Ostafe, Raluca

Link to this page

Authority KeyName Variants
orcid::0000-0003-0479-152757a2f6db4
  • Ostafe, Raluca (18)
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 Ministry of Education, Science and Technological Development, Republic of Serbia, Grant no. 451-03-68/2020-14/200168 (University of Belgrade, Faculty of Chemistry)
Excellence Initiative by the German federal government Harvard MRSEC [DMR-0820484]
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) Cluster of Excellence “Tailor-made Fuels from Biomass”.
DAAD bilateral project 451-03-01038/2015-09/21 Deutsche Forschungsgemeinschaft (DFG)
Engineering and Physical Sciences Research Council [EP/C535456/1] EPSRC [EP/C535456/1]
Fulbright Foundation Fulbright Foundation.
Fulbright Foundation [N0009552407] 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.
internal program MEF from the Fraunhofer Gesellschaft [125-600156] National Institute of Health [R01 EB014703, P01GM096971]
National Science Foundation [DMR-1310266] National Science Foundation (NSF) [DMR-1310266]
National Science Foundation (NSF) (DMR-1310266) and Harvard MRSEC (DMR-0820484). National Science Foundation under NSF Award [ECS-0335765]
National Science Foundation under NSF Award No. ECS-0335765. NSERC PGS D
RWTH Aachen University RWTH Aachen University.
US Office of Naval Research (ONR) [N00014-03-1-0026]

Author's Bibliography

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

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

(Springer, 2021)

TY  - JOUR
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/123456789/4101
AB  - The enzymatic degradation of azo dyes is a promising alternative to ineffective chemical and physical remediation methods. Lignin peroxidase (LiP) from Phanerochaete chrysosporium is a heme-containing lignin-degrading oxidoreductase that catalyzes the peroxide-dependent oxidation of diverse molecules, including industrial dyes. This enzyme is therefore ideal as a starting point for protein engineering. Accordingly, we subjected two positions (165 and 264) in the environment of the catalytic Trp171 residue to saturation mutagenesis, and the resulting library of 104 independent clones was expressed on the surface of yeast cells. This yeast display library was used for the selection of variants with the ability to break down structurally-distinct azo dyes more efficiently. We identified mutants with up to 10-fold greater affinity than wild-type LiP for three diverse azo dyes (Evans blue, amido black 10B and Guinea green) and up to 13-fold higher catalytic activity. Additionally, cell wall fragments displaying mutant LiP enzymes were prepared by toluene-induced cell lysis, achieving significant increases in both enzyme activity and stability compared to a whole-cell biocatalyst. LiP-coated cell wall fragments retained their initial dye degradation activity after 10 reaction cycles each lasting 8 h. The best-performing mutants removed up to 2.5-fold more of each dye than the wild-type LiP in multiple reaction cycles.
PB  - Springer
T2  - Frontiers of Environmental Science & Engineering
T2  - Frontiers of Environmental Science & EngineeringFront. Environ. Sci. Eng.
T1  - 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
VL  - 15
IS  - 2
SP  - 19
DO  - 10.1007/s11783-020-1311-4
ER  - 
@article{
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",
url = "http://cherry.chem.bg.ac.rs/handle/123456789/4101",
abstract = "The enzymatic degradation of azo dyes is a promising alternative to ineffective chemical and physical remediation methods. Lignin peroxidase (LiP) from Phanerochaete chrysosporium is a heme-containing lignin-degrading oxidoreductase that catalyzes the peroxide-dependent oxidation of diverse molecules, including industrial dyes. This enzyme is therefore ideal as a starting point for protein engineering. Accordingly, we subjected two positions (165 and 264) in the environment of the catalytic Trp171 residue to saturation mutagenesis, and the resulting library of 104 independent clones was expressed on the surface of yeast cells. This yeast display library was used for the selection of variants with the ability to break down structurally-distinct azo dyes more efficiently. We identified mutants with up to 10-fold greater affinity than wild-type LiP for three diverse azo dyes (Evans blue, amido black 10B and Guinea green) and up to 13-fold higher catalytic activity. Additionally, cell wall fragments displaying mutant LiP enzymes were prepared by toluene-induced cell lysis, achieving significant increases in both enzyme activity and stability compared to a whole-cell biocatalyst. LiP-coated cell wall fragments retained their initial dye degradation activity after 10 reaction cycles each lasting 8 h. The best-performing mutants removed up to 2.5-fold more of each dye than the wild-type LiP in multiple reaction cycles.",
publisher = "Springer",
journal = "Frontiers of Environmental Science & Engineering, Frontiers of Environmental Science & EngineeringFront. Environ. Sci. Eng.",
title = "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",
volume = "15",
number = "2",
pages = "19",
doi = "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). 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.
Frontiers of Environmental Science & EngineeringFront. Environ. Sci. Eng.
Springer., 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. 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. Frontiers of Environmental Science & EngineeringFront. Environ. Sci. Eng.. 2021;15(2):19
Ilić Đurđić Karla, Ostafe Raluca, Prodanović Olivera, Đurđević Đelmaš Aleksandra, Popović Nikolina, Fischer Rainer, Schillberg Stefan, Prodanović Radivoje, "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" Frontiers of Environmental Science & EngineeringFront. Environ. Sci. Eng., 15, no. 2 (2021):19,
https://doi.org/10.1007/s11783-020-1311-4 .
4
2

A High-Throughput Screening System Based on Fluorescence-Activated Cell Sorting for the Directed Evolution of Chitinase A

Menghiu, Gheorghita; Ostafe, Vasile; Prodanović, Radivoje; Fischer, Rainer; Ostafe, Raluca

(MDPI, 2021)

TY  - JOUR
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/123456789/4393
AB  - Chitinases catalyze the degradation of chitin, a polymer of N-acetylglucosamine found in crustacean shells, insect cuticles, and fungal cell walls. There is great interest in the development of improved chitinases to address the environmental burden of chitin waste from the food processing industry as well as the potential medical, agricultural, and industrial uses of partially deacetylated chitin (chitosan) and its products (chito-oligosaccharides). The depolymerization of chitin can be achieved using chemical and physical treatments, but an enzymatic process would be more environmentally friendly and more sustainable. However, chitinases are slow-acting enzymes, limiting their biotechnological exploitation, although this can be overcome by molecular evolution approaches to enhance the features required for specific applications. The two main goals of this study were the development of a high-throughput screening system for chitinase activity (which could be extrapolated to other hydrolytic enzymes), and the deployment of this new method to select improved chitinase variants. We therefore cloned and expressed the Bacillus licheniformis DSM8785 chitinase A (chiA) gene in Escherichia coli BL21 (DE3) cells and generated a mutant library by error-prone PCR. We then developed a screening method based on fluorescence-activated cell sorting (FACS) using the model substrate 4-methylumbelliferyl β-d-N,N′,N″-triacetyl chitotrioside to identify improved enzymes. We prevented cross-talk between emulsion compartments caused by the hydrophobicity of 4-methylumbelliferone, the fluorescent product of the enzymatic reaction, by incorporating cyclodextrins into the aqueous phases. We also addressed the toxicity of long-term chiA expression in E. coli by limiting the reaction time. We identified 12 mutants containing 2–8 mutations per gene resulting in up to twofold higher activity than wild-type ChiA.
PB  - MDPI
T2  - International Journal of Molecular Sciences
T1  - A High-Throughput Screening System Based on Fluorescence-Activated Cell Sorting for the Directed Evolution of Chitinase A
VL  - 22
IS  - 6
SP  - 3041
DO  - 10.3390/ijms22063041
ER  - 
@article{
author = "Menghiu, Gheorghita and Ostafe, Vasile and Prodanović, Radivoje and Fischer, Rainer and Ostafe, Raluca",
year = "2021",
url = "https://www.mdpi.com/1422-0067/22/6/3041, http://cherry.chem.bg.ac.rs/handle/123456789/4393",
abstract = "Chitinases catalyze the degradation of chitin, a polymer of N-acetylglucosamine found in crustacean shells, insect cuticles, and fungal cell walls. There is great interest in the development of improved chitinases to address the environmental burden of chitin waste from the food processing industry as well as the potential medical, agricultural, and industrial uses of partially deacetylated chitin (chitosan) and its products (chito-oligosaccharides). The depolymerization of chitin can be achieved using chemical and physical treatments, but an enzymatic process would be more environmentally friendly and more sustainable. However, chitinases are slow-acting enzymes, limiting their biotechnological exploitation, although this can be overcome by molecular evolution approaches to enhance the features required for specific applications. The two main goals of this study were the development of a high-throughput screening system for chitinase activity (which could be extrapolated to other hydrolytic enzymes), and the deployment of this new method to select improved chitinase variants. We therefore cloned and expressed the Bacillus licheniformis DSM8785 chitinase A (chiA) gene in Escherichia coli BL21 (DE3) cells and generated a mutant library by error-prone PCR. We then developed a screening method based on fluorescence-activated cell sorting (FACS) using the model substrate 4-methylumbelliferyl β-d-N,N′,N″-triacetyl chitotrioside to identify improved enzymes. We prevented cross-talk between emulsion compartments caused by the hydrophobicity of 4-methylumbelliferone, the fluorescent product of the enzymatic reaction, by incorporating cyclodextrins into the aqueous phases. We also addressed the toxicity of long-term chiA expression in E. coli by limiting the reaction time. We identified 12 mutants containing 2–8 mutations per gene resulting in up to twofold higher activity than wild-type ChiA.",
publisher = "MDPI",
journal = "International Journal of Molecular Sciences",
title = "A High-Throughput Screening System Based on Fluorescence-Activated Cell Sorting for the Directed Evolution of Chitinase A",
volume = "22",
number = "6",
pages = "3041",
doi = "10.3390/ijms22063041"
}
Menghiu, G., Ostafe, V., Prodanović, R., Fischer, R.,& Ostafe, R. (2021). A High-Throughput Screening System Based on Fluorescence-Activated Cell Sorting for the Directed Evolution of Chitinase A.
International Journal of Molecular Sciences
MDPI., 22(6), 3041.
https://doi.org/10.3390/ijms22063041
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
Menghiu Gheorghita, Ostafe Vasile, Prodanović Radivoje, Fischer Rainer, Ostafe Raluca, "A High-Throughput Screening System Based on Fluorescence-Activated Cell Sorting for the Directed Evolution of Chitinase A" International Journal of Molecular Sciences, 22, no. 6 (2021):3041,
https://doi.org/10.3390/ijms22063041 .

Immobilization of yeast cell walls with surface displayed laccase from Streptomyces cyaneus within dopamine-alginate beads for dye decolorization

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

(2021)

TY  - JOUR
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/123456789/4404
AB  - High amounts of toxic textile dyes are released into the environment due to coloring and wastewaters treatment processes' inefficiency. To remove dyes from the environment and wastewaters, researchers focused on applying immobilized enzymes due to mild reaction conditions and enzyme nontoxicity. Laccases are oxidases with wide substrate specificity, capable of degradation of many different dye types. Laccase from Streptomyces cyaneus was expressed on the surface of Saccharomyces cerevisiae EBY100 cells. The specific activity of surface-displayed laccase was increased by toluene-induced lysis to 3.1 U/g of cell walls. For cell wall laccase immobilization within hydrogel beads, alginate was modified by dopamine using periodate oxidation and reductive amination and characterized by UV–Vis, FTIR, and NMR spectroscopy. Cell wall laccase was immobilized within alginate and dopamine-alginate beads additionally cross-linked by oxygen and laccase. The immobilized enzyme's specific activity was two times higher using dopamine-alginate compared to native alginate beads, and immobilization yield increased 16 times. Cell wall laccase immobilized within dopamine-alginate beads decolorized Amido Black 10B, Reactive Black 5, Evans Blue, and Remazol Brilliant Blue with 100% efficiency and after ten rounds of multiple-use retained decolorization efficiency of 90% with Evans Blue and 61% with Amido Black.
T2  - International Journal of Biological Macromolecules
T1  - Immobilization of yeast cell walls with surface displayed laccase from Streptomyces cyaneus within dopamine-alginate beads for dye decolorization
VL  - 181
SP  - 1072
EP  - 1080
DO  - 10.1016/j.ijbiomac.2021.04.115
ER  - 
@article{
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",
url = "https://www.sciencedirect.com/science/article/pii/S0141813021008813, http://cherry.chem.bg.ac.rs/handle/123456789/4404",
abstract = "High amounts of toxic textile dyes are released into the environment due to coloring and wastewaters treatment processes' inefficiency. To remove dyes from the environment and wastewaters, researchers focused on applying immobilized enzymes due to mild reaction conditions and enzyme nontoxicity. Laccases are oxidases with wide substrate specificity, capable of degradation of many different dye types. Laccase from Streptomyces cyaneus was expressed on the surface of Saccharomyces cerevisiae EBY100 cells. The specific activity of surface-displayed laccase was increased by toluene-induced lysis to 3.1 U/g of cell walls. For cell wall laccase immobilization within hydrogel beads, alginate was modified by dopamine using periodate oxidation and reductive amination and characterized by UV–Vis, FTIR, and NMR spectroscopy. Cell wall laccase was immobilized within alginate and dopamine-alginate beads additionally cross-linked by oxygen and laccase. The immobilized enzyme's specific activity was two times higher using dopamine-alginate compared to native alginate beads, and immobilization yield increased 16 times. Cell wall laccase immobilized within dopamine-alginate beads decolorized Amido Black 10B, Reactive Black 5, Evans Blue, and Remazol Brilliant Blue with 100% efficiency and after ten rounds of multiple-use retained decolorization efficiency of 90% with Evans Blue and 61% with Amido Black.",
journal = "International Journal of Biological Macromolecules",
title = "Immobilization of yeast cell walls with surface displayed laccase from Streptomyces cyaneus within dopamine-alginate beads for dye decolorization",
volume = "181",
pages = "1072-1080",
doi = "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). Immobilization of yeast cell walls with surface displayed laccase from Streptomyces cyaneus within dopamine-alginate beads for dye decolorization.
International Journal of Biological Macromolecules, 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. 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
Popović Nikolina, Pržulj Dunja, Mladenović Maja, Prodanović Olivera, Ece Selin, Ilić Đurđić Karla, Ostafe Raluca, Fischer Rainer, Prodanović Radivoje, "Immobilization of yeast cell walls with surface displayed laccase from Streptomyces cyaneus within dopamine-alginate beads for dye decolorization" International Journal of Biological Macromolecules, 181 (2021):1072-1080,
https://doi.org/10.1016/j.ijbiomac.2021.04.115 .
3

Immobilization of yeast cell walls with surface displayed laccase from Streptomyces cyaneus within dopamine-alginate beads for dye decolorization

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

(2021)

TY  - JOUR
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/123456789/4406
AB  - High amounts of toxic textile dyes are released into the environment due to coloring and wastewaters treatment processes' inefficiency. To remove dyes from the environment and wastewaters, researchers focused on applying immobilized enzymes due to mild reaction conditions and enzyme nontoxicity. Laccases are oxidases with wide substrate specificity, capable of degradation of many different dye types. Laccase from Streptomyces cyaneus was expressed on the surface of Saccharomyces cerevisiae EBY100 cells. The specific activity of surface-displayed laccase was increased by toluene-induced lysis to 3.1 U/g of cell walls. For cell wall laccase immobilization within hydrogel beads, alginate was modified by dopamine using periodate oxidation and reductive amination and characterized by UV–Vis, FTIR, and NMR spectroscopy. Cell wall laccase was immobilized within alginate and dopamine-alginate beads additionally cross-linked by oxygen and laccase. The immobilized enzyme's specific activity was two times higher using dopamine-alginate compared to native alginate beads, and immobilization yield increased 16 times. Cell wall laccase immobilized within dopamine-alginate beads decolorized Amido Black 10B, Reactive Black 5, Evans Blue, and Remazol Brilliant Blue with 100% efficiency and after ten rounds of multiple-use retained decolorization efficiency of 90% with Evans Blue and 61% with Amido Black.
T2  - International Journal of Biological Macromolecules
T1  - Immobilization of yeast cell walls with surface displayed laccase from Streptomyces cyaneus within dopamine-alginate beads for dye decolorization
VL  - 181
SP  - 1072
EP  - 1080
DO  - 10.1016/j.ijbiomac.2021.04.115
ER  - 
@article{
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",
url = "https://www.sciencedirect.com/science/article/pii/S0141813021008813, http://cherry.chem.bg.ac.rs/handle/123456789/4406",
abstract = "High amounts of toxic textile dyes are released into the environment due to coloring and wastewaters treatment processes' inefficiency. To remove dyes from the environment and wastewaters, researchers focused on applying immobilized enzymes due to mild reaction conditions and enzyme nontoxicity. Laccases are oxidases with wide substrate specificity, capable of degradation of many different dye types. Laccase from Streptomyces cyaneus was expressed on the surface of Saccharomyces cerevisiae EBY100 cells. The specific activity of surface-displayed laccase was increased by toluene-induced lysis to 3.1 U/g of cell walls. For cell wall laccase immobilization within hydrogel beads, alginate was modified by dopamine using periodate oxidation and reductive amination and characterized by UV–Vis, FTIR, and NMR spectroscopy. Cell wall laccase was immobilized within alginate and dopamine-alginate beads additionally cross-linked by oxygen and laccase. The immobilized enzyme's specific activity was two times higher using dopamine-alginate compared to native alginate beads, and immobilization yield increased 16 times. Cell wall laccase immobilized within dopamine-alginate beads decolorized Amido Black 10B, Reactive Black 5, Evans Blue, and Remazol Brilliant Blue with 100% efficiency and after ten rounds of multiple-use retained decolorization efficiency of 90% with Evans Blue and 61% with Amido Black.",
journal = "International Journal of Biological Macromolecules",
title = "Immobilization of yeast cell walls with surface displayed laccase from Streptomyces cyaneus within dopamine-alginate beads for dye decolorization",
volume = "181",
pages = "1072-1080",
doi = "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). Immobilization of yeast cell walls with surface displayed laccase from Streptomyces cyaneus within dopamine-alginate beads for dye decolorization.
International Journal of Biological Macromolecules, 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. 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
Popović Nikolina, Pržulj Dunja, Mladenović Maja, Prodanović Olivera, Ece Selin, Ilić Đurđić Karla, Ostafe Raluca, Fischer Rainer, Prodanović Radivoje, "Immobilization of yeast cell walls with surface displayed laccase from Streptomyces cyaneus within dopamine-alginate beads for dye decolorization" International Journal of Biological Macromolecules, 181 (2021):1072-1080,
https://doi.org/10.1016/j.ijbiomac.2021.04.115 .
3

Flow cytometry-based system for screening of lignin peroxidase mutants with higher oxidative stability

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

(Elsevier, 2020)

TY  - JOUR
AU  - Ilić Đurđić, Karla
AU  - Ece, Selin
AU  - Ostafe, Raluca
AU  - Vogel, Simon
AU  - Balaž, Ana Marija
AU  - Schillberg, Stefan
AU  - Fischer, Rainer
AU  - Prodanović, Radivoje
PY  - 2020
UR  - http://cherry.chem.bg.ac.rs/handle/123456789/3974
AB  - Lignin peroxidase (LiP) is a heme-containing oxidoreductase that oxidizes structurally diverse substrates in an H2O2-dependent manner. Its ability to oxidize many pollutants makes it suitable for bioremediation applications and an ideal candidate for optimization by mutagenesis and selection. In order to increase oxidative stability of LiP we generated a random mutagenesis library comprising 106 mutated LiP genes and screened for expressed enzymes with higher than wild-type activity after incubation in 30 mM H2O2 by flow cytometry with fluorescein-tyramide as a substrate. To preserve the genotype-phenotype connection, the LiP mutants were displayed on the yeast cell surface. Two rounds of sorting were performed, recovered colonies were then screened in microtiter plates, and activity analysis revealed a significant increase in the percentage of cells expressing LiP variants with higher oxidative stability than wtLiP. Two rounds of sorting increased the proportion of more-stable variants from 1.4% in the original library to 52.3%. The most stable variants after two rounds of sorting featured between two and four mutations and retained up to 80% of initial activity after 1 h incubation in 30 mM H2O2. We for the first-time applied flow cytometry for screening of any ligninolytic peroxidase library. Obtained results suggest that developed system may be applied for improvement of industrially important characteristics of lignin peroxidase.
PB  - Elsevier
T2  - Journal of Bioscience and Bioengineering
T1  - Flow cytometry-based system for screening of lignin peroxidase mutants with higher oxidative stability
VL  - 129
IS  - 6
SP  - 664
EP  - 671
DO  - 10.1016/j.jbiosc.2019.12.009
ER  - 
@article{
author = "Ilić Đurđić, Karla and Ece, Selin and Ostafe, Raluca and Vogel, Simon and Balaž, Ana Marija and Schillberg, Stefan and Fischer, Rainer and Prodanović, Radivoje",
year = "2020",
url = "http://cherry.chem.bg.ac.rs/handle/123456789/3974",
abstract = "Lignin peroxidase (LiP) is a heme-containing oxidoreductase that oxidizes structurally diverse substrates in an H2O2-dependent manner. Its ability to oxidize many pollutants makes it suitable for bioremediation applications and an ideal candidate for optimization by mutagenesis and selection. In order to increase oxidative stability of LiP we generated a random mutagenesis library comprising 106 mutated LiP genes and screened for expressed enzymes with higher than wild-type activity after incubation in 30 mM H2O2 by flow cytometry with fluorescein-tyramide as a substrate. To preserve the genotype-phenotype connection, the LiP mutants were displayed on the yeast cell surface. Two rounds of sorting were performed, recovered colonies were then screened in microtiter plates, and activity analysis revealed a significant increase in the percentage of cells expressing LiP variants with higher oxidative stability than wtLiP. Two rounds of sorting increased the proportion of more-stable variants from 1.4% in the original library to 52.3%. The most stable variants after two rounds of sorting featured between two and four mutations and retained up to 80% of initial activity after 1 h incubation in 30 mM H2O2. We for the first-time applied flow cytometry for screening of any ligninolytic peroxidase library. Obtained results suggest that developed system may be applied for improvement of industrially important characteristics of lignin peroxidase.",
publisher = "Elsevier",
journal = "Journal of Bioscience and Bioengineering",
title = "Flow cytometry-based system for screening of lignin peroxidase mutants with higher oxidative stability",
volume = "129",
number = "6",
pages = "664-671",
doi = "10.1016/j.jbiosc.2019.12.009"
}
Ilić Đurđić, K., Ece, S., Ostafe, R., Vogel, S., Balaž, A. M., Schillberg, S., Fischer, R.,& Prodanović, R. (2020). Flow cytometry-based system for screening of lignin peroxidase mutants with higher oxidative stability.
Journal of Bioscience and Bioengineering
Elsevier., 129(6), 664-671.
https://doi.org/10.1016/j.jbiosc.2019.12.009
Ilić Đurđić K, Ece S, Ostafe R, Vogel S, Balaž AM, Schillberg S, Fischer R, Prodanović R. Flow cytometry-based system for screening of lignin peroxidase mutants with higher oxidative stability. Journal of Bioscience and Bioengineering. 2020;129(6):664-671
Ilić Đurđić Karla, Ece Selin, Ostafe Raluca, Vogel Simon, Balaž Ana Marija, Schillberg Stefan, Fischer Rainer, Prodanović Radivoje, "Flow cytometry-based system for screening of lignin peroxidase mutants with higher oxidative stability" Journal of Bioscience and Bioengineering, 129, no. 6 (2020):664-671,
https://doi.org/10.1016/j.jbiosc.2019.12.009 .
4
1

Semi-rational design of cellobiose dehydrogenase for increased stability in the presence of peroxide

Balaž, Ana Marija; Stevanović, Jelena; Ostafe, Raluca; Blazić, Marija; Ilić Đurđić, Karla; Fischer, Rainer; Prodanović, Radivoje

(2020)

TY  - JOUR
AU  - Balaž, Ana Marija
AU  - Stevanović, Jelena
AU  - Ostafe, Raluca
AU  - Blazić, Marija
AU  - Ilić Đurđić, Karla
AU  - Fischer, Rainer
AU  - Prodanović, Radivoje
PY  - 2020
UR  - http://cherry.chem.bg.ac.rs/handle/123456789/4011
AB  - Cellobiose dehydrogenase (CDH, EC 1.1.99.18) from white rot fungi Phanerochaete chrysosporium can be used for constructing biosensors and biofuel cells, for bleaching cotton in textile industry, and recently, the enzyme has found an important application in biomedicine as an antimicrobial and antibiofilm agent. Stability and activity of the wild-type (wt) CDH and mutants at methionine residues in the presence of hydrogen peroxide were investigated. Saturation mutagenesis libraries were made at the only methionine in heme domain M65 and two methionines M685 and M738 in the flavin domain that were closest to the active site. After screening the libraries, three mutants with increased activity and stability in the presence of peroxide were found, M65F with 70% of residual activity after 6 h of incubation in 0.3 M hydrogen peroxide, M738S with 80% of residual activity and M685Y with over 90% of residual activity compared to wild-type CDH that retained 40% of original activity. Combined mutants showed no activity. The most stable mutant M685Y with 5.8 times increased half-life in the presence of peroxide showed also 2.5 times increased kcat for lactose compared to wtCDH and could be good candidate for applications in biofuel cells and biocatalysis for lactobionic acid production.
T2  - Molecular Diversity
T1  - Semi-rational design of cellobiose dehydrogenase for increased stability in the presence of peroxide
VL  - 24
IS  - 3
SP  - 593
EP  - 601
DO  - 10.1007/s11030-019-09965-0
ER  - 
@article{
author = "Balaž, Ana Marija and Stevanović, Jelena and Ostafe, Raluca and Blazić, Marija and Ilić Đurđić, Karla and Fischer, Rainer and Prodanović, Radivoje",
year = "2020",
url = "http://cherry.chem.bg.ac.rs/handle/123456789/4011",
abstract = "Cellobiose dehydrogenase (CDH, EC 1.1.99.18) from white rot fungi Phanerochaete chrysosporium can be used for constructing biosensors and biofuel cells, for bleaching cotton in textile industry, and recently, the enzyme has found an important application in biomedicine as an antimicrobial and antibiofilm agent. Stability and activity of the wild-type (wt) CDH and mutants at methionine residues in the presence of hydrogen peroxide were investigated. Saturation mutagenesis libraries were made at the only methionine in heme domain M65 and two methionines M685 and M738 in the flavin domain that were closest to the active site. After screening the libraries, three mutants with increased activity and stability in the presence of peroxide were found, M65F with 70% of residual activity after 6 h of incubation in 0.3 M hydrogen peroxide, M738S with 80% of residual activity and M685Y with over 90% of residual activity compared to wild-type CDH that retained 40% of original activity. Combined mutants showed no activity. The most stable mutant M685Y with 5.8 times increased half-life in the presence of peroxide showed also 2.5 times increased kcat for lactose compared to wtCDH and could be good candidate for applications in biofuel cells and biocatalysis for lactobionic acid production.",
journal = "Molecular Diversity",
title = "Semi-rational design of cellobiose dehydrogenase for increased stability in the presence of peroxide",
volume = "24",
number = "3",
pages = "593-601",
doi = "10.1007/s11030-019-09965-0"
}
Balaž, A. M., Stevanović, J., Ostafe, R., Blazić, M., Ilić Đurđić, K., Fischer, R.,& Prodanović, R. (2020). Semi-rational design of cellobiose dehydrogenase for increased stability in the presence of peroxide.
Molecular Diversity, 24(3), 593-601.
https://doi.org/10.1007/s11030-019-09965-0
Balaž AM, Stevanović J, Ostafe R, Blazić M, Ilić Đurđić K, Fischer R, Prodanović R. Semi-rational design of cellobiose dehydrogenase for increased stability in the presence of peroxide. Molecular Diversity. 2020;24(3):593-601
Balaž Ana Marija, Stevanović Jelena, Ostafe Raluca, Blazić Marija, Ilić Đurđić Karla, Fischer Rainer, Prodanović Radivoje, "Semi-rational design of cellobiose dehydrogenase for increased stability in the presence of peroxide" Molecular Diversity, 24, no. 3 (2020):593-601,
https://doi.org/10.1007/s11030-019-09965-0 .
4
2

Expression, purification and characterization of cellobiose dehydrogenase mutants from Phanerochaete chrysosporium in Pichia pastoris KM71H strain

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

(Belgrade : Serbian Chemical Society, 2020)

TY  - JOUR
AU  - Balaž, Ana Marija
AU  - Blažić, Marija
AU  - Popović, Nikolina
AU  - Prodanović, Olivera
AU  - Ostafe, Raluca
AU  - Fischer, Rainer
AU  - Prodanović, Radivoje
PY  - 2020
UR  - http://cherry.chem.bg.ac.rs/handle/123456789/4270
AB  - Production of soluble cellobiose dehydrogenase (CDH) mutant proteins previously evolved on the surface of S. cerevisiae yeast cells was established for use in biosensors and biofuel cells. For this purpose, mutant cdh genes tm (D20N, A64T, V592M), H5 (D20N, V22A, A64T, V592M) and H9 (D20N, A64T, T84A, A261P, V592M, E674G, N715S) were cloned to pPICZα plasmid and transformed into Pichia pastoris KM71H strain for high expression in a soluble form and kinetic characterization. After 6 days of expression under methanol induction, the CDHs were purified by ultrafiltration, ion- -exchange chromatography and gel filtration. Sodium dodecyl sulfate electrophoresis confirmed the purity and presence of a single protein band at a molecular weight of 100 kDa. Kinetic characterization showed that the H5 mutant had the highest catalytic constant of 43.5 s-1 for lactose, while the mutant H9 showed the highest specificity constant for lactose of 132 mM-1 s-1. All three mutant proteins did not change the pH optimum that was between 4.5 and 5.5. Compared to the previously obtained wild types and mutants of CDH from Phanerochaete chrysosporium, the variants reported in this article had higher activity and specificity that together with high protein expression rate in P. pastoris, makes them good candidates for use in biotechnology for lactobionic acid production and biosensor manufacture.
AB  - У циљу употребе у биосензорима и биогоривним ћелијама, успостављена је производњарастворних облика целобиоза дехидрогеназе (CDH) претходно еволуираних на површиниквашчевих ћелија S. cerevisiae. У ту сврху су мутанти CDH, tm (D20N, A64T, V592M), H5(D20N, V22A, A64T, V592M) и H9 (D20N, A64T, T84A, A261P, V592M, E674G, N715S)клонирани у pPICZα плазмид и трансформисани у Pichia pastoris KM71H сој за високуекспресију у растворном облику и кинетичку карактеризацију. После 6 дана експресије подиндукцијом метанолом, мутанти су пречишћени ултрафилтрацијом, јоноизмењивачкомхроматографијом и гел-филтрацијом. SDS електрофореза је потврдила чистоћу уз присуствоједне протеинске траке молекулскe масe од 100 kDa. Кинетичка карактеризација је показалада H5 мутирани протеин поседује највећу каталитичку константу од 43,5 s-1 за лактозу, докје H9 имао највећу константу специфичности за лактозу од 132 mM-1 s-1. Сва три мутиранапротеина су имала неизмењен pH оптимум који је био у опсегу од 4,5 до 5,5. У поређењу сапретходно добијеним природним и мутантним облицима CDH протеина из Phanerochaetechrysosporium, облици приказани у овом раду имају већу активност и специфичност, што их,повезано са високом експресијом протеина у P. Pastoris, чини добрим кандидатима за упо-требу у биотехнологији за производњу лактобионске киселине и биосензора.
PB  - Belgrade : Serbian Chemical Society
T2  - Journal of the Serbian Chemical Society
T1  - Expression, purification and characterization of cellobiose dehydrogenase mutants from Phanerochaete chrysosporium in Pichia pastoris KM71H strain
T1  - Ekspresija, prečišćavanje i karakterizacija mutanata celobioza - dehidrogenaze iz Phanerochaete chrysosporium u Pichia pastoris KM71H soju
VL  - 85
IS  - 1
SP  - 25
EP  - 35
DO  - 10.2298/JSC190320058B
ER  - 
@article{
author = "Balaž, Ana Marija and Blažić, Marija and Popović, Nikolina and Prodanović, Olivera and Ostafe, Raluca and Fischer, Rainer and Prodanović, Radivoje",
year = "2020",
url = "http://cherry.chem.bg.ac.rs/handle/123456789/4270",
abstract = "Production of soluble cellobiose dehydrogenase (CDH) mutant proteins previously evolved on the surface of S. cerevisiae yeast cells was established for use in biosensors and biofuel cells. For this purpose, mutant cdh genes tm (D20N, A64T, V592M), H5 (D20N, V22A, A64T, V592M) and H9 (D20N, A64T, T84A, A261P, V592M, E674G, N715S) were cloned to pPICZα plasmid and transformed into Pichia pastoris KM71H strain for high expression in a soluble form and kinetic characterization. After 6 days of expression under methanol induction, the CDHs were purified by ultrafiltration, ion- -exchange chromatography and gel filtration. Sodium dodecyl sulfate electrophoresis confirmed the purity and presence of a single protein band at a molecular weight of 100 kDa. Kinetic characterization showed that the H5 mutant had the highest catalytic constant of 43.5 s-1 for lactose, while the mutant H9 showed the highest specificity constant for lactose of 132 mM-1 s-1. All three mutant proteins did not change the pH optimum that was between 4.5 and 5.5. Compared to the previously obtained wild types and mutants of CDH from Phanerochaete chrysosporium, the variants reported in this article had higher activity and specificity that together with high protein expression rate in P. pastoris, makes them good candidates for use in biotechnology for lactobionic acid production and biosensor manufacture., У циљу употребе у биосензорима и биогоривним ћелијама, успостављена је производњарастворних облика целобиоза дехидрогеназе (CDH) претходно еволуираних на површиниквашчевих ћелија S. cerevisiae. У ту сврху су мутанти CDH, tm (D20N, A64T, V592M), H5(D20N, V22A, A64T, V592M) и H9 (D20N, A64T, T84A, A261P, V592M, E674G, N715S)клонирани у pPICZα плазмид и трансформисани у Pichia pastoris KM71H сој за високуекспресију у растворном облику и кинетичку карактеризацију. После 6 дана експресије подиндукцијом метанолом, мутанти су пречишћени ултрафилтрацијом, јоноизмењивачкомхроматографијом и гел-филтрацијом. SDS електрофореза је потврдила чистоћу уз присуствоједне протеинске траке молекулскe масe од 100 kDa. Кинетичка карактеризација је показалада H5 мутирани протеин поседује највећу каталитичку константу од 43,5 s-1 за лактозу, докје H9 имао највећу константу специфичности за лактозу од 132 mM-1 s-1. Сва три мутиранапротеина су имала неизмењен pH оптимум који је био у опсегу од 4,5 до 5,5. У поређењу сапретходно добијеним природним и мутантним облицима CDH протеина из Phanerochaetechrysosporium, облици приказани у овом раду имају већу активност и специфичност, што их,повезано са високом експресијом протеина у P. Pastoris, чини добрим кандидатима за упо-требу у биотехнологији за производњу лактобионске киселине и биосензора.",
publisher = "Belgrade : Serbian Chemical Society",
journal = "Journal of the Serbian Chemical Society",
title = "Expression, purification and characterization of cellobiose dehydrogenase mutants from Phanerochaete chrysosporium in Pichia pastoris KM71H strain, Ekspresija, prečišćavanje i karakterizacija mutanata celobioza - dehidrogenaze iz Phanerochaete chrysosporium u Pichia pastoris KM71H soju",
volume = "85",
number = "1",
pages = "25-35",
doi = "10.2298/JSC190320058B"
}
Balaž, A. M., Blažić, M., Popović, N., Prodanović, O., Ostafe, R., Fischer, R.,& Prodanović, R. (2020). Ekspresija, prečišćavanje i karakterizacija mutanata celobioza - dehidrogenaze iz Phanerochaete chrysosporium u Pichia pastoris KM71H soju.
Journal of the Serbian Chemical Society
Belgrade : Serbian Chemical Society., 85(1), 25-35.
https://doi.org/10.2298/JSC190320058B
Balaž AM, Blažić M, Popović N, Prodanović O, Ostafe R, Fischer R, Prodanović R. Ekspresija, prečišćavanje i karakterizacija mutanata celobioza - dehidrogenaze iz Phanerochaete chrysosporium u Pichia pastoris KM71H soju. Journal of the Serbian Chemical Society. 2020;85(1):25-35
Balaž Ana Marija, Blažić Marija, Popović Nikolina, Prodanović Olivera, Ostafe Raluca, Fischer Rainer, Prodanović Radivoje, "Ekspresija, prečišćavanje i karakterizacija mutanata celobioza - dehidrogenaze iz Phanerochaete chrysosporium u Pichia pastoris KM71H soju" Journal of the Serbian Chemical Society, 85, no. 1 (2020):25-35,
https://doi.org/10.2298/JSC190320058B .

Improvement in oxidative stability of versatile peroxidase by flow cytometry-based high-throughput screening system

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

(Elsevier, 2020)

TY  - JOUR
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/123456789/3888
AB  - Pleurotus eryngii wild-type versatile peroxidase (wtVP) oxidizes structurally diverse substrates in an H2O2-dependent manner, but its ability to oxidize many pollutants is limited by suicidal enzyme inactivation in the presence of excess H2O2. To address this drawback, we generated random mutagenesis libraries containing 3 × 106 mutated VP genes and screened for enzymes with higher oxidative stability expressed on the surface of yeast cells. This was achieved by flow cytometry using the substrate fluorescein tyramide. After two rounds of sorting, the percentage of cells expressing variants with improved oxidative stability had increased from 1 % to 56 %. The most stable variants featured 3–5 amino acid substitutions and retained up to 70 % of their initial activity after incubation for 1 h in 30 mM H2O2 (conditions that completely inactivate wtVP). Selected variants were extracted from yeast cell walls and purified for kinetic characterization. We also prepared yeast cell walls with wtVP and the three most stable VP variants for multiple cycles of azo dye (Reactive black 5) degradation. After 10 cycles of 12 h, two of the variants retained more than 97 % of their initial activity, whereas the activity of wtVP declined by ∼30 %. These results confirm that our high-throughput screening system can improve the oxidative stability of versatile peroxidase, providing a source of novel enzymes for remediation applications.
PB  - Elsevier
T2  - Biochemical Engineering Journal
T1  - Improvement in oxidative stability of versatile peroxidase by flow cytometry-based high-throughput screening system
VL  - 157
DO  - 10.1016/j.bej.2020.107555
ER  - 
@article{
author = "Ilić Đurđić, Karla and Ece, Selin and Ostafe, Raluca and Vogel, Simon and Schillberg, Stefan and Fischer, Rainer and Prodanović, Radivoje",
year = "2020",
url = "http://cherry.chem.bg.ac.rs/handle/123456789/3888",
abstract = "Pleurotus eryngii wild-type versatile peroxidase (wtVP) oxidizes structurally diverse substrates in an H2O2-dependent manner, but its ability to oxidize many pollutants is limited by suicidal enzyme inactivation in the presence of excess H2O2. To address this drawback, we generated random mutagenesis libraries containing 3 × 106 mutated VP genes and screened for enzymes with higher oxidative stability expressed on the surface of yeast cells. This was achieved by flow cytometry using the substrate fluorescein tyramide. After two rounds of sorting, the percentage of cells expressing variants with improved oxidative stability had increased from 1 % to 56 %. The most stable variants featured 3–5 amino acid substitutions and retained up to 70 % of their initial activity after incubation for 1 h in 30 mM H2O2 (conditions that completely inactivate wtVP). Selected variants were extracted from yeast cell walls and purified for kinetic characterization. We also prepared yeast cell walls with wtVP and the three most stable VP variants for multiple cycles of azo dye (Reactive black 5) degradation. After 10 cycles of 12 h, two of the variants retained more than 97 % of their initial activity, whereas the activity of wtVP declined by ∼30 %. These results confirm that our high-throughput screening system can improve the oxidative stability of versatile peroxidase, providing a source of novel enzymes for remediation applications.",
publisher = "Elsevier",
journal = "Biochemical Engineering Journal",
title = "Improvement in oxidative stability of versatile peroxidase by flow cytometry-based high-throughput screening system",
volume = "157",
doi = "10.1016/j.bej.2020.107555"
}
Ilić Đurđić, K., Ece, S., Ostafe, R., Vogel, S., Schillberg, S., Fischer, R.,& Prodanović, R. (2020). Improvement in oxidative stability of versatile peroxidase by flow cytometry-based high-throughput screening system.
Biochemical Engineering Journal
Elsevier., 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. Improvement in oxidative stability of versatile peroxidase by flow cytometry-based high-throughput screening system. Biochemical Engineering Journal. 2020;157
Ilić Đurđić Karla, Ece Selin, Ostafe Raluca, Vogel Simon, Schillberg Stefan, Fischer Rainer, Prodanović Radivoje, "Improvement in oxidative stability of versatile peroxidase by flow cytometry-based high-throughput screening system" Biochemical Engineering Journal, 157 (2020),
https://doi.org/10.1016/j.bej.2020.107555 .
1
1

Improvement in oxidative stability of versatile peroxidase by flow cytometry-based high-throughput screening system

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

(Elsevier, 2020)

TY  - JOUR
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/123456789/3898
AB  - Pleurotus eryngii wild-type versatile peroxidase (wtVP) oxidizes structurally diverse substrates in an H2O2-dependent manner, but its ability to oxidize many pollutants is limited by suicidal enzyme inactivation in the presence of excess H2O2. To address this drawback, we generated random mutagenesis libraries containing 3 × 106 mutated VP genes and screened for enzymes with higher oxidative stability expressed on the surface of yeast cells. This was achieved by flow cytometry using the substrate fluorescein tyramide. After two rounds of sorting, the percentage of cells expressing variants with improved oxidative stability had increased from 1 % to 56 %. The most stable variants featured 3–5 amino acid substitutions and retained up to 70 % of their initial activity after incubation for 1 h in 30 mM H2O2 (conditions that completely inactivate wtVP). Selected variants were extracted from yeast cell walls and purified for kinetic characterization. We also prepared yeast cell walls with wtVP and the three most stable VP variants for multiple cycles of azo dye (Reactive black 5) degradation. After 10 cycles of 12 h, two of the variants retained more than 97 % of their initial activity, whereas the activity of wtVP declined by ∼30 %. These results confirm that our high-throughput screening system can improve the oxidative stability of versatile peroxidase, providing a source of novel enzymes for remediation applications.
PB  - Elsevier
T2  - Biochemical Engineering Journal
T1  - Improvement in oxidative stability of versatile peroxidase by flow cytometry-based high-throughput screening system
VL  - 157
DO  - 10.1016/j.bej.2020.107555
ER  - 
@article{
author = "Ilić Đurđić, Karla and Ece, Selin and Ostafe, Raluca and Vogel, Simon and Schillberg, Stefan and Fischer, Rainer and Prodanović, Radivoje",
year = "2020",
url = "http://cherry.chem.bg.ac.rs/handle/123456789/3898",
abstract = "Pleurotus eryngii wild-type versatile peroxidase (wtVP) oxidizes structurally diverse substrates in an H2O2-dependent manner, but its ability to oxidize many pollutants is limited by suicidal enzyme inactivation in the presence of excess H2O2. To address this drawback, we generated random mutagenesis libraries containing 3 × 106 mutated VP genes and screened for enzymes with higher oxidative stability expressed on the surface of yeast cells. This was achieved by flow cytometry using the substrate fluorescein tyramide. After two rounds of sorting, the percentage of cells expressing variants with improved oxidative stability had increased from 1 % to 56 %. The most stable variants featured 3–5 amino acid substitutions and retained up to 70 % of their initial activity after incubation for 1 h in 30 mM H2O2 (conditions that completely inactivate wtVP). Selected variants were extracted from yeast cell walls and purified for kinetic characterization. We also prepared yeast cell walls with wtVP and the three most stable VP variants for multiple cycles of azo dye (Reactive black 5) degradation. After 10 cycles of 12 h, two of the variants retained more than 97 % of their initial activity, whereas the activity of wtVP declined by ∼30 %. These results confirm that our high-throughput screening system can improve the oxidative stability of versatile peroxidase, providing a source of novel enzymes for remediation applications.",
publisher = "Elsevier",
journal = "Biochemical Engineering Journal",
title = "Improvement in oxidative stability of versatile peroxidase by flow cytometry-based high-throughput screening system",
volume = "157",
doi = "10.1016/j.bej.2020.107555"
}
Ilić Đurđić, K., Ece, S., Ostafe, R., Vogel, S., Schillberg, S., Fischer, R.,& Prodanović, R. (2020). Improvement in oxidative stability of versatile peroxidase by flow cytometry-based high-throughput screening system.
Biochemical Engineering Journal
Elsevier., 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. Improvement in oxidative stability of versatile peroxidase by flow cytometry-based high-throughput screening system. Biochemical Engineering Journal. 2020;157
Ilić Đurđić Karla, Ece Selin, Ostafe Raluca, Vogel Simon, Schillberg Stefan, Fischer Rainer, Prodanović Radivoje, "Improvement in oxidative stability of versatile peroxidase by flow cytometry-based high-throughput screening system" Biochemical Engineering Journal, 157 (2020),
https://doi.org/10.1016/j.bej.2020.107555 .
1
1

A high.throughput screening system based on droplet microfluidics for glucose oxidase gene libraries

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

(MDPI, 2020)

TY  - JOUR
AU  - Prodanović, Radivoje
AU  - Lloyd Ung, W.
AU  - Ilić Đurđić, Karla
AU  - Fischer, Rainer
AU  - Weitz, David A.
AU  - Ostafe, Raluca
PY  - 2020
UR  - http://cherry.chem.bg.ac.rs/handle/123456789/3950
AB  - Glucose oxidase (GOx) is an important industrial enzyme that can be optimized for specific applications by mutagenesis and activity.based screening. To increase the efficiency of this approach, we have developed a new ultrahigh.throughput screening platform based on a microfluidic lab.on.chip device that allows the sorting of GOx mutants from a saturation mutagenesis library expressed on the surface of yeast cells. GOx activity was measured by monitoring the fluorescence of water microdroplets dispersed in perfluorinated oil. The signal was generated via a series of coupled enzyme reactions leading to the formation of fluorescein. Using this new method, we were able to enrich the yeast cell population by more than 35.fold for GOx mutants with higher than wild.type activity after two rounds of sorting, almost double the efficiency of our previously described flow cytometry platform. We identified and characterized novel GOx mutants, the most promising of which (M6) contained a combination of six point mutations that increased the catalytic constant kcat by 2.1.fold compared to wild.type GOx and by 1.4.fold compared to a parental GOx variant. The new microfluidic platform for GOx was therefore more sensitive than flow cytometry and supports comprehensive screens of gene libraries containing multiple mutations per gene. © 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
PB  - MDPI
T2  - Molecules
T1  - A high.throughput screening system based on droplet microfluidics for glucose oxidase gene libraries
VL  - 25
IS  - 10
DO  - 10.3390/molecules25102418
ER  - 
@article{
author = "Prodanović, Radivoje and Lloyd Ung, W. and Ilić Đurđić, Karla and Fischer, Rainer and Weitz, David A. and Ostafe, Raluca",
year = "2020",
url = "http://cherry.chem.bg.ac.rs/handle/123456789/3950",
abstract = "Glucose oxidase (GOx) is an important industrial enzyme that can be optimized for specific applications by mutagenesis and activity.based screening. To increase the efficiency of this approach, we have developed a new ultrahigh.throughput screening platform based on a microfluidic lab.on.chip device that allows the sorting of GOx mutants from a saturation mutagenesis library expressed on the surface of yeast cells. GOx activity was measured by monitoring the fluorescence of water microdroplets dispersed in perfluorinated oil. The signal was generated via a series of coupled enzyme reactions leading to the formation of fluorescein. Using this new method, we were able to enrich the yeast cell population by more than 35.fold for GOx mutants with higher than wild.type activity after two rounds of sorting, almost double the efficiency of our previously described flow cytometry platform. We identified and characterized novel GOx mutants, the most promising of which (M6) contained a combination of six point mutations that increased the catalytic constant kcat by 2.1.fold compared to wild.type GOx and by 1.4.fold compared to a parental GOx variant. The new microfluidic platform for GOx was therefore more sensitive than flow cytometry and supports comprehensive screens of gene libraries containing multiple mutations per gene. © 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).",
publisher = "MDPI",
journal = "Molecules",
title = "A high.throughput screening system based on droplet microfluidics for glucose oxidase gene libraries",
volume = "25",
number = "10",
doi = "10.3390/molecules25102418"
}
Prodanović, R., Lloyd Ung, W., Ilić Đurđić, K., Fischer, R., Weitz, D. A.,& Ostafe, R. (2020). A high.throughput screening system based on droplet microfluidics for glucose oxidase gene libraries.
Molecules
MDPI., 25(10).
https://doi.org/10.3390/molecules25102418
Prodanović R, Lloyd Ung W, Ilić Đurđić K, Fischer R, Weitz DA, Ostafe R. A high.throughput screening system based on droplet microfluidics for glucose oxidase gene libraries. Molecules. 2020;25(10)
Prodanović Radivoje, Lloyd Ung W., Ilić Đurđić Karla, Fischer Rainer, Weitz David A., Ostafe Raluca, "A high.throughput screening system based on droplet microfluidics for glucose oxidase gene libraries" Molecules, 25, no. 10 (2020),
https://doi.org/10.3390/molecules25102418 .
2
2
1
2

Supplementary data for the article: Kostić, A. Ž.; Gašić, U. M.; Pešić, M. B.; Stanojević, S. P.; Barać, M. B.; Mačukanović-Jocić, M. P.; Avramov, S. N.; Tešić, Ž. L. Phytochemical Analysis and Total Antioxidant Capacity of Rhizome, Above-Ground Vegetative Parts and Flower of Three Iris Species. Chemistry and Biodiversity 2019, 16 (3), 1–17. https://doi.org/10.1002/cbdv.201800565

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

(Elsevier, 2019)

TY  - BOOK
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/123456789/2900
PB  - Elsevier
T2  - Biochemical Engineering Journal
T1  - Supplementary data for the article: Kostić, A. Ž.; Gašić, U. M.; Pešić, M. B.; Stanojević, S. P.; Barać, M. B.; Mačukanović-Jocić, M. P.; Avramov, S. N.; Tešić, Ž. L. Phytochemical Analysis and Total Antioxidant Capacity of Rhizome, Above-Ground Vegetative Parts and Flower of Three Iris Species. Chemistry and Biodiversity 2019, 16 (3), 1–17. https://doi.org/10.1002/cbdv.201800565
ER  - 
@book{
author = "Blažić, Marija and Balaž, Ana Marija and Tadić, Vojin and Draganić, Bojana and Ostafe, Raluca and Fischer, Rainer and Prodanović, Radivoje",
year = "2019",
url = "http://cherry.chem.bg.ac.rs/handle/123456789/2900",
publisher = "Elsevier",
journal = "Biochemical Engineering Journal",
title = "Supplementary data for the article: Kostić, A. Ž.; Gašić, U. M.; Pešić, M. B.; Stanojević, S. P.; Barać, M. B.; Mačukanović-Jocić, M. P.; Avramov, S. N.; Tešić, Ž. L. Phytochemical Analysis and Total Antioxidant Capacity of Rhizome, Above-Ground Vegetative Parts and Flower of Three Iris Species. Chemistry and Biodiversity 2019, 16 (3), 1–17. https://doi.org/10.1002/cbdv.201800565"
}
Blažić, M., Balaž, A. M., Tadić, V., Draganić, B., Ostafe, R., Fischer, R.,& Prodanović, R. (2019). Supplementary data for the article: Kostić, A. Ž.; Gašić, U. M.; Pešić, M. B.; Stanojević, S. P.; Barać, M. B.; Mačukanović-Jocić, M. P.; Avramov, S. N.; Tešić, Ž. L. Phytochemical Analysis and Total Antioxidant Capacity of Rhizome, Above-Ground Vegetative Parts and Flower of Three Iris Species. Chemistry and Biodiversity 2019, 16 (3), 1–17. https://doi.org/10.1002/cbdv.201800565.
Biochemical Engineering Journal
Elsevier..
Blažić M, Balaž AM, Tadić V, Draganić B, Ostafe R, Fischer R, Prodanović R. Supplementary data for the article: Kostić, A. Ž.; Gašić, U. M.; Pešić, M. B.; Stanojević, S. P.; Barać, M. B.; Mačukanović-Jocić, M. P.; Avramov, S. N.; Tešić, Ž. L. Phytochemical Analysis and Total Antioxidant Capacity of Rhizome, Above-Ground Vegetative Parts and Flower of Three Iris Species. Chemistry and Biodiversity 2019, 16 (3), 1–17. https://doi.org/10.1002/cbdv.201800565. Biochemical Engineering Journal. 2019;
Blažić Marija, Balaž Ana Marija, Tadić Vojin, Draganić Bojana, Ostafe Raluca, Fischer Rainer, Prodanović Radivoje, "Supplementary data for the article: Kostić, A. Ž.; Gašić, U. M.; Pešić, M. B.; Stanojević, S. P.; Barać, M. B.; Mačukanović-Jocić, M. P.; Avramov, S. N.; Tešić, Ž. L. Phytochemical Analysis and Total Antioxidant Capacity of Rhizome, Above-Ground Vegetative Parts and Flower of Three Iris Species. Chemistry and Biodiversity 2019, 16 (3), 1–17. https://doi.org/10.1002/cbdv.201800565" Biochemical Engineering Journal (2019)

Supplementary data for the article: Blažić, M.; Balaž, A. M.; 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. Applied Sciences (Switzerland) 2019, 9 (7). https://doi.org/10.3390/app9071413

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

(Applied sciences, 2019)

TY  - BOOK
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/123456789/2912
PB  - Applied sciences
T2  - Applied Sciences (Switzerland)
T1  - Supplementary data for the article: Blažić, M.; Balaž, A. M.; 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. Applied Sciences (Switzerland) 2019, 9 (7). https://doi.org/10.3390/app9071413
ER  - 
@book{
author = "Blažić, Marija and Balaž, Ana Marija and Prodanović, Olivera and Popović, Nikolina and Ostafe, Raluca and Fischer, Rainer and Prodanović, Radivoje",
year = "2019",
url = "http://cherry.chem.bg.ac.rs/handle/123456789/2912",
publisher = "Applied sciences",
journal = "Applied Sciences (Switzerland)",
title = "Supplementary data for the article: Blažić, M.; Balaž, A. M.; 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. Applied Sciences (Switzerland) 2019, 9 (7). https://doi.org/10.3390/app9071413"
}
Blažić, M., Balaž, A. M., Prodanović, O., Popović, N., Ostafe, R., Fischer, R.,& Prodanović, R. (2019). Supplementary data for the article: Blažić, M.; Balaž, A. M.; 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. Applied Sciences (Switzerland) 2019, 9 (7). https://doi.org/10.3390/app9071413.
Applied Sciences (Switzerland)
Applied sciences..
Blažić M, Balaž AM, Prodanović O, Popović N, Ostafe R, Fischer R, Prodanović R. Supplementary data for the article: Blažić, M.; Balaž, A. M.; 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. Applied Sciences (Switzerland) 2019, 9 (7). https://doi.org/10.3390/app9071413. Applied Sciences (Switzerland). 2019;
Blažić Marija, Balaž Ana Marija, Prodanović Olivera, Popović Nikolina, Ostafe Raluca, Fischer Rainer, Prodanović Radivoje, "Supplementary data for the article: Blažić, M.; Balaž, A. M.; 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. Applied Sciences (Switzerland) 2019, 9 (7). https://doi.org/10.3390/app9071413" Applied Sciences (Switzerland) (2019)

Supplementary data for the article: Kovačević, G.; Ostafe, R.; Balaž, A. M.; Fischer, R.; Prodanović, R. Development of GFP-Based High-Throughput Screening System for Directed Evolution of Glucose Oxidase. Journal of Bioscience and Bioengineering 2019, 127 (1), 30–37. https://doi.org/10.1016/j.jbiosc.2018.07.002

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

(Elsevier, 2018)

TY  - BOOK
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/123456789/3013
PB  - Elsevier
T2  - Journal of Bioscience and Bioengineering
T1  - Supplementary data for the article: Kovačević, G.; Ostafe, R.; Balaž, A. M.; Fischer, R.; Prodanović, R. Development of GFP-Based High-Throughput Screening System for Directed Evolution of Glucose Oxidase. Journal of Bioscience and Bioengineering 2019, 127 (1), 30–37. https://doi.org/10.1016/j.jbiosc.2018.07.002
ER  - 
@book{
author = "Kovačević, Gordana and Ostafe, Raluca and Balaž, Ana Marija and Fischer, Rainer and Prodanović, Radivoje",
year = "2018",
url = "http://cherry.chem.bg.ac.rs/handle/123456789/3013",
publisher = "Elsevier",
journal = "Journal of Bioscience and Bioengineering",
title = "Supplementary data for the article: Kovačević, G.; Ostafe, R.; Balaž, A. M.; Fischer, R.; Prodanović, R. Development of GFP-Based High-Throughput Screening System for Directed Evolution of Glucose Oxidase. Journal of Bioscience and Bioengineering 2019, 127 (1), 30–37. https://doi.org/10.1016/j.jbiosc.2018.07.002"
}
Kovačević, G., Ostafe, R., Balaž, A. M., Fischer, R.,& Prodanović, R. (2018). Supplementary data for the article: Kovačević, G.; Ostafe, R.; Balaž, A. M.; Fischer, R.; Prodanović, R. Development of GFP-Based High-Throughput Screening System for Directed Evolution of Glucose Oxidase. Journal of Bioscience and Bioengineering 2019, 127 (1), 30–37. https://doi.org/10.1016/j.jbiosc.2018.07.002.
Journal of Bioscience and Bioengineering
Elsevier..
Kovačević G, Ostafe R, Balaž AM, Fischer R, Prodanović R. Supplementary data for the article: Kovačević, G.; Ostafe, R.; Balaž, A. M.; Fischer, R.; Prodanović, R. Development of GFP-Based High-Throughput Screening System for Directed Evolution of Glucose Oxidase. Journal of Bioscience and Bioengineering 2019, 127 (1), 30–37. https://doi.org/10.1016/j.jbiosc.2018.07.002. Journal of Bioscience and Bioengineering. 2018;
Kovačević Gordana, Ostafe Raluca, Balaž Ana Marija, Fischer Rainer, Prodanović Radivoje, "Supplementary data for the article: Kovačević, G.; Ostafe, R.; Balaž, A. M.; Fischer, R.; Prodanović, R. Development of GFP-Based High-Throughput Screening System for Directed Evolution of Glucose Oxidase. Journal of Bioscience and Bioengineering 2019, 127 (1), 30–37. https://doi.org/10.1016/j.jbiosc.2018.07.002" Journal of Bioscience and Bioengineering (2018)

Supplementary data for article: Utech, S.; Prodanovic, R.; Mao, A. S.; Ostafe, R.; Mooney, D. J.; Weitz, D. A. Microfluidic Generation of Monodisperse, Structurally Homogeneous Alginate Microgels for Cell Encapsulation and 3D Cell Culture. Advanced Healthcare Materials 2015, 4 (11), 1628–1633. https://doi.org/10.1002/adhm.201500021

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

(Wiley-Blackwell, Hoboken, 2015)

TY  - BOOK
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/123456789/3383
PB  - Wiley-Blackwell, Hoboken
T2  - Advanced Healthcare Materials
T1  - Supplementary data for article: Utech, S.; Prodanovic, R.; Mao, A. S.; Ostafe, R.; Mooney, D. J.; Weitz, D. A. Microfluidic Generation of Monodisperse, Structurally Homogeneous Alginate Microgels for Cell Encapsulation and 3D Cell Culture. Advanced Healthcare Materials 2015, 4 (11), 1628–1633. https://doi.org/10.1002/adhm.201500021
ER  - 
@book{
author = "Utech, Stefanie and Prodanović, Radivoje and Mao, Angelo S. and Ostafe, Raluca and Mooney, David J. and Weitz, David A.",
year = "2015",
url = "http://cherry.chem.bg.ac.rs/handle/123456789/3383",
publisher = "Wiley-Blackwell, Hoboken",
journal = "Advanced Healthcare Materials",
title = "Supplementary data for article: Utech, S.; Prodanovic, R.; Mao, A. S.; Ostafe, R.; Mooney, D. J.; Weitz, D. A. Microfluidic Generation of Monodisperse, Structurally Homogeneous Alginate Microgels for Cell Encapsulation and 3D Cell Culture. Advanced Healthcare Materials 2015, 4 (11), 1628–1633. https://doi.org/10.1002/adhm.201500021"
}
Utech, S., Prodanović, R., Mao, A. S., Ostafe, R., Mooney, D. J.,& Weitz, D. A. (2015). Supplementary data for article: Utech, S.; Prodanovic, R.; Mao, A. S.; Ostafe, R.; Mooney, D. J.; Weitz, D. A. Microfluidic Generation of Monodisperse, Structurally Homogeneous Alginate Microgels for Cell Encapsulation and 3D Cell Culture. Advanced Healthcare Materials 2015, 4 (11), 1628–1633. https://doi.org/10.1002/adhm.201500021.
Advanced Healthcare Materials
Wiley-Blackwell, Hoboken..
Utech S, Prodanović R, Mao AS, Ostafe R, Mooney DJ, Weitz DA. Supplementary data for article: Utech, S.; Prodanovic, R.; Mao, A. S.; Ostafe, R.; Mooney, D. J.; Weitz, D. A. Microfluidic Generation of Monodisperse, Structurally Homogeneous Alginate Microgels for Cell Encapsulation and 3D Cell Culture. Advanced Healthcare Materials 2015, 4 (11), 1628–1633. https://doi.org/10.1002/adhm.201500021. Advanced Healthcare Materials. 2015;
Utech Stefanie, Prodanović Radivoje, Mao Angelo S., Ostafe Raluca, Mooney David J., Weitz David A., "Supplementary data for article: Utech, S.; Prodanovic, R.; Mao, A. S.; Ostafe, R.; Mooney, D. J.; Weitz, D. A. Microfluidic Generation of Monodisperse, Structurally Homogeneous Alginate Microgels for Cell Encapsulation and 3D Cell Culture. Advanced Healthcare Materials 2015, 4 (11), 1628–1633. https://doi.org/10.1002/adhm.201500021" Advanced Healthcare Materials (2015)

Supplementary data for the article: Ostafe, R.; Prodanovic, R.; Ung, W. L.; Weitz, D. A.; Fischer, R. A High-Throughput Cellulase Screening System Based on Droplet Microfluidics. Biomicrofluidics 2014, 8 (4). https://doi.org/10.1063/1.4886771

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

(Amer Inst Physics, Melville, 2014)

TY  - BOOK
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/123456789/3666
PB  - Amer Inst Physics, Melville
T2  - Biomicrofluidics
T1  - Supplementary data for the article: Ostafe, R.; Prodanovic, R.; Ung, W. L.; Weitz, D. A.; Fischer, R. A High-Throughput Cellulase Screening System Based on Droplet Microfluidics. Biomicrofluidics 2014, 8 (4). https://doi.org/10.1063/1.4886771
ER  - 
@book{
author = "Ostafe, Raluca and Prodanović, Radivoje and Ung, Lloyd W. and Weitz, David A. and Fischer, Rainer",
year = "2014",
url = "http://cherry.chem.bg.ac.rs/handle/123456789/3666",
publisher = "Amer Inst Physics, Melville",
journal = "Biomicrofluidics",
title = "Supplementary data for the article: Ostafe, R.; Prodanovic, R.; Ung, W. L.; Weitz, D. A.; Fischer, R. A High-Throughput Cellulase Screening System Based on Droplet Microfluidics. Biomicrofluidics 2014, 8 (4). https://doi.org/10.1063/1.4886771"
}
Ostafe, R., Prodanović, R., Ung, L. W., Weitz, D. A.,& Fischer, R. (2014). Supplementary data for the article: Ostafe, R.; Prodanovic, R.; Ung, W. L.; Weitz, D. A.; Fischer, R. A High-Throughput Cellulase Screening System Based on Droplet Microfluidics. Biomicrofluidics 2014, 8 (4). https://doi.org/10.1063/1.4886771.
Biomicrofluidics
Amer Inst Physics, Melville..
Ostafe R, Prodanović R, Ung LW, Weitz DA, Fischer R. Supplementary data for the article: Ostafe, R.; Prodanovic, R.; Ung, W. L.; Weitz, D. A.; Fischer, R. A High-Throughput Cellulase Screening System Based on Droplet Microfluidics. Biomicrofluidics 2014, 8 (4). https://doi.org/10.1063/1.4886771. Biomicrofluidics. 2014;
Ostafe Raluca, Prodanović Radivoje, Ung Lloyd W., Weitz David A., Fischer Rainer, "Supplementary data for the article: Ostafe, R.; Prodanovic, R.; Ung, W. L.; Weitz, D. A.; Fischer, R. A High-Throughput Cellulase Screening System Based on Droplet Microfluidics. Biomicrofluidics 2014, 8 (4). https://doi.org/10.1063/1.4886771" Biomicrofluidics (2014)

Supplementary data for article: 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. Protein Expression and Purification 2013, 89 (2), 175–180. https://doi.org/10.1016/j.pep.2013.03.014

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

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

TY  - BOOK
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/123456789/3568
PB  - Academic Press Inc Elsevier Science, San Diego
T2  - Protein Expression and Purification
T1  - Supplementary data for article: 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. Protein Expression and Purification 2013, 89 (2), 175–180. https://doi.org/10.1016/j.pep.2013.03.014
ER  - 
@book{
author = "Blažić, Marija and Kovačević, Gordana and Prodanović, Olivera and Ostafe, Raluca and Gavrović-Jankulović, Marija and Fischer, Rainer and Prodanović, Radivoje",
year = "2013",
url = "http://cherry.chem.bg.ac.rs/handle/123456789/3568",
publisher = "Academic Press Inc Elsevier Science, San Diego",
journal = "Protein Expression and Purification",
title = "Supplementary data for article: 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. Protein Expression and Purification 2013, 89 (2), 175–180. https://doi.org/10.1016/j.pep.2013.03.014"
}
Blažić, M., Kovačević, G., Prodanović, O., Ostafe, R., Gavrović-Jankulović, M., Fischer, R.,& Prodanović, R. (2013). Supplementary data for article: 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. Protein Expression and Purification 2013, 89 (2), 175–180. https://doi.org/10.1016/j.pep.2013.03.014.
Protein Expression and Purification
Academic Press Inc Elsevier Science, San Diego..
Blažić M, Kovačević G, Prodanović O, Ostafe R, Gavrović-Jankulović M, Fischer R, Prodanović R. Supplementary data for article: 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. Protein Expression and Purification 2013, 89 (2), 175–180. https://doi.org/10.1016/j.pep.2013.03.014. Protein Expression and Purification. 2013;
Blažić Marija, Kovačević Gordana, Prodanović Olivera, Ostafe Raluca, Gavrović-Jankulović Marija, Fischer Rainer, Prodanović Radivoje, "Supplementary data for article: 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. Protein Expression and Purification 2013, 89 (2), 175–180. https://doi.org/10.1016/j.pep.2013.03.014" Protein Expression and Purification (2013)

Flow cytometry-based ultra-high-throughput screening assay for cellulase activity

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

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

TY  - JOUR
AU  - Ostafe, Raluca
AU  - Prodanović, Radivoje
AU  - Commandeur, Ulrich
AU  - Fischer, Rainer
PY  - 2013
UR  - http://cherry.chem.bg.ac.rs/handle/123456789/1606
AB  - We have developed a novel, ultra-high-throughput screening assay for the detection of cellulase activity based on fluorescence-activated cell sorting (FACS) and double emulsion technology. Cellulase activity is detected using a series of coupled enzymes, including hexose oxidase (HOx), which generates hydrogen peroxide from the reducing sugars released by cellulases in the presence of any natural or artificial substrate. The assay can be adapted to suit a microtiter plate format, but the highest throughput is achieved by using FACS to screen high-complexity cellulase clone libraries. Using this approach, we achieved a 12-fold enrichment of positive (cellulase-expressing) cells in cellulase reference libraries after just one sorting round.
PB  - Academic Press Inc Elsevier Science, San Diego
T2  - Analytical Biochemistry
T1  - Flow cytometry-based ultra-high-throughput screening assay for cellulase activity
VL  - 435
IS  - 1
SP  - 93
EP  - 98
DO  - 10.1016/j.ab.2012.10.043
ER  - 
@article{
author = "Ostafe, Raluca and Prodanović, Radivoje and Commandeur, Ulrich and Fischer, Rainer",
year = "2013",
url = "http://cherry.chem.bg.ac.rs/handle/123456789/1606",
abstract = "We have developed a novel, ultra-high-throughput screening assay for the detection of cellulase activity based on fluorescence-activated cell sorting (FACS) and double emulsion technology. Cellulase activity is detected using a series of coupled enzymes, including hexose oxidase (HOx), which generates hydrogen peroxide from the reducing sugars released by cellulases in the presence of any natural or artificial substrate. The assay can be adapted to suit a microtiter plate format, but the highest throughput is achieved by using FACS to screen high-complexity cellulase clone libraries. Using this approach, we achieved a 12-fold enrichment of positive (cellulase-expressing) cells in cellulase reference libraries after just one sorting round.",
publisher = "Academic Press Inc Elsevier Science, San Diego",
journal = "Analytical Biochemistry",
title = "Flow cytometry-based ultra-high-throughput screening assay for cellulase activity",
volume = "435",
number = "1",
pages = "93-98",
doi = "10.1016/j.ab.2012.10.043"
}
Ostafe, R., Prodanović, R., Commandeur, U.,& Fischer, R. (2013). Flow cytometry-based ultra-high-throughput screening assay for cellulase activity.
Analytical Biochemistry
Academic Press Inc Elsevier Science, San Diego., 435(1), 93-98.
https://doi.org/10.1016/j.ab.2012.10.043
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
Ostafe Raluca, Prodanović Radivoje, Commandeur Ulrich, Fischer Rainer, "Flow cytometry-based ultra-high-throughput screening assay for cellulase activity" Analytical Biochemistry, 435, no. 1 (2013):93-98,
https://doi.org/10.1016/j.ab.2012.10.043 .
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Supplementary data for article: Yu, E. H.; Prodanović, R.; Gueven, G.; Ostafe, R.; Schwaneberg, U. Electrochemical Oxidation of Glucose Using Mutant Glucose Oxidase from Directed Protein Evolution for Biosensor and Biofuel Cell Applications. Applied Biochemistry and Biotechnology 2011, 165 (7–8), 1448–1457. https://doi.org/10.1007/s12010-011-9366-0

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

(Humana Press Inc, Totowa, 2011)

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