Technology 2020, 136. https://doi.org/10.1016/j.enzmictec.2020.109509

Ilić Đurđić, Karla; Ostafe, Raluca; Đurđević Đelmaš, Aleksandra; Popović, Nikolina; Schillberg, Stefan; Fischer, Rainer; Prodanović, Radivoje

(Elsevier, 2020)

TY  - DATA
AU  - Ilić Đurđić, Karla
AU  - Ostafe, Raluca
AU  - Đurđević Đelmaš, Aleksandra
AU  - Popović, Nikolina
AU  - Schillberg, Stefan
AU  - Fischer, Rainer
AU  - Prodanović, Radivoje
PY  - 2020
UR  - http://cherry.chem.bg.ac.rs/handle/123456789/3836
PB  - Elsevier
T2  - Enzyme and Microbial Technology
T1  - Supplementary data for article: Ilić Đurđić, K.; Ostafe, R.; Đurđević Đelmaš, A.; Popović, N.; Schillberg, S.; Fischer, R.;  Prodanović, R. Saturation Mutagenesis to Improve the Degradation of Azo Dyes by Versatile Peroxidase and Application in Form of VP-Coated Yeast Cell Walls. Enzyme and Microbial Technology 2020, 136. https://doi.org/10.1016/j.enzmictec.2020.109509
ER  - 
@misc{
author = "Ilić Đurđić, Karla and Ostafe, Raluca and Đurđević Đelmaš, Aleksandra and Popović, Nikolina and Schillberg, Stefan and Fischer, Rainer and Prodanović, Radivoje",
year = "2020",
publisher = "Elsevier",
journal = "Enzyme and Microbial Technology",
title = "Supplementary data for article: Ilić Đurđić, K.; Ostafe, R.; Đurđević Đelmaš, A.; Popović, N.; Schillberg, S.; Fischer, R.;  Prodanović, R. Saturation Mutagenesis to Improve the Degradation of Azo Dyes by Versatile Peroxidase and Application in Form of VP-Coated Yeast Cell Walls. Enzyme and Microbial Technology 2020, 136. https://doi.org/10.1016/j.enzmictec.2020.109509"
}
Ilić Đurđić, K., Ostafe, R., Đurđević Đelmaš, A., Popović, N., Schillberg, S., Fischer, R.,& Prodanović, R.. (2020). Supplementary data for article: Ilić Đurđić, K.; Ostafe, R.; Đurđević Đelmaš, A.; Popović, N.; Schillberg, S.; Fischer, R.;  Prodanović, R. Saturation Mutagenesis to Improve the Degradation of Azo Dyes by Versatile Peroxidase and Application in Form of VP-Coated Yeast Cell Walls. Enzyme and Microbial Technology 2020, 136. https://doi.org/10.1016/j.enzmictec.2020.109509. in Enzyme and Microbial Technology
Elsevier..
Ilić Đurđić K, Ostafe R, Đurđević Đelmaš A, Popović N, Schillberg S, Fischer R, Prodanović R. Supplementary data for article: Ilić Đurđić, K.; Ostafe, R.; Đurđević Đelmaš, A.; Popović, N.; Schillberg, S.; Fischer, R.;  Prodanović, R. Saturation Mutagenesis to Improve the Degradation of Azo Dyes by Versatile Peroxidase and Application in Form of VP-Coated Yeast Cell Walls. Enzyme and Microbial Technology 2020, 136. https://doi.org/10.1016/j.enzmictec.2020.109509. in Enzyme and Microbial Technology. 2020;..
Ilić Đurđić, Karla, Ostafe, Raluca, Đurđević Đelmaš, Aleksandra, Popović, Nikolina, Schillberg, Stefan, Fischer, Rainer, Prodanović, Radivoje, "Supplementary data for article: Ilić Đurđić, K.; Ostafe, R.; Đurđević Đelmaš, A.; Popović, N.; Schillberg, S.; Fischer, R.;  Prodanović, R. Saturation Mutagenesis to Improve the Degradation of Azo Dyes by Versatile Peroxidase and Application in Form of VP-Coated Yeast Cell Walls. Enzyme and Microbial Technology 2020, 136. https://doi.org/10.1016/j.enzmictec.2020.109509" in Enzyme and Microbial Technology (2020).

Supplementary data for the article: Ilić Đurđić, K.; Ece, S.; Ostafe, R.; Vogel, S.; Schillberg, S.; Fischer, R.; Prodanović, R. Improvement in Oxidative Stability of Versatile Peroxidase by Flow Cytometry-Based High-Throughput Screening System. Biochemical Engineering Journal 2020, 157. https://doi.org/10.1016/j.bej.2020.107555

Ilić Đurđić, Karla; Ece, Selin; Ostafe, Raluca; Vogel, Simon; Schillberg, Stefan; Fischer, Rainer; Prodanović, Radivoje

(Elsevier, 2020)

TY  - DATA
AU  - Ilić Đurđić, Karla
AU  - Ece, Selin
AU  - Ostafe, Raluca
AU  - Vogel, Simon
AU  - Schillberg, Stefan
AU  - Fischer, Rainer
AU  - Prodanović, Radivoje
PY  - 2020
UR  - http://cherry.chem.bg.ac.rs/handle/123456789/3899
PB  - Elsevier
T2  - Biochemical Engineering Journal
T1  - Supplementary data for the article: Ilić Đurđić, K.; Ece, S.; Ostafe, R.; Vogel, S.; Schillberg, S.; Fischer, R.; Prodanović, R. Improvement in Oxidative Stability of Versatile Peroxidase by Flow Cytometry-Based High-Throughput Screening System. Biochemical Engineering Journal 2020, 157. https://doi.org/10.1016/j.bej.2020.107555
ER  - 
@misc{
author = "Ilić Đurđić, Karla and Ece, Selin and Ostafe, Raluca and Vogel, Simon and Schillberg, Stefan and Fischer, Rainer and Prodanović, Radivoje",
year = "2020",
publisher = "Elsevier",
journal = "Biochemical Engineering Journal",
title = "Supplementary data for the article: Ilić Đurđić, K.; Ece, S.; Ostafe, R.; Vogel, S.; Schillberg, S.; Fischer, R.; Prodanović, R. Improvement in Oxidative Stability of Versatile Peroxidase by Flow Cytometry-Based High-Throughput Screening System. Biochemical Engineering Journal 2020, 157. https://doi.org/10.1016/j.bej.2020.107555"
}
Ilić Đurđić, K., Ece, S., Ostafe, R., Vogel, S., Schillberg, S., Fischer, R.,& Prodanović, R.. (2020). Supplementary data for the article: Ilić Đurđić, K.; Ece, S.; Ostafe, R.; Vogel, S.; Schillberg, S.; Fischer, R.; Prodanović, R. Improvement in Oxidative Stability of Versatile Peroxidase by Flow Cytometry-Based High-Throughput Screening System. Biochemical Engineering Journal 2020, 157. https://doi.org/10.1016/j.bej.2020.107555. in Biochemical Engineering Journal
Elsevier..
Ilić Đurđić K, Ece S, Ostafe R, Vogel S, Schillberg S, Fischer R, Prodanović R. Supplementary data for the article: Ilić Đurđić, K.; Ece, S.; Ostafe, R.; Vogel, S.; Schillberg, S.; Fischer, R.; Prodanović, R. Improvement in Oxidative Stability of Versatile Peroxidase by Flow Cytometry-Based High-Throughput Screening System. Biochemical Engineering Journal 2020, 157. https://doi.org/10.1016/j.bej.2020.107555. in Biochemical Engineering Journal. 2020;..
Ilić Đurđić, Karla, Ece, Selin, Ostafe, Raluca, Vogel, Simon, Schillberg, Stefan, Fischer, Rainer, Prodanović, Radivoje, "Supplementary data for the article: Ilić Đurđić, K.; Ece, S.; Ostafe, R.; Vogel, S.; Schillberg, S.; Fischer, R.; Prodanović, R. Improvement in Oxidative Stability of Versatile Peroxidase by Flow Cytometry-Based High-Throughput Screening System. Biochemical Engineering Journal 2020, 157. https://doi.org/10.1016/j.bej.2020.107555" in Biochemical Engineering Journal (2020).

Protein engineering of cellobiose dehydrogenase from Phanerochaete chrysosporium in yeast Saccharomyces cerevisiae InvSc1 for increased activity and stability

Blažić, Marija; Balaž, Ana Marija; Tadić, Vojin; Draganić, Bojana; Ostafe, Raluca; Fischer, Rainer; Prodanović, Radivoje

(Elsevier, 2019)

TY  - JOUR
AU  - Blažić, Marija
AU  - Balaž, Ana Marija
AU  - Tadić, Vojin
AU  - Draganić, Bojana
AU  - Ostafe, Raluca
AU  - Fischer, Rainer
AU  - Prodanović, Radivoje
PY  - 2019
UR  - http://cherry.chem.bg.ac.rs/handle/123456789/2898
AB  - Cellobiose dehydrogenase (CDH) can be used in industry for lactobionic acid production, as a part of biosensors for disaccharides and in wound healing. In fungi it is involved in lignocellulose degradation. CDH gene from Phanerochaete chrysosporium has been cloned in pYES2 plasmid for extracellular expression and protein engineering in yeast Saccharomyces cerevisiae InvSC1 for the first time. A CDH gene library was generated using error-prone PCR and screened by spectrophotometric enzymatic assay based on 2,6-dichloroindophenol reduction detection in microtiter plates. Several mutants with increased activity and specificity towards lactose and cellobiose were found, purified and characterized in detail. Recombinant CDH enzymes showed a broad molecular weight between 120 and 150 KDa due to hyper-glycosylation and the best S137 N mutant showed 2.2 times increased k cat and 1.5 and 2 times increased specificity constant for lactose and cellobiose compared to the wild type enzyme. pH optimum of mutants was not changed while thermostability of selected mutants improved and S137 N mutant retained 30% of it's original activity after 15 min at 70 °C compared to 10% of activity that the wild type enzyme retained. Mutants M65S and S137 N showed also 1.6 and 1.5 times increased productivity of hydrogen peroxide in the presence of 30 mM lactose compared to the wild type.
PB  - Elsevier
T2  - Biochemical Engineering Journal
T1  - Protein engineering of cellobiose dehydrogenase from Phanerochaete chrysosporium in yeast Saccharomyces cerevisiae InvSc1 for increased activity and stability
VL  - 146
SP  - 179
EP  - 185
DO  - 10.1016/j.bej.2019.03.025
ER  - 
@article{
author = "Blažić, Marija and Balaž, Ana Marija and Tadić, Vojin and Draganić, Bojana and Ostafe, Raluca and Fischer, Rainer and Prodanović, Radivoje",
year = "2019",
abstract = "Cellobiose dehydrogenase (CDH) can be used in industry for lactobionic acid production, as a part of biosensors for disaccharides and in wound healing. In fungi it is involved in lignocellulose degradation. CDH gene from Phanerochaete chrysosporium has been cloned in pYES2 plasmid for extracellular expression and protein engineering in yeast Saccharomyces cerevisiae InvSC1 for the first time. A CDH gene library was generated using error-prone PCR and screened by spectrophotometric enzymatic assay based on 2,6-dichloroindophenol reduction detection in microtiter plates. Several mutants with increased activity and specificity towards lactose and cellobiose were found, purified and characterized in detail. Recombinant CDH enzymes showed a broad molecular weight between 120 and 150 KDa due to hyper-glycosylation and the best S137 N mutant showed 2.2 times increased k cat and 1.5 and 2 times increased specificity constant for lactose and cellobiose compared to the wild type enzyme. pH optimum of mutants was not changed while thermostability of selected mutants improved and S137 N mutant retained 30% of it's original activity after 15 min at 70 °C compared to 10% of activity that the wild type enzyme retained. Mutants M65S and S137 N showed also 1.6 and 1.5 times increased productivity of hydrogen peroxide in the presence of 30 mM lactose compared to the wild type.",
publisher = "Elsevier",
journal = "Biochemical Engineering Journal",
title = "Protein engineering of cellobiose dehydrogenase from Phanerochaete chrysosporium in yeast Saccharomyces cerevisiae InvSc1 for increased activity and stability",
volume = "146",
pages = "179-185",
doi = "10.1016/j.bej.2019.03.025"
}
Blažić, M., Balaž, A. M., Tadić, V., Draganić, B., Ostafe, R., Fischer, R.,& Prodanović, R.. (2019). Protein engineering of cellobiose dehydrogenase from Phanerochaete chrysosporium in yeast Saccharomyces cerevisiae InvSc1 for increased activity and stability. in Biochemical Engineering Journal
Elsevier., 146, 179-185.
https://doi.org/10.1016/j.bej.2019.03.025
Blažić M, Balaž AM, Tadić V, Draganić B, Ostafe R, Fischer R, Prodanović R. Protein engineering of cellobiose dehydrogenase from Phanerochaete chrysosporium in yeast Saccharomyces cerevisiae InvSc1 for increased activity and stability. in Biochemical Engineering Journal. 2019;146:179-185.
doi:10.1016/j.bej.2019.03.025 .
Blažić, Marija, Balaž, Ana Marija, Tadić, Vojin, Draganić, Bojana, Ostafe, Raluca, Fischer, Rainer, Prodanović, Radivoje, "Protein engineering of cellobiose dehydrogenase from Phanerochaete chrysosporium in yeast Saccharomyces cerevisiae InvSc1 for increased activity and stability" in Biochemical Engineering Journal, 146 (2019):179-185,
https://doi.org/10.1016/j.bej.2019.03.025 . .
7
6
6

Directed evolution of cellobiose dehydrogenase on the surface of yeast cells using resazurin-based fluorescent assay

Blažić, Marija; Balaž, Ana Marija; Prodanović, Olivera; Popović, Nikolina; Ostafe, Raluca; Fischer, Rainer; Prodanović, Radivoje

(Applied sciences, 2019)

TY  - JOUR
AU  - Blažić, Marija
AU  - Balaž, Ana Marija
AU  - Prodanović, Olivera
AU  - Popović, Nikolina
AU  - Ostafe, Raluca
AU  - Fischer, Rainer
AU  - Prodanović, Radivoje
PY  - 2019
UR  - http://cherry.chem.bg.ac.rs/handle/123456789/2909
AB  - Cellobiose dehydrogenase (CDH) from Phanerochaete chrysosporium can be used in lactobionic acid production, biosensor for lactose, biofuel cells, lignocellulose degradation, and wound-healing applications. To make it a better biocatalyst, CDH with higher activity in an immobilized form is desirable. For this purpose, CDH was expressed for the first time on the surface of S. cerevisiae EBY100 cells in an active form as a triple mutant tmCDH (D20N, A64T, V592M) and evolved further for higher activity using resazurin-based fluorescent assay. In order to decrease blank reaction of resazurin with yeast cells and to have linear correlation between enzyme activity on the cell surface and fluorescence signal, the assay was optimized with respect to resazurin concentration (0.1 mM), substrate concentration (10mMlactose and 0.08mMcellobiose), and pH (6.0). Using optimized assay an error prone PCR gene library of tmCDH was screened. Two mutants with 5 (H5) and 7 mutations (H9) were found having two times higher activity than the parent tmCDH enzyme that already had improved activity compared to wild type CDH whose activity could not be detected on the surface of yeast cells.
PB  - Applied sciences
T2  - Applied Sciences (Switzerland)
T1  - Directed evolution of cellobiose dehydrogenase on the surface of yeast cells using resazurin-based fluorescent assay
VL  - 9
IS  - 7
SP  - 1
EP  - 15
DO  - 10.3390/app9071413
ER  - 
@article{
author = "Blažić, Marija and Balaž, Ana Marija and Prodanović, Olivera and Popović, Nikolina and Ostafe, Raluca and Fischer, Rainer and Prodanović, Radivoje",
year = "2019",
abstract = "Cellobiose dehydrogenase (CDH) from Phanerochaete chrysosporium can be used in lactobionic acid production, biosensor for lactose, biofuel cells, lignocellulose degradation, and wound-healing applications. To make it a better biocatalyst, CDH with higher activity in an immobilized form is desirable. For this purpose, CDH was expressed for the first time on the surface of S. cerevisiae EBY100 cells in an active form as a triple mutant tmCDH (D20N, A64T, V592M) and evolved further for higher activity using resazurin-based fluorescent assay. In order to decrease blank reaction of resazurin with yeast cells and to have linear correlation between enzyme activity on the cell surface and fluorescence signal, the assay was optimized with respect to resazurin concentration (0.1 mM), substrate concentration (10mMlactose and 0.08mMcellobiose), and pH (6.0). Using optimized assay an error prone PCR gene library of tmCDH was screened. Two mutants with 5 (H5) and 7 mutations (H9) were found having two times higher activity than the parent tmCDH enzyme that already had improved activity compared to wild type CDH whose activity could not be detected on the surface of yeast cells.",
publisher = "Applied sciences",
journal = "Applied Sciences (Switzerland)",
title = "Directed evolution of cellobiose dehydrogenase on the surface of yeast cells using resazurin-based fluorescent assay",
volume = "9",
number = "7",
pages = "1-15",
doi = "10.3390/app9071413"
}
Blažić, M., Balaž, A. M., Prodanović, O., Popović, N., Ostafe, R., Fischer, R.,& Prodanović, R.. (2019). Directed evolution of cellobiose dehydrogenase on the surface of yeast cells using resazurin-based fluorescent assay. in Applied Sciences (Switzerland)
Applied sciences., 9(7), 1-15.
https://doi.org/10.3390/app9071413
Blažić M, Balaž AM, Prodanović O, Popović N, Ostafe R, Fischer R, Prodanović R. Directed evolution of cellobiose dehydrogenase on the surface of yeast cells using resazurin-based fluorescent assay. in Applied Sciences (Switzerland). 2019;9(7):1-15.
doi:10.3390/app9071413 .
Blažić, Marija, Balaž, Ana Marija, Prodanović, Olivera, Popović, Nikolina, Ostafe, Raluca, Fischer, Rainer, Prodanović, Radivoje, "Directed evolution of cellobiose dehydrogenase on the surface of yeast cells using resazurin-based fluorescent assay" in Applied Sciences (Switzerland), 9, no. 7 (2019):1-15,
https://doi.org/10.3390/app9071413 . .
6
6
6

Influence of methionine residue position on oxidative stability of glucose oxidase from Aspergillus niger

Kovačević, Gordana; Ostafe, Raluca; Fischer, Rainer; Prodanović, Radivoje

(Elsevier, 2019)

TY  - JOUR
AU  - Kovačević, Gordana
AU  - Ostafe, Raluca
AU  - Fischer, Rainer
AU  - Prodanović, Radivoje
PY  - 2019
AB  - Glucose oxidase (GOx) is a promising candidate for construction of implantable miniature biofuel cells and biosensors for continuous glucose monitoring. The main drawback that limits current application of GOx in these devices is its low stability, especially sensitivity to reactive oxygen species. In order to address this problem, we performed saturation mutagenesis at all 11 methionine residues as their interaction with reactive oxygen species inactivates enzymes. For successful screening of these libraries a method based on yeast surface display (YSD) systems was developed. Mutations at methionine positions close to the GOx active site contributed the most to the oxidative stability, and combinations of the four best single mutations were tested. Combined mutants did not show higher stability or activity compared to the parental single mutants. To confirm oxidative stability of YSD expressed GOx mutants they were re-cloned in Pichia pastoris, purified and immobilized on macroporous copolymer. The additional kinetic analysis of immobilized GOx mutants confirmed that the best mutant with only one mutation close to the active site (M561S) has 2.5 times increased half-life in the presence of hydrogen peroxide compared to the wild-type variant.
PB  - Elsevier
T2  - Biochemical Engineering Journal
T1  - Influence of methionine residue position on oxidative stability of glucose oxidase from Aspergillus niger
VL  - 146
SP  - 143
EP  - 149
DO  - 10.1016/j.bej.2019.03.016
ER  - 
@article{
author = "Kovačević, Gordana and Ostafe, Raluca and Fischer, Rainer and Prodanović, Radivoje",
year = "2019",
abstract = "Glucose oxidase (GOx) is a promising candidate for construction of implantable miniature biofuel cells and biosensors for continuous glucose monitoring. The main drawback that limits current application of GOx in these devices is its low stability, especially sensitivity to reactive oxygen species. In order to address this problem, we performed saturation mutagenesis at all 11 methionine residues as their interaction with reactive oxygen species inactivates enzymes. For successful screening of these libraries a method based on yeast surface display (YSD) systems was developed. Mutations at methionine positions close to the GOx active site contributed the most to the oxidative stability, and combinations of the four best single mutations were tested. Combined mutants did not show higher stability or activity compared to the parental single mutants. To confirm oxidative stability of YSD expressed GOx mutants they were re-cloned in Pichia pastoris, purified and immobilized on macroporous copolymer. The additional kinetic analysis of immobilized GOx mutants confirmed that the best mutant with only one mutation close to the active site (M561S) has 2.5 times increased half-life in the presence of hydrogen peroxide compared to the wild-type variant.",
publisher = "Elsevier",
journal = "Biochemical Engineering Journal",
title = "Influence of methionine residue position on oxidative stability of glucose oxidase from Aspergillus niger",
volume = "146",
pages = "143-149",
doi = "10.1016/j.bej.2019.03.016"
}
Kovačević, G., Ostafe, R., Fischer, R.,& Prodanović, R.. (2019). Influence of methionine residue position on oxidative stability of glucose oxidase from Aspergillus niger. in Biochemical Engineering Journal
Elsevier., 146, 143-149.
https://doi.org/10.1016/j.bej.2019.03.016
Kovačević G, Ostafe R, Fischer R, Prodanović R. Influence of methionine residue position on oxidative stability of glucose oxidase from Aspergillus niger. in Biochemical Engineering Journal. 2019;146:143-149.
doi:10.1016/j.bej.2019.03.016 .
Kovačević, Gordana, Ostafe, Raluca, Fischer, Rainer, Prodanović, Radivoje, "Influence of methionine residue position on oxidative stability of glucose oxidase from Aspergillus niger" in Biochemical Engineering Journal, 146 (2019):143-149,
https://doi.org/10.1016/j.bej.2019.03.016 . .
9
7
8

Biochemical characterization of chitinase A from Bacillus licheniformis DSM8785 expressed in Pichia pastoris KM71H

Menghiu, G.; Ostafe, V.; Prodanović, Radivoje; Fischer, Rainer; Ostafe, Raluca

(2019)

TY  - JOUR
AU  - Menghiu, G.
AU  - Ostafe, V.
AU  - Prodanović, Radivoje
AU  - Fischer, Rainer
AU  - Ostafe, Raluca
PY  - 2019
UR  - http://cherry.chem.bg.ac.rs/handle/123456789/2798
AB  - Chitin is an abundant biopolymer found mainly in the exoskeleton of crustaceans and insects. The degradation of chitin using chitinases is one way to address the accumulation of chitin waste streams in the environment, and research has therefore focused on the identification, improvement and expression of suitable enzymes. Here we describe the production, purification and characterization of Bacillus licheniformis chitinase A in the Pichia pastoris expression system. Optimal enzyme activity occurred at pH 4.0–5.0 and within the temperature range 50–60 °C. With colloidal chitin as the substrate, the Km (2.307 mM) and Vmax (0.024 mM min−1) of the enzyme were determined using a 3,5-dinitrosalicylic acid assay. The degradation products of colloidal chitin and hexa-N-acetylchitohexaose were compared by thin-layer chromatography. The activity of the glycosylated enzyme produced in P. pastoris was compared with the in vitro deglycosylated and aglycosylated version produced in Escherichia coli. We showed that the glycosylated chitinase was more active than the deglycosylated and aglycosylated variants. © 2018 Elsevier Inc.
T2  - Protein Expression and Purification
T1  - Biochemical characterization of chitinase A from Bacillus licheniformis DSM8785 expressed in Pichia pastoris KM71H
VL  - 154
SP  - 25
EP  - 32
DO  - 10.1016/j.pep.2018.09.007
ER  - 
@article{
author = "Menghiu, G. and Ostafe, V. and Prodanović, Radivoje and Fischer, Rainer and Ostafe, Raluca",
year = "2019",
abstract = "Chitin is an abundant biopolymer found mainly in the exoskeleton of crustaceans and insects. The degradation of chitin using chitinases is one way to address the accumulation of chitin waste streams in the environment, and research has therefore focused on the identification, improvement and expression of suitable enzymes. Here we describe the production, purification and characterization of Bacillus licheniformis chitinase A in the Pichia pastoris expression system. Optimal enzyme activity occurred at pH 4.0–5.0 and within the temperature range 50–60 °C. With colloidal chitin as the substrate, the Km (2.307 mM) and Vmax (0.024 mM min−1) of the enzyme were determined using a 3,5-dinitrosalicylic acid assay. The degradation products of colloidal chitin and hexa-N-acetylchitohexaose were compared by thin-layer chromatography. The activity of the glycosylated enzyme produced in P. pastoris was compared with the in vitro deglycosylated and aglycosylated version produced in Escherichia coli. We showed that the glycosylated chitinase was more active than the deglycosylated and aglycosylated variants. © 2018 Elsevier Inc.",
journal = "Protein Expression and Purification",
title = "Biochemical characterization of chitinase A from Bacillus licheniformis DSM8785 expressed in Pichia pastoris KM71H",
volume = "154",
pages = "25-32",
doi = "10.1016/j.pep.2018.09.007"
}
Menghiu, G., Ostafe, V., Prodanović, R., Fischer, R.,& Ostafe, R.. (2019). Biochemical characterization of chitinase A from Bacillus licheniformis DSM8785 expressed in Pichia pastoris KM71H. in Protein Expression and Purification, 154, 25-32.
https://doi.org/10.1016/j.pep.2018.09.007
Menghiu G, Ostafe V, Prodanović R, Fischer R, Ostafe R. Biochemical characterization of chitinase A from Bacillus licheniformis DSM8785 expressed in Pichia pastoris KM71H. in Protein Expression and Purification. 2019;154:25-32.
doi:10.1016/j.pep.2018.09.007 .
Menghiu, G., Ostafe, V., Prodanović, Radivoje, Fischer, Rainer, Ostafe, Raluca, "Biochemical characterization of chitinase A from Bacillus licheniformis DSM8785 expressed in Pichia pastoris KM71H" in Protein Expression and Purification, 154 (2019):25-32,
https://doi.org/10.1016/j.pep.2018.09.007 . .
13
10
9

Biochemical characterization of chitinase A from Bacillus licheniformis DSM8785 expressed in Pichia pastoris KM71H

Menghiu, G.; Ostafe, V.; Prodanović, Radivoje; Fischer, Rainer; Ostafe, Raluca

(Elsevier, 2019)

TY  - JOUR
AU  - Menghiu, G.
AU  - Ostafe, V.
AU  - Prodanović, Radivoje
AU  - Fischer, Rainer
AU  - Ostafe, Raluca
PY  - 2019
UR  - http://cherry.chem.bg.ac.rs/handle/123456789/348
AB  - Chitin is an abundant biopolymer found mainly in the exoskeleton of crustaceans and insects. The degradation of chitin using chitinases is one way to address the accumulation of chitin waste streams in the environment, and research has therefore focused on the identification, improvement and expression of suitable enzymes. Here we describe the production, purification and characterization of Bacillus licheniformis chitinase A in the Pichia pastoris expression system. Optimal enzyme activity occurred at pH 4.0–5.0 and within the temperature range 50–60 °C. With colloidal chitin as the substrate, the Km (2.307 mM) and Vmax (0.024 mM min−1) of the enzyme were determined using a 3,5-dinitrosalicylic acid assay. The degradation products of colloidal chitin and hexa-N-acetylchitohexaose were compared by thin-layer chromatography. The activity of the glycosylated enzyme produced in P. pastoris was compared with the in vitro deglycosylated and aglycosylated version produced in Escherichia coli. We showed that the glycosylated chitinase was more active than the deglycosylated and aglycosylated variants. © 2018 Elsevier Inc.
PB  - Elsevier
T2  - Protein Expression and Purification
T1  - Biochemical characterization of chitinase A from Bacillus licheniformis DSM8785 expressed in Pichia pastoris KM71H
VL  - 154
SP  - 25
EP  - 32
DO  - 10.1016/j.pep.2018.09.007
UR  - Kon_1319
ER  - 
@article{
author = "Menghiu, G. and Ostafe, V. and Prodanović, Radivoje and Fischer, Rainer and Ostafe, Raluca",
year = "2019",
abstract = "Chitin is an abundant biopolymer found mainly in the exoskeleton of crustaceans and insects. The degradation of chitin using chitinases is one way to address the accumulation of chitin waste streams in the environment, and research has therefore focused on the identification, improvement and expression of suitable enzymes. Here we describe the production, purification and characterization of Bacillus licheniformis chitinase A in the Pichia pastoris expression system. Optimal enzyme activity occurred at pH 4.0–5.0 and within the temperature range 50–60 °C. With colloidal chitin as the substrate, the Km (2.307 mM) and Vmax (0.024 mM min−1) of the enzyme were determined using a 3,5-dinitrosalicylic acid assay. The degradation products of colloidal chitin and hexa-N-acetylchitohexaose were compared by thin-layer chromatography. The activity of the glycosylated enzyme produced in P. pastoris was compared with the in vitro deglycosylated and aglycosylated version produced in Escherichia coli. We showed that the glycosylated chitinase was more active than the deglycosylated and aglycosylated variants. © 2018 Elsevier Inc.",
publisher = "Elsevier",
journal = "Protein Expression and Purification",
title = "Biochemical characterization of chitinase A from Bacillus licheniformis DSM8785 expressed in Pichia pastoris KM71H",
volume = "154",
pages = "25-32",
doi = "10.1016/j.pep.2018.09.007",
url = "Kon_1319"
}
Menghiu, G., Ostafe, V., Prodanović, R., Fischer, R.,& Ostafe, R.. (2019). Biochemical characterization of chitinase A from Bacillus licheniformis DSM8785 expressed in Pichia pastoris KM71H. in Protein Expression and Purification
Elsevier., 154, 25-32.
https://doi.org/10.1016/j.pep.2018.09.007
Kon_1319
Menghiu G, Ostafe V, Prodanović R, Fischer R, Ostafe R. Biochemical characterization of chitinase A from Bacillus licheniformis DSM8785 expressed in Pichia pastoris KM71H. in Protein Expression and Purification. 2019;154:25-32.
doi:10.1016/j.pep.2018.09.007
Kon_1319 .
Menghiu, G., Ostafe, V., Prodanović, Radivoje, Fischer, Rainer, Ostafe, Raluca, "Biochemical characterization of chitinase A from Bacillus licheniformis DSM8785 expressed in Pichia pastoris KM71H" in Protein Expression and Purification, 154 (2019):25-32,
https://doi.org/10.1016/j.pep.2018.09.007 .,
Kon_1319 .
13
10
10

Supplementary data for the article: Kovačević, G.; Ostafe, R.; Fischer, R.; Prodanović, R. Influence of Methionine Residue Position on Oxidative Stability of Glucose Oxidase from Aspergillus Niger. Biochemical Engineering Journal 2019, 146, 143–149. https://doi.org/10.1016/j.bej.2019.03.016

Kovačević, Gordana; Ostafe, Raluca; Fischer, Rainer; Prodanović, Radivoje

(Elsevier, 2019)

TY  - DATA
AU  - Kovačević, Gordana
AU  - Ostafe, Raluca
AU  - Fischer, Rainer
AU  - Prodanović, Radivoje
PY  - 2019
UR  - http://cherry.chem.bg.ac.rs/handle/123456789/2882
PB  - Elsevier
T2  - Biochemical Engineering Journal
T1  - Supplementary data for the article: Kovačević, G.; Ostafe, R.; Fischer, R.; Prodanović, R. Influence of Methionine Residue Position on Oxidative Stability of Glucose Oxidase from Aspergillus Niger. Biochemical Engineering Journal 2019, 146, 143–149. https://doi.org/10.1016/j.bej.2019.03.016
ER  - 
@misc{
author = "Kovačević, Gordana and Ostafe, Raluca and Fischer, Rainer and Prodanović, Radivoje",
year = "2019",
publisher = "Elsevier",
journal = "Biochemical Engineering Journal",
title = "Supplementary data for the article: Kovačević, G.; Ostafe, R.; Fischer, R.; Prodanović, R. Influence of Methionine Residue Position on Oxidative Stability of Glucose Oxidase from Aspergillus Niger. Biochemical Engineering Journal 2019, 146, 143–149. https://doi.org/10.1016/j.bej.2019.03.016"
}
Kovačević, G., Ostafe, R., Fischer, R.,& Prodanović, R.. (2019). Supplementary data for the article: Kovačević, G.; Ostafe, R.; Fischer, R.; Prodanović, R. Influence of Methionine Residue Position on Oxidative Stability of Glucose Oxidase from Aspergillus Niger. Biochemical Engineering Journal 2019, 146, 143–149. https://doi.org/10.1016/j.bej.2019.03.016. in Biochemical Engineering Journal
Elsevier..
Kovačević G, Ostafe R, Fischer R, Prodanović R. Supplementary data for the article: Kovačević, G.; Ostafe, R.; Fischer, R.; Prodanović, R. Influence of Methionine Residue Position on Oxidative Stability of Glucose Oxidase from Aspergillus Niger. Biochemical Engineering Journal 2019, 146, 143–149. https://doi.org/10.1016/j.bej.2019.03.016. in Biochemical Engineering Journal. 2019;..
Kovačević, Gordana, Ostafe, Raluca, Fischer, Rainer, Prodanović, Radivoje, "Supplementary data for the article: Kovačević, G.; Ostafe, R.; Fischer, R.; Prodanović, R. Influence of Methionine Residue Position on Oxidative Stability of Glucose Oxidase from Aspergillus Niger. Biochemical Engineering Journal 2019, 146, 143–149. https://doi.org/10.1016/j.bej.2019.03.016" in Biochemical Engineering Journal (2019).

Development of GFP-based high-throughput screening system for directed evolution of glucose oxidase

Kovačević, Gordana; Ostafe, Raluca; Balaž, Ana Marija; Fischer, Rainer; Prodanović, Radivoje

(Elsevier, 2018)

TY  - JOUR
AU  - Kovačević, Gordana
AU  - Ostafe, Raluca
AU  - Balaž, Ana Marija
AU  - Fischer, Rainer
AU  - Prodanović, Radivoje
PY  - 2018
UR  - http://cherry.chem.bg.ac.rs/handle/123456789/336
AB  - Glucose oxidase (GOx) mutants with higher activity or stability have important role in industry and in the development of biosensors and biofuel cells. Discovering these mutants can be time-consuming if appropriate high-throughput screening (HTS) systems are not available. GOx gene libraries were successfully screened and sorted using a HTS system based on GOx activity dependent fluorescent labeling of yeast cells with tyramids and quantification of the amount of expressed enzyme by yeast enhanced green fluorescent protein (yGFP) tagging and flow cytometry. For this purpose, we expressed wild type and a mutant GOx as a chimera with the yGFP to confirm differences in catalytic activity between wild-type and mutant GOx. Fluorescence of yGFP is preserved during expression of chimera, and also after the oxidative enzymatic reaction. We have obtained a 2.5-fold enrichment in population of cells expressing active enzyme, and percentage of enzyme variants with enzymatic mean activity higher than wild type activity was increased to 44% after a single round of GOx gene library sorting. We have found two mutants with 1.3 and 2.3-fold increase in Vmax values compared to the wtGOx. By simultaneous detection of protein expression level and enzyme activity we have increased the likelihood of finding GOx variants with increased activity in a single round of flow cytometry sorting. © 2018 The Society for Biotechnology, Japan
PB  - Elsevier
T2  - Journal of Bioscience and Bioengineering
T1  - Development of GFP-based high-throughput screening system for directed evolution of glucose oxidase
DO  - 10.1016/j.jbiosc.2018.07.002
UR  - Kon_1307
ER  - 
@article{
author = "Kovačević, Gordana and Ostafe, Raluca and Balaž, Ana Marija and Fischer, Rainer and Prodanović, Radivoje",
year = "2018",
abstract = "Glucose oxidase (GOx) mutants with higher activity or stability have important role in industry and in the development of biosensors and biofuel cells. Discovering these mutants can be time-consuming if appropriate high-throughput screening (HTS) systems are not available. GOx gene libraries were successfully screened and sorted using a HTS system based on GOx activity dependent fluorescent labeling of yeast cells with tyramids and quantification of the amount of expressed enzyme by yeast enhanced green fluorescent protein (yGFP) tagging and flow cytometry. For this purpose, we expressed wild type and a mutant GOx as a chimera with the yGFP to confirm differences in catalytic activity between wild-type and mutant GOx. Fluorescence of yGFP is preserved during expression of chimera, and also after the oxidative enzymatic reaction. We have obtained a 2.5-fold enrichment in population of cells expressing active enzyme, and percentage of enzyme variants with enzymatic mean activity higher than wild type activity was increased to 44% after a single round of GOx gene library sorting. We have found two mutants with 1.3 and 2.3-fold increase in Vmax values compared to the wtGOx. By simultaneous detection of protein expression level and enzyme activity we have increased the likelihood of finding GOx variants with increased activity in a single round of flow cytometry sorting. © 2018 The Society for Biotechnology, Japan",
publisher = "Elsevier",
journal = "Journal of Bioscience and Bioengineering",
title = "Development of GFP-based high-throughput screening system for directed evolution of glucose oxidase",
doi = "10.1016/j.jbiosc.2018.07.002",
url = "Kon_1307"
}
Kovačević, G., Ostafe, R., Balaž, A. M., Fischer, R.,& Prodanović, R.. (2018). Development of GFP-based high-throughput screening system for directed evolution of glucose oxidase. in Journal of Bioscience and Bioengineering
Elsevier..
https://doi.org/10.1016/j.jbiosc.2018.07.002
Kon_1307
Kovačević G, Ostafe R, Balaž AM, Fischer R, Prodanović R. Development of GFP-based high-throughput screening system for directed evolution of glucose oxidase. in Journal of Bioscience and Bioengineering. 2018;.
doi:10.1016/j.jbiosc.2018.07.002
Kon_1307 .
Kovačević, Gordana, Ostafe, Raluca, Balaž, Ana Marija, Fischer, Rainer, Prodanović, Radivoje, "Development of GFP-based high-throughput screening system for directed evolution of glucose oxidase" in Journal of Bioscience and Bioengineering (2018),
https://doi.org/10.1016/j.jbiosc.2018.07.002 .,
Kon_1307 .
1
13
11
12

Yeast surface display is a novel tool for the rapid immunological characterization of plant-derived food allergens

Popović, Milica; Prodanović, Radivoje; Ostafe, Raluca; Schillberg, Stefan; Fischer, Rainer; Gavrović-Jankulović, Marija

(Humana Press Inc, Totowa, 2015)

TY  - JOUR
AU  - Popović, Milica
AU  - Prodanović, Radivoje
AU  - Ostafe, Raluca
AU  - Schillberg, Stefan
AU  - Fischer, Rainer
AU  - Gavrović-Jankulović, Marija
PY  - 2015
UR  - http://cherry.chem.bg.ac.rs/handle/123456789/1668
AB  - High-throughput characterization of allergens relies often on phage display technique which is subject to the limitations of a prokaryotic expression system. Substituting the phage display platform with a yeast surface display could lead to fast immunological characterization of allergens with complex structures. Our objective was to evaluate the potential of yeast surface display for characterization of plant-derived food allergens. The coding sequence of mature actinidin (Act d 1) was cloned into pCTCON2 surface display vector. Flow cytometry was used to confirm localization of recombinant Act d 1 on the surface of yeast cells using rabbit polyclonal antisera IgG and IgE from sera of kiwifruit-allergic individuals. Immunological (dot blot, immunoblot ELISA and ELISA inhibition), biochemical (enzymatic activity in gel) and biological (basophil activation) characterization of Act d 1 after solubilization from the yeast cell confirmed that recombinant Act d 1 produced on the surface of yeast cell is similar to its natural counterpart isolated from green kiwifruit. Yeast surface display is a potent technique that enables fast immunochemical characterization of allergens in situ without the need for protein purification and offers an alternative that could lead to improvement of standard immunodiagnostic and immunotherapeutic approaches.
PB  - Humana Press Inc, Totowa
T2  - Immunologic Research
T1  - Yeast surface display is a novel tool for the rapid immunological characterization of plant-derived food allergens
VL  - 61
IS  - 3
SP  - 230
EP  - 239
DO  - 10.1007/s12026-014-8614-0
UR  - Kon_2814
ER  - 
@article{
author = "Popović, Milica and Prodanović, Radivoje and Ostafe, Raluca and Schillberg, Stefan and Fischer, Rainer and Gavrović-Jankulović, Marija",
year = "2015",
abstract = "High-throughput characterization of allergens relies often on phage display technique which is subject to the limitations of a prokaryotic expression system. Substituting the phage display platform with a yeast surface display could lead to fast immunological characterization of allergens with complex structures. Our objective was to evaluate the potential of yeast surface display for characterization of plant-derived food allergens. The coding sequence of mature actinidin (Act d 1) was cloned into pCTCON2 surface display vector. Flow cytometry was used to confirm localization of recombinant Act d 1 on the surface of yeast cells using rabbit polyclonal antisera IgG and IgE from sera of kiwifruit-allergic individuals. Immunological (dot blot, immunoblot ELISA and ELISA inhibition), biochemical (enzymatic activity in gel) and biological (basophil activation) characterization of Act d 1 after solubilization from the yeast cell confirmed that recombinant Act d 1 produced on the surface of yeast cell is similar to its natural counterpart isolated from green kiwifruit. Yeast surface display is a potent technique that enables fast immunochemical characterization of allergens in situ without the need for protein purification and offers an alternative that could lead to improvement of standard immunodiagnostic and immunotherapeutic approaches.",
publisher = "Humana Press Inc, Totowa",
journal = "Immunologic Research",
title = "Yeast surface display is a novel tool for the rapid immunological characterization of plant-derived food allergens",
volume = "61",
number = "3",
pages = "230-239",
doi = "10.1007/s12026-014-8614-0",
url = "Kon_2814"
}
Popović, M., Prodanović, R., Ostafe, R., Schillberg, S., Fischer, R.,& Gavrović-Jankulović, M.. (2015). Yeast surface display is a novel tool for the rapid immunological characterization of plant-derived food allergens. in Immunologic Research
Humana Press Inc, Totowa., 61(3), 230-239.
https://doi.org/10.1007/s12026-014-8614-0
Kon_2814
Popović M, Prodanović R, Ostafe R, Schillberg S, Fischer R, Gavrović-Jankulović M. Yeast surface display is a novel tool for the rapid immunological characterization of plant-derived food allergens. in Immunologic Research. 2015;61(3):230-239.
doi:10.1007/s12026-014-8614-0
Kon_2814 .
Popović, Milica, Prodanović, Radivoje, Ostafe, Raluca, Schillberg, Stefan, Fischer, Rainer, Gavrović-Jankulović, Marija, "Yeast surface display is a novel tool for the rapid immunological characterization of plant-derived food allergens" in Immunologic Research, 61, no. 3 (2015):230-239,
https://doi.org/10.1007/s12026-014-8614-0 .,
Kon_2814 .
1
5
4
4

Microfluidic Generation of Monodisperse, Structurally Homogeneous Alginate Microgels for Cell Encapsulation and 3D Cell Culture

Utech, Stefanie; Prodanović, Radivoje; Mao, Angelo S.; Ostafe, Raluca; Mooney, David J.; Weitz, David A.

(Wiley-Blackwell, Hoboken, 2015)

TY  - JOUR
AU  - Utech, Stefanie
AU  - Prodanović, Radivoje
AU  - Mao, Angelo S.
AU  - Ostafe, Raluca
AU  - Mooney, David J.
AU  - Weitz, David A.
PY  - 2015
UR  - http://cherry.chem.bg.ac.rs/handle/123456789/2785
AB  - Monodisperse alginate microgels (10–50 μm) are created via droplet‐based microfluidics by a novel crosslinking procedure. Ionic crosslinking of alginate is induced by release of chelated calcium ions. The process separates droplet formation and gelation reaction enabling excellent control over size and homogeneity under mild reaction conditions. Living mesenchymal stem cells are encapsulated and cultured in the generated 3D microenvironments.
PB  - Wiley-Blackwell, Hoboken
T2  - Advanced Healthcare Materials
T1  - Microfluidic Generation of Monodisperse, Structurally Homogeneous Alginate Microgels for Cell Encapsulation and 3D Cell Culture
VL  - 4
IS  - 11
SP  - 1628
EP  - 1633
DO  - 10.1002/adhm.201500021
ER  - 
@article{
author = "Utech, Stefanie and Prodanović, Radivoje and Mao, Angelo S. and Ostafe, Raluca and Mooney, David J. and Weitz, David A.",
year = "2015",
abstract = "Monodisperse alginate microgels (10–50 μm) are created via droplet‐based microfluidics by a novel crosslinking procedure. Ionic crosslinking of alginate is induced by release of chelated calcium ions. The process separates droplet formation and gelation reaction enabling excellent control over size and homogeneity under mild reaction conditions. Living mesenchymal stem cells are encapsulated and cultured in the generated 3D microenvironments.",
publisher = "Wiley-Blackwell, Hoboken",
journal = "Advanced Healthcare Materials",
title = "Microfluidic Generation of Monodisperse, Structurally Homogeneous Alginate Microgels for Cell Encapsulation and 3D Cell Culture",
volume = "4",
number = "11",
pages = "1628-1633",
doi = "10.1002/adhm.201500021"
}
Utech, S., Prodanović, R., Mao, A. S., Ostafe, R., Mooney, D. J.,& Weitz, D. A.. (2015). Microfluidic Generation of Monodisperse, Structurally Homogeneous Alginate Microgels for Cell Encapsulation and 3D Cell Culture. in Advanced Healthcare Materials
Wiley-Blackwell, Hoboken., 4(11), 1628-1633.
https://doi.org/10.1002/adhm.201500021
Utech S, Prodanović R, Mao AS, Ostafe R, Mooney DJ, Weitz DA. Microfluidic Generation of Monodisperse, Structurally Homogeneous Alginate Microgels for Cell Encapsulation and 3D Cell Culture. in Advanced Healthcare Materials. 2015;4(11):1628-1633.
doi:10.1002/adhm.201500021 .
Utech, Stefanie, Prodanović, Radivoje, Mao, Angelo S., Ostafe, Raluca, Mooney, David J., Weitz, David A., "Microfluidic Generation of Monodisperse, Structurally Homogeneous Alginate Microgels for Cell Encapsulation and 3D Cell Culture" in Advanced Healthcare Materials, 4, no. 11 (2015):1628-1633,
https://doi.org/10.1002/adhm.201500021 . .
187
167
169

Microfluidic Generation of Monodisperse, Structurally Homogeneous Alginate Microgels for Cell Encapsulation and 3D Cell Culture

Utech, Stefanie; Prodanović, Radivoje; Mao, Angelo S.; Ostafe, Raluca; Mooney, David J.; Weitz, David A.

(Wiley-Blackwell, Hoboken, 2015)

TY  - JOUR
AU  - Utech, Stefanie
AU  - Prodanović, Radivoje
AU  - Mao, Angelo S.
AU  - Ostafe, Raluca
AU  - Mooney, David J.
AU  - Weitz, David A.
PY  - 2015
UR  - http://cherry.chem.bg.ac.rs/handle/123456789/1751
PB  - Wiley-Blackwell, Hoboken
T2  - Advanced Healthcare Materials
T1  - Microfluidic Generation of Monodisperse, Structurally Homogeneous Alginate Microgels for Cell Encapsulation and 3D Cell Culture
VL  - 4
IS  - 11
SP  - 1628
EP  - 1633
DO  - 10.1002/adhm.201500021
UR  - Kon_2897
ER  - 
@article{
author = "Utech, Stefanie and Prodanović, Radivoje and Mao, Angelo S. and Ostafe, Raluca and Mooney, David J. and Weitz, David A.",
year = "2015",
publisher = "Wiley-Blackwell, Hoboken",
journal = "Advanced Healthcare Materials",
title = "Microfluidic Generation of Monodisperse, Structurally Homogeneous Alginate Microgels for Cell Encapsulation and 3D Cell Culture",
volume = "4",
number = "11",
pages = "1628-1633",
doi = "10.1002/adhm.201500021",
url = "Kon_2897"
}
Utech, S., Prodanović, R., Mao, A. S., Ostafe, R., Mooney, D. J.,& Weitz, D. A.. (2015). Microfluidic Generation of Monodisperse, Structurally Homogeneous Alginate Microgels for Cell Encapsulation and 3D Cell Culture. in Advanced Healthcare Materials
Wiley-Blackwell, Hoboken., 4(11), 1628-1633.
https://doi.org/10.1002/adhm.201500021
Kon_2897
Utech S, Prodanović R, Mao AS, Ostafe R, Mooney DJ, Weitz DA. Microfluidic Generation of Monodisperse, Structurally Homogeneous Alginate Microgels for Cell Encapsulation and 3D Cell Culture. in Advanced Healthcare Materials. 2015;4(11):1628-1633.
doi:10.1002/adhm.201500021
Kon_2897 .
Utech, Stefanie, Prodanović, Radivoje, Mao, Angelo S., Ostafe, Raluca, Mooney, David J., Weitz, David A., "Microfluidic Generation of Monodisperse, Structurally Homogeneous Alginate Microgels for Cell Encapsulation and 3D Cell Culture" in Advanced Healthcare Materials, 4, no. 11 (2015):1628-1633,
https://doi.org/10.1002/adhm.201500021 .,
Kon_2897 .
187
167
172

Preventing oxidative damage at the early phase: The case of glucose oxidase

Petrović, D.; Kovačević, Gordana; Ostafe, Raluca; Fischer, Rainer; Strodel, B.; Prodanović, Radivoje

(Wiley-Blackwell, Hoboken, 2015)

TY  - CONF
AU  - Petrović, D.
AU  - Kovačević, Gordana
AU  - Ostafe, Raluca
AU  - Fischer, Rainer
AU  - Strodel, B.
AU  - Prodanović, Radivoje
PY  - 2015
UR  - http://cherry.chem.bg.ac.rs/handle/123456789/1978
PB  - Wiley-Blackwell, Hoboken
C3  - FEBS Journal / Federation of European of Biochemical Societies
T1  - Preventing oxidative damage at the early phase: The case of glucose oxidase
VL  - 282
SP  - 339
EP  - 340
UR  - Kon_2933
ER  - 
@conference{
author = "Petrović, D. and Kovačević, Gordana and Ostafe, Raluca and Fischer, Rainer and Strodel, B. and Prodanović, Radivoje",
year = "2015",
publisher = "Wiley-Blackwell, Hoboken",
journal = "FEBS Journal / Federation of European of Biochemical Societies",
title = "Preventing oxidative damage at the early phase: The case of glucose oxidase",
volume = "282",
pages = "339-340",
url = "Kon_2933"
}
Petrović, D., Kovačević, G., Ostafe, R., Fischer, R., Strodel, B.,& Prodanović, R.. (2015). Preventing oxidative damage at the early phase: The case of glucose oxidase. in FEBS Journal / Federation of European of Biochemical Societies
Wiley-Blackwell, Hoboken., 282, 339-340.
Kon_2933
Petrović D, Kovačević G, Ostafe R, Fischer R, Strodel B, Prodanović R. Preventing oxidative damage at the early phase: The case of glucose oxidase. in FEBS Journal / Federation of European of Biochemical Societies. 2015;282:339-340.
Kon_2933 .
Petrović, D., Kovačević, Gordana, Ostafe, Raluca, Fischer, Rainer, Strodel, B., Prodanović, Radivoje, "Preventing oxidative damage at the early phase: The case of glucose oxidase" in FEBS Journal / Federation of European of Biochemical Societies, 282 (2015):339-340,
Kon_2933 .

A high-throughput cellulase screening system based on droplet microfluidics

Ostafe, Raluca; Prodanović, Radivoje; Ung, Lloyd W.; Weitz, David A.; Fischer, Rainer

(Amer Inst Physics, Melville, 2014)

TY  - JOUR
AU  - Ostafe, Raluca
AU  - Prodanović, Radivoje
AU  - Ung, Lloyd W.
AU  - Weitz, David A.
AU  - Fischer, Rainer
PY  - 2014
UR  - http://cherry.chem.bg.ac.rs/handle/123456789/1873
AB  - A new ultra-high-throughput screening assay for the detection of cellulase activity was developed based on microfluidic sorting. Cellulase activity is detected using a series of coupled enzymes leading to the formation of a fluorescent product that can be detected on a chip. Using this method, we have achieved up to 300-fold enrichments of the active population of cells and greater than 90% purity after just one sorting round. In addition, we proved that we can sort the cellulase-expressing cells from mixtures containing less than 1% active cells. (C) 2014 Author(s). All article content, except where otherwise noted, is licensed under a Creative Commons Attribution 3.0 Unported License.
PB  - Amer Inst Physics, Melville
T2  - Biomicrofluidics
T1  - A high-throughput cellulase screening system based on droplet microfluidics
VL  - 8
IS  - 4
DO  - 10.1063/1.4886771
UR  - Kon_2756
ER  - 
@article{
author = "Ostafe, Raluca and Prodanović, Radivoje and Ung, Lloyd W. and Weitz, David A. and Fischer, Rainer",
year = "2014",
abstract = "A new ultra-high-throughput screening assay for the detection of cellulase activity was developed based on microfluidic sorting. Cellulase activity is detected using a series of coupled enzymes leading to the formation of a fluorescent product that can be detected on a chip. Using this method, we have achieved up to 300-fold enrichments of the active population of cells and greater than 90% purity after just one sorting round. In addition, we proved that we can sort the cellulase-expressing cells from mixtures containing less than 1% active cells. (C) 2014 Author(s). All article content, except where otherwise noted, is licensed under a Creative Commons Attribution 3.0 Unported License.",
publisher = "Amer Inst Physics, Melville",
journal = "Biomicrofluidics",
title = "A high-throughput cellulase screening system based on droplet microfluidics",
volume = "8",
number = "4",
doi = "10.1063/1.4886771",
url = "Kon_2756"
}
Ostafe, R., Prodanović, R., Ung, L. W., Weitz, D. A.,& Fischer, R.. (2014). A high-throughput cellulase screening system based on droplet microfluidics. in Biomicrofluidics
Amer Inst Physics, Melville., 8(4).
https://doi.org/10.1063/1.4886771
Kon_2756
Ostafe R, Prodanović R, Ung LW, Weitz DA, Fischer R. A high-throughput cellulase screening system based on droplet microfluidics. in Biomicrofluidics. 2014;8(4).
doi:10.1063/1.4886771
Kon_2756 .
Ostafe, Raluca, Prodanović, Radivoje, Ung, Lloyd W., Weitz, David A., Fischer, Rainer, "A high-throughput cellulase screening system based on droplet microfluidics" in Biomicrofluidics, 8, no. 4 (2014),
https://doi.org/10.1063/1.4886771 .,
Kon_2756 .
4
44
38
41

Ultra-High-Throughput Screening Method for the Directed Evolution of Glucose Oxidase

Ostafe, Raluca; Prodanović, Radivoje; Nazor, Jovana; Fischer, Rainer

(Cell Press, Cambridge, 2014)

TY  - JOUR
AU  - Ostafe, Raluca
AU  - Prodanović, Radivoje
AU  - Nazor, Jovana
AU  - Fischer, Rainer
PY  - 2014
UR  - http://cherry.chem.bg.ac.rs/handle/123456789/1519
AB  - Glucose oxidase (GOx) is used in many industrial processes that could benefit from improved versions of the enzyme. Some improvements like higher activity under physiological conditions and thermal stability could be useful for GOx applications in biosensors and biofuel cells. Directed evolution is one of the currently available methods to engineer improved GOx variants. Here, we describe an ultra-high-throughput screening system for sorting the best enzyme variants generated by directed evolution that incorporates several methodological refinements: flow cytometry, in vitro compartmentalization, yeast surface display, fluorescent labeling of the expressed enzyme, delivery of glucose substrate to the reaction mixture through the oil phase, and covalent labeling of the cells with fluorescein-tyramide. The method enables quantitative screening of gene libraries to identify clones with improved activity and it also allows cells to be selected based not only on the overall activity but also on the specific activity of the enzyme.
PB  - Cell Press, Cambridge
T2  - Chemistry and Biology
T1  - Ultra-High-Throughput Screening Method for the Directed Evolution of Glucose Oxidase
VL  - 21
IS  - 3
SP  - 414
EP  - 421
DO  - 10.1016/j.chembiol.2014.01.010
UR  - Kon_2639
ER  - 
@article{
author = "Ostafe, Raluca and Prodanović, Radivoje and Nazor, Jovana and Fischer, Rainer",
year = "2014",
abstract = "Glucose oxidase (GOx) is used in many industrial processes that could benefit from improved versions of the enzyme. Some improvements like higher activity under physiological conditions and thermal stability could be useful for GOx applications in biosensors and biofuel cells. Directed evolution is one of the currently available methods to engineer improved GOx variants. Here, we describe an ultra-high-throughput screening system for sorting the best enzyme variants generated by directed evolution that incorporates several methodological refinements: flow cytometry, in vitro compartmentalization, yeast surface display, fluorescent labeling of the expressed enzyme, delivery of glucose substrate to the reaction mixture through the oil phase, and covalent labeling of the cells with fluorescein-tyramide. The method enables quantitative screening of gene libraries to identify clones with improved activity and it also allows cells to be selected based not only on the overall activity but also on the specific activity of the enzyme.",
publisher = "Cell Press, Cambridge",
journal = "Chemistry and Biology",
title = "Ultra-High-Throughput Screening Method for the Directed Evolution of Glucose Oxidase",
volume = "21",
number = "3",
pages = "414-421",
doi = "10.1016/j.chembiol.2014.01.010",
url = "Kon_2639"
}
Ostafe, R., Prodanović, R., Nazor, J.,& Fischer, R.. (2014). Ultra-High-Throughput Screening Method for the Directed Evolution of Glucose Oxidase. in Chemistry and Biology
Cell Press, Cambridge., 21(3), 414-421.
https://doi.org/10.1016/j.chembiol.2014.01.010
Kon_2639
Ostafe R, Prodanović R, Nazor J, Fischer R. Ultra-High-Throughput Screening Method for the Directed Evolution of Glucose Oxidase. in Chemistry and Biology. 2014;21(3):414-421.
doi:10.1016/j.chembiol.2014.01.010
Kon_2639 .
Ostafe, Raluca, Prodanović, Radivoje, Nazor, Jovana, Fischer, Rainer, "Ultra-High-Throughput Screening Method for the Directed Evolution of Glucose Oxidase" in Chemistry and Biology, 21, no. 3 (2014):414-421,
https://doi.org/10.1016/j.chembiol.2014.01.010 .,
Kon_2639 .
48
46
46

Cloning, Heterologous Expression, Purification and Characterization of M12 Mutant of Aspergillus niger Glucose Oxidase in Yeast Pichia pastoris KM71H

Kovačević, Gordana; Blažić, Marija; Draganić, Bojana; Ostafe, Raluca; Gavrović-Jankulović, Marija; Fischer, Rainer; Prodanović, Radivoje

(Humana Press Inc, Totowa, 2014)

TY  - JOUR
AU  - Kovačević, Gordana
AU  - Blažić, Marija
AU  - Draganić, Bojana
AU  - Ostafe, Raluca
AU  - Gavrović-Jankulović, Marija
AU  - Fischer, Rainer
AU  - Prodanović, Radivoje
PY  - 2014
UR  - http://cherry.chem.bg.ac.rs/handle/123456789/1517
AB  - Aspergillus niger glucose oxidase (GOx) genes for wild-type (GenBank accession no. X16061, swiss-Prot; P13006) and M12 mutant (N2Y, K13E, T30 V, I94 V, K152R) were cloned into pPICZ alpha A vector for expression in Pichia pastoris KM71H strain. The highest expression level of 17.5 U/mL of fermentation media was obtained in 0.5 % (v/v) methanol after 9 days of fermentation. The recombinant GOx was purified by cross-flow ultrafiltration using membranes of 30 kDa molecular cutoff and DEAE ion-exchange chromatography at pH 6.0. Purified wt GOx had k (cat) of 189.4 s(-1) and K (m) of 28.26 mM while M12 GOx had k (cat) of 352.0 s(-1) and K (m) of 13.33 mM for glucose at pH 5.5. Specificity constants k (cat)/K (m) of wt (6.70 mM(-1) s(-1)) and M12 GOx (26.7 mM(-1) s(-1)) expressed in P. pastoris KM71H were around three times higher than for the same enzymes previously expressed in Saccharomyces cerevisiae InvSc1 strain. The pH optimum and sugar specificity of M12 mutant of GOx remained similar to the wild-type form of the enzyme, while thermostability was slightly decreased. M12 GOx expressed in P. pastoris showed three times higher activity compared to the wt GOx toward redox mediators like N,N-dimethyl-nitroso-aniline used for glucose strips manufacturing. M12 mutant of GOx produced in P. pastoris KM71H could be useful for manufacturing of glucose biosensors and biofuel cells.
PB  - Humana Press Inc, Totowa
T2  - Molecular Biotechnology
T1  - Cloning, Heterologous Expression, Purification and Characterization of M12 Mutant of Aspergillus niger Glucose Oxidase in Yeast Pichia pastoris KM71H
VL  - 56
IS  - 4
SP  - 305
EP  - 311
DO  - 10.1007/s12033-013-9709-x
UR  - Kon_2637
ER  - 
@article{
author = "Kovačević, Gordana and Blažić, Marija and Draganić, Bojana and Ostafe, Raluca and Gavrović-Jankulović, Marija and Fischer, Rainer and Prodanović, Radivoje",
year = "2014",
abstract = "Aspergillus niger glucose oxidase (GOx) genes for wild-type (GenBank accession no. X16061, swiss-Prot; P13006) and M12 mutant (N2Y, K13E, T30 V, I94 V, K152R) were cloned into pPICZ alpha A vector for expression in Pichia pastoris KM71H strain. The highest expression level of 17.5 U/mL of fermentation media was obtained in 0.5 % (v/v) methanol after 9 days of fermentation. The recombinant GOx was purified by cross-flow ultrafiltration using membranes of 30 kDa molecular cutoff and DEAE ion-exchange chromatography at pH 6.0. Purified wt GOx had k (cat) of 189.4 s(-1) and K (m) of 28.26 mM while M12 GOx had k (cat) of 352.0 s(-1) and K (m) of 13.33 mM for glucose at pH 5.5. Specificity constants k (cat)/K (m) of wt (6.70 mM(-1) s(-1)) and M12 GOx (26.7 mM(-1) s(-1)) expressed in P. pastoris KM71H were around three times higher than for the same enzymes previously expressed in Saccharomyces cerevisiae InvSc1 strain. The pH optimum and sugar specificity of M12 mutant of GOx remained similar to the wild-type form of the enzyme, while thermostability was slightly decreased. M12 GOx expressed in P. pastoris showed three times higher activity compared to the wt GOx toward redox mediators like N,N-dimethyl-nitroso-aniline used for glucose strips manufacturing. M12 mutant of GOx produced in P. pastoris KM71H could be useful for manufacturing of glucose biosensors and biofuel cells.",
publisher = "Humana Press Inc, Totowa",
journal = "Molecular Biotechnology",
title = "Cloning, Heterologous Expression, Purification and Characterization of M12 Mutant of Aspergillus niger Glucose Oxidase in Yeast Pichia pastoris KM71H",
volume = "56",
number = "4",
pages = "305-311",
doi = "10.1007/s12033-013-9709-x",
url = "Kon_2637"
}
Kovačević, G., Blažić, M., Draganić, B., Ostafe, R., Gavrović-Jankulović, M., Fischer, R.,& Prodanović, R.. (2014). Cloning, Heterologous Expression, Purification and Characterization of M12 Mutant of Aspergillus niger Glucose Oxidase in Yeast Pichia pastoris KM71H. in Molecular Biotechnology
Humana Press Inc, Totowa., 56(4), 305-311.
https://doi.org/10.1007/s12033-013-9709-x
Kon_2637
Kovačević G, Blažić M, Draganić B, Ostafe R, Gavrović-Jankulović M, Fischer R, Prodanović R. Cloning, Heterologous Expression, Purification and Characterization of M12 Mutant of Aspergillus niger Glucose Oxidase in Yeast Pichia pastoris KM71H. in Molecular Biotechnology. 2014;56(4):305-311.
doi:10.1007/s12033-013-9709-x
Kon_2637 .
Kovačević, Gordana, Blažić, Marija, Draganić, Bojana, Ostafe, Raluca, Gavrović-Jankulović, Marija, Fischer, Rainer, Prodanović, Radivoje, "Cloning, Heterologous Expression, Purification and Characterization of M12 Mutant of Aspergillus niger Glucose Oxidase in Yeast Pichia pastoris KM71H" in Molecular Biotechnology, 56, no. 4 (2014):305-311,
https://doi.org/10.1007/s12033-013-9709-x .,
Kon_2637 .
18
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Supplementary data for article: Ostafe, R.; Prodanović, R.; Commandeur, U.; Fischer, R. Flow Cytometry-Based Ultra-High-Throughput Screening Assay for Cellulase Activity. Analytical Biochemistry 2013, 435 (1), 93–98. https://doi.org/10.1016/j.ab.2012.10.043

Ostafe, Raluca; Prodanović, Radivoje; Commandeur, Ulrich; Fischer, Rainer

(Academic Press Inc Elsevier Science, San Diego, 2013)

TY  - DATA
AU  - Ostafe, Raluca
AU  - Prodanović, Radivoje
AU  - Commandeur, Ulrich
AU  - Fischer, Rainer
PY  - 2013
UR  - http://cherry.chem.bg.ac.rs/handle/123456789/3494
PB  - Academic Press Inc Elsevier Science, San Diego
T2  - Analytical Biochemistry
T1  - Supplementary data for article: Ostafe, R.; Prodanović, R.; Commandeur, U.; Fischer, R. Flow Cytometry-Based Ultra-High-Throughput Screening Assay for Cellulase Activity. Analytical Biochemistry 2013, 435 (1), 93–98. https://doi.org/10.1016/j.ab.2012.10.043
ER  - 
@misc{
author = "Ostafe, Raluca and Prodanović, Radivoje and Commandeur, Ulrich and Fischer, Rainer",
year = "2013",
publisher = "Academic Press Inc Elsevier Science, San Diego",
journal = "Analytical Biochemistry",
title = "Supplementary data for article: Ostafe, R.; Prodanović, R.; Commandeur, U.; Fischer, R. Flow Cytometry-Based Ultra-High-Throughput Screening Assay for Cellulase Activity. Analytical Biochemistry 2013, 435 (1), 93–98. https://doi.org/10.1016/j.ab.2012.10.043"
}
Ostafe, R., Prodanović, R., Commandeur, U.,& Fischer, R.. (2013). Supplementary data for article: Ostafe, R.; Prodanović, R.; Commandeur, U.; Fischer, R. Flow Cytometry-Based Ultra-High-Throughput Screening Assay for Cellulase Activity. Analytical Biochemistry 2013, 435 (1), 93–98. https://doi.org/10.1016/j.ab.2012.10.043. in Analytical Biochemistry
Academic Press Inc Elsevier Science, San Diego..
Ostafe R, Prodanović R, Commandeur U, Fischer R. Supplementary data for article: Ostafe, R.; Prodanović, R.; Commandeur, U.; Fischer, R. Flow Cytometry-Based Ultra-High-Throughput Screening Assay for Cellulase Activity. Analytical Biochemistry 2013, 435 (1), 93–98. https://doi.org/10.1016/j.ab.2012.10.043. in Analytical Biochemistry. 2013;..
Ostafe, Raluca, Prodanović, Radivoje, Commandeur, Ulrich, Fischer, Rainer, "Supplementary data for article: Ostafe, R.; Prodanović, R.; Commandeur, U.; Fischer, R. Flow Cytometry-Based Ultra-High-Throughput Screening Assay for Cellulase Activity. Analytical Biochemistry 2013, 435 (1), 93–98. https://doi.org/10.1016/j.ab.2012.10.043" in Analytical Biochemistry (2013).

Yeast surface display for the expression, purification and characterization of wild-type and B11 mutant glucose oxidases

Blažić, Marija; Kovačević, Gordana; Prodanović, Olivera; Ostafe, Raluca; Gavrović-Jankulović, Marija; Fischer, Rainer; Prodanović, Radivoje

(Academic Press Inc Elsevier Science, San Diego, 2013)

TY  - JOUR
AU  - Blažić, Marija
AU  - Kovačević, Gordana
AU  - Prodanović, Olivera
AU  - Ostafe, Raluca
AU  - Gavrović-Jankulović, Marija
AU  - Fischer, Rainer
AU  - Prodanović, Radivoje
PY  - 2013
UR  - http://cherry.chem.bg.ac.rs/handle/123456789/1357
AB  - Glucose oxidase (GOx) catalyzes the oxidation of glucose to form gluconic acid and hydrogen peroxide, a reaction with important applications in food preservation, the manufacture of cosmetics and pharmaceuticals, and the development of glucose monitoring devices and biofuel cells. We expressed Aspergillus niger wild type GOx and the B11 mutant, which has twice the activity of the wild type enzyme at pH 5.5, as C-terminal fusions with the Saccharomyces cerevisiae Aga2 protein, allowing the fusion proteins to be displayed on the surface of yeast EBY100 cells. After expression, we extracted the proteins from the yeast cell wall and purified them by ion-exchange chromatography and ultrafiltration. This produced a broad 100-140 kDa band by denaturing SDS-PAGE and a high-molecular-weight band by native PAGE corresponding to the activity band revealed by zymography. The wild type and B11 fusion proteins had k(cat) values of 33.3 and 61.3 s(-1) and K-m values for glucose of 33.4 and 27.9 mM, respectively. The pH optimum for both enzymes was 5.0. The kinetic properties of the fusion proteins displayed the same ratio as their native counterparts, confirming that yeast surface display is suitable for the high-throughput directed evolution of GOx using flow cytometry for selection. Aga2-GOx fusion proteins in the yeast cell wall could also be used as immobilized catalysts for the production of gluconic acid.
PB  - Academic Press Inc Elsevier Science, San Diego
T2  - Protein Expression and Purification
T1  - Yeast surface display for the expression, purification and characterization of wild-type and B11 mutant glucose oxidases
VL  - 89
IS  - 2
SP  - 175
EP  - 180
DO  - 10.1016/j.pep.2013.03.014
UR  - Kon_2477
ER  - 
@article{
author = "Blažić, Marija and Kovačević, Gordana and Prodanović, Olivera and Ostafe, Raluca and Gavrović-Jankulović, Marija and Fischer, Rainer and Prodanović, Radivoje",
year = "2013",
abstract = "Glucose oxidase (GOx) catalyzes the oxidation of glucose to form gluconic acid and hydrogen peroxide, a reaction with important applications in food preservation, the manufacture of cosmetics and pharmaceuticals, and the development of glucose monitoring devices and biofuel cells. We expressed Aspergillus niger wild type GOx and the B11 mutant, which has twice the activity of the wild type enzyme at pH 5.5, as C-terminal fusions with the Saccharomyces cerevisiae Aga2 protein, allowing the fusion proteins to be displayed on the surface of yeast EBY100 cells. After expression, we extracted the proteins from the yeast cell wall and purified them by ion-exchange chromatography and ultrafiltration. This produced a broad 100-140 kDa band by denaturing SDS-PAGE and a high-molecular-weight band by native PAGE corresponding to the activity band revealed by zymography. The wild type and B11 fusion proteins had k(cat) values of 33.3 and 61.3 s(-1) and K-m values for glucose of 33.4 and 27.9 mM, respectively. The pH optimum for both enzymes was 5.0. The kinetic properties of the fusion proteins displayed the same ratio as their native counterparts, confirming that yeast surface display is suitable for the high-throughput directed evolution of GOx using flow cytometry for selection. Aga2-GOx fusion proteins in the yeast cell wall could also be used as immobilized catalysts for the production of gluconic acid.",
publisher = "Academic Press Inc Elsevier Science, San Diego",
journal = "Protein Expression and Purification",
title = "Yeast surface display for the expression, purification and characterization of wild-type and B11 mutant glucose oxidases",
volume = "89",
number = "2",
pages = "175-180",
doi = "10.1016/j.pep.2013.03.014",
url = "Kon_2477"
}
Blažić, M., Kovačević, G., Prodanović, O., Ostafe, R., Gavrović-Jankulović, M., Fischer, R.,& Prodanović, R.. (2013). Yeast surface display for the expression, purification and characterization of wild-type and B11 mutant glucose oxidases. in Protein Expression and Purification
Academic Press Inc Elsevier Science, San Diego., 89(2), 175-180.
https://doi.org/10.1016/j.pep.2013.03.014
Kon_2477
Blažić M, Kovačević G, Prodanović O, Ostafe R, Gavrović-Jankulović M, Fischer R, Prodanović R. Yeast surface display for the expression, purification and characterization of wild-type and B11 mutant glucose oxidases. in Protein Expression and Purification. 2013;89(2):175-180.
doi:10.1016/j.pep.2013.03.014
Kon_2477 .
Blažić, Marija, Kovačević, Gordana, Prodanović, Olivera, Ostafe, Raluca, Gavrović-Jankulović, Marija, Fischer, Rainer, Prodanović, Radivoje, "Yeast surface display for the expression, purification and characterization of wild-type and B11 mutant glucose oxidases" in Protein Expression and Purification, 89, no. 2 (2013):175-180,
https://doi.org/10.1016/j.pep.2013.03.014 .,
Kon_2477 .
24
24
27

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
UR  - Kon_2356
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",
url = "Kon_2356"
}
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
Kon_2356
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
Kon_2356 .
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 .,
Kon_2356 .
22
20
19