TY  - JOUR
AU  - Balaž, Ana Marija
AU  - Stevanović, Jelena
AU  - Ostafe, Raluca
AU  - Blažić, Marija
AU  - Ilić Đurđić, Karla
AU  - Fischer, Rainer
AU  - Prodanović, Radivoje
PY  - 2020
UR  - https://cer.ihtm.bg.ac.rs/handle/123456789/3128
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.
PB  - Springer International Publishing
T2  - Molecular Diversity
T1  - Semi‑rational design of cellobiose dehydrogenase for increased stability in the presence of peroxide
VL  - 24
SP  - 593
EP  - 601
DO  - 10.1007/s11030-019-09965-0
ER  - 

@article{
author = "Balaž, Ana Marija and Stevanović, Jelena and Ostafe, Raluca and Blažić, Marija and Ilić Đurđić, Karla and Fischer, Rainer and Prodanović, Radivoje",
year = "2020",
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.",
publisher = "Springer International Publishing",
journal = "Molecular Diversity",
title = "Semi‑rational design of cellobiose dehydrogenase for increased stability in the presence of peroxide",
volume = "24",
pages = "593-601",
doi = "10.1007/s11030-019-09965-0"
}

Balaž, A. M., Stevanović, J., Ostafe, R., Blažić, M., Ilić Đurđić, K., Fischer, R.,& Prodanović, R.. (2020). Semi‑rational design of cellobiose dehydrogenase for increased stability in the presence of peroxide. in Molecular Diversity
Springer International Publishing., 24, 593-601.
https://doi.org/10.1007/s11030-019-09965-0

Balaž AM, Stevanović J, Ostafe R, Blažić M, Ilić Đurđić K, Fischer R, Prodanović R. Semi‑rational design of cellobiose dehydrogenase for increased stability in the presence of peroxide. in Molecular Diversity. 2020;24:593-601.
doi:10.1007/s11030-019-09965-0 .

Balaž, Ana Marija, Stevanović, Jelena, Ostafe, Raluca, Blažić, Marija, Ilić Đurđić, Karla, Fischer, Rainer, Prodanović, Radivoje, "Semi‑rational design of cellobiose dehydrogenase for increased stability in the presence of peroxide" in Molecular Diversity, 24 (2020):593-601,
https://doi.org/10.1007/s11030-019-09965-0 . .

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
UR  - https://cer.ihtm.bg.ac.rs/handle/123456789/3580
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",
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. in 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. in Journal of Bioscience and Bioengineering. 2020;129(6):664-671.
doi:10.1016/j.jbiosc.2019.12.009 .

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" in Journal of Bioscience and Bioengineering, 129, no. 6 (2020):664-671,
https://doi.org/10.1016/j.jbiosc.2019.12.009 . .

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  - https://cer.ihtm.bg.ac.rs/handle/123456789/3880
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 производња
растворних облика целобиоза дехидрогеназе (CDH) претходно еволуираних на површини
квашчевих 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 високу
експресију у растворном облику и кинетичку карактеризацију. После 6 дана експресије под
индукцијом метанолом, мутанти су пречишћени ултрафилтрацијом, јоноизмењивачком
хроматографијом и гел-филтрацијом. SDS електрофореза је потврдила чистоћу уз присуство
једне протеинске траке молекулскe масe од 100 kDa. Кинетичка карактеризација је показала
да H5 мутирани протеин поседује највећу каталитичку константу од 43,5 s-1 за лактозу, док
је H9 имао највећу константу специфичности за лактозу од 132 mM-1 s-1. Сва три мутирана
протеина су имала неизмењен pH оптимум који је био у опсегу од 4,5 до 5,5. У поређењу са
претходно добијеним природним и мутантним облицима CDH протеина из Phanerochaete
chrysosporium, 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",
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 производња
растворних облика целобиоза дехидрогеназе (CDH) претходно еволуираних на површини
квашчевих 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 високу
експресију у растворном облику и кинетичку карактеризацију. После 6 дана експресије под
индукцијом метанолом, мутанти су пречишћени ултрафилтрацијом, јоноизмењивачком
хроматографијом и гел-филтрацијом. SDS електрофореза је потврдила чистоћу уз присуство
једне протеинске траке молекулскe масe од 100 kDa. Кинетичка карактеризација је показала
да H5 мутирани протеин поседује највећу каталитичку константу од 43,5 s-1 за лактозу, док
је H9 имао највећу константу специфичности за лактозу од 132 mM-1 s-1. Сва три мутирана
протеина су имала неизмењен pH оптимум који је био у опсегу од 4,5 до 5,5. У поређењу са
претходно добијеним природним и мутантним облицима CDH протеина из Phanerochaete
chrysosporium, 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). Expression, purification and characterization of cellobiose dehydrogenase mutants from Phanerochaete chrysosporium in Pichia pastoris KM71H strain. in 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. Expression, purification and characterization of cellobiose dehydrogenase mutants from Phanerochaete chrysosporium in Pichia pastoris KM71H strain. in Journal of the Serbian Chemical Society. 2020;85(1):25-35.
doi:10.2298/JSC190320058B .

Balaž, Ana Marija, Blažić, Marija, Popović, Nikolina, Prodanović, Olivera, Ostafe, Raluca, Fischer, Rainer, Prodanović, Radivoje, "Expression, purification and characterization of cellobiose dehydrogenase mutants from Phanerochaete chrysosporium in Pichia pastoris KM71H strain" in Journal of the Serbian Chemical Society, 85, no. 1 (2020):25-35,
https://doi.org/10.2298/JSC190320058B . .

TY  - JOUR
AU  - Kovačević, Gordana
AU  - Ostafe, Raluca
AU  - Balaž, Ana Marija
AU  - Fischer, Rainer
AU  - Prodanović, Radivoje
PY  - 2019
UR  - https://cer.ihtm.bg.ac.rs/handle/123456789/2497
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.
PB  - Elsevier
T2  - Journal of Bioscience and Bioengineering
T1  - Development of GFP-based high-throughput screening system for directed evolution of glucose oxidase
VL  - 127
IS  - 1
SP  - 30
EP  - 37
DO  - 10.1016/j.jbiosc.2018.07.002
ER  - 

@article{
author = "Kovačević, Gordana and Ostafe, Raluca and Balaž, Ana Marija and Fischer, Rainer and Prodanović, Radivoje",
year = "2019",
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.",
publisher = "Elsevier",
journal = "Journal of Bioscience and Bioengineering",
title = "Development of GFP-based high-throughput screening system for directed evolution of glucose oxidase",
volume = "127",
number = "1",
pages = "30-37",
doi = "10.1016/j.jbiosc.2018.07.002"
}

Kovačević, G., Ostafe, R., Balaž, A. M., Fischer, R.,& Prodanović, R.. (2019). Development of GFP-based high-throughput screening system for directed evolution of glucose oxidase. in Journal of Bioscience and Bioengineering
Elsevier., 127(1), 30-37.
https://doi.org/10.1016/j.jbiosc.2018.07.002

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. 2019;127(1):30-37.
doi:10.1016/j.jbiosc.2018.07.002 .

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, 127, no. 1 (2019):30-37,
https://doi.org/10.1016/j.jbiosc.2018.07.002 . .

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  - https://cer.ihtm.bg.ac.rs/handle/123456789/2875
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  - MDPI
T2  - Applied Sciences
T1  - Directed Evolution of Cellobiose Dehydrogenase on the Surface of Yeast Cells Using Resazurin-Based Fluorescent Assay
VL  - 9
IS  - 7
SP  - 1413
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 = "MDPI",
journal = "Applied Sciences",
title = "Directed Evolution of Cellobiose Dehydrogenase on the Surface of Yeast Cells Using Resazurin-Based Fluorescent Assay",
volume = "9",
number = "7",
pages = "1413",
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
MDPI., 9(7), 1413.
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. 2019;9(7):1413.
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, 9, no. 7 (2019):1413,
https://doi.org/10.3390/app9071413 . .

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  - https://cer.ihtm.bg.ac.rs/handle/123456789/2655
UR  - https://cer.ihtm.bg.ac.rs/handle/123456789/2656
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 assaybased 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 hyperglycosylation and the best S137N mutant showed 2.2 times increased kcat 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 S137N mutant retained 30% of it’s original activity after 15 minutes at 70oC compared to 10% of activity that the wild type enzyme retained. Mutants M65S and S137N showed also 1.6 and 1.5 times increased productivity of hydrogen peroxide in the presence of 30mM 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 assaybased 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 hyperglycosylation and the best S137N mutant showed 2.2 times increased kcat 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 S137N mutant retained 30% of it’s original activity after 15 minutes at 70oC compared to 10% of activity that the wild type enzyme retained. Mutants M65S and S137N showed also 1.6 and 1.5 times increased productivity of hydrogen peroxide in the presence of 30mM 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 . .

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  - https://cer.ihtm.bg.ac.rs/handle/123456789/2655
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 hyperglycosylation and the best S137N mutant showed 2.2 times increased kcat 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 S137N mutant retained 30% of it’s original activity after 15 minutes at 70oC compared to 10% of activity that the wild type enzyme retained. Mutants M65S and S137N showed also 1.6 and 1.5 times increased productivity of hydrogen peroxide in the presence of 30mM 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 hyperglycosylation and the best S137N mutant showed 2.2 times increased kcat 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 S137N mutant retained 30% of it’s original activity after 15 minutes at 70oC compared to 10% of activity that the wild type enzyme retained. Mutants M65S and S137N showed also 1.6 and 1.5 times increased productivity of hydrogen peroxide in the presence of 30mM 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 . .

TY  - CONF
AU  - Balaž, Ana Marija
AU  - Ostafe, Raluca
AU  - Fischer, Rainer
AU  - Prodanović, Radivoje
PY  - 2017
UR  - http://www.bds.org.rs/en/conferences.php
UR  - https://cer.ihtm.bg.ac.rs/handle/123456789/3279
AB  - Cellobiose dehydrogenase (CDH, EC 1.1.99.18) from Phanerochaete chrysosporium
 belongs to a group of oxidoreductases and has the ability to degrade different components
 of woody plants. CDH is secreted by wood degrading, phytopathogenic and saprotrophic
 fungi and this widespread appearance implies hers important function and makes her an
 important enzyme for applications in industrial and biotechnological processes, as well as
 biosensors and biofuel cells. CDH is also used in industry for bleaching cotton and in
 food industry for lactose detection. CDH is monomeric enzyme consisting of two
 domains, flavin domain containing FAD as cofactor and smaller hem b containing
 cytochrome domain, connected via flexible linker. Physiological role of CDH is reflected
 in the degradation of cellulose and lignin in cooperation with other cellulolytic enzymes,
 because CDH catalyzes oxidation of celobiose (Glc - β - 1,4 Glc) and other β - 1,4 - linked
 disaccharides and oligosaccharides to the corresponding lactons. Enzymes used in
 biosensors and for bleaching cotton should have high stability, especially toward reactive
 oxygen species. In order to improve oxidative stability of CDH, we have mutated CDH
 and tested its stability in presence of hydrogen peroxide. After successful cloning of the
 CDH gene in pYES2 vector, saturation mutagenesis was used to make library mutants
 where tree methionine residues were mutated. Residual activity of mutants was measured
 after the enzyme incubation in 0.3 M hydrogen peroxide for 0, 2 and 6h. After analysis of
 large number of mutants, it was observed that three mutants are showing higher oxidative
 stability compared to the wild - type enzyme. Residual activities of these mutants after 6
 hour incubation in the hydrogen peroxide were over 50%, whereas wild-type has 30%.
 Selected mutants were expressed in S. cerevisiae and purified on DEAE column. Purity
 and activity of the enzymes were detected on the electrophoresis gel, oxidative stability of
 purified mutants was measured once again and characterization of these mutants was done. Mutants showing increased oxidative stability were sequenced and we have decided to
 combine these mutations with each other in order to make combined mutants that will be
 tested for oxidative stability. Screening library mutants for improved features in
 microtitatar plates is a long time process, in order to shorten the time necessary for
 screening libraries with 106 mutants we are developing fluorescent assay for flou
 cytometry.
PB  - Faculty of Chemistry, Serbian Biochemical Society
C3  - Serbian Biochemical Society Seventh Conference "Biochemistry of Control in Life and Technology" - Proceedings
T1  - Semi - rational design of cellobiose dehydrogenase from Phanerochaete chrysosporium for increased oxidative stability and high-throughput screening of library mutants
UR  - https://hdl.handle.net/21.15107/rcub_cer_3279
ER  - 

@conference{
author = "Balaž, Ana Marija and Ostafe, Raluca and Fischer, Rainer and Prodanović, Radivoje",
year = "2017",
abstract = "Cellobiose dehydrogenase (CDH, EC 1.1.99.18) from Phanerochaete chrysosporium
 belongs to a group of oxidoreductases and has the ability to degrade different components
 of woody plants. CDH is secreted by wood degrading, phytopathogenic and saprotrophic
 fungi and this widespread appearance implies hers important function and makes her an
 important enzyme for applications in industrial and biotechnological processes, as well as
 biosensors and biofuel cells. CDH is also used in industry for bleaching cotton and in
 food industry for lactose detection. CDH is monomeric enzyme consisting of two
 domains, flavin domain containing FAD as cofactor and smaller hem b containing
 cytochrome domain, connected via flexible linker. Physiological role of CDH is reflected
 in the degradation of cellulose and lignin in cooperation with other cellulolytic enzymes,
 because CDH catalyzes oxidation of celobiose (Glc - β - 1,4 Glc) and other β - 1,4 - linked
 disaccharides and oligosaccharides to the corresponding lactons. Enzymes used in
 biosensors and for bleaching cotton should have high stability, especially toward reactive
 oxygen species. In order to improve oxidative stability of CDH, we have mutated CDH
 and tested its stability in presence of hydrogen peroxide. After successful cloning of the
 CDH gene in pYES2 vector, saturation mutagenesis was used to make library mutants
 where tree methionine residues were mutated. Residual activity of mutants was measured
 after the enzyme incubation in 0.3 M hydrogen peroxide for 0, 2 and 6h. After analysis of
 large number of mutants, it was observed that three mutants are showing higher oxidative
 stability compared to the wild - type enzyme. Residual activities of these mutants after 6
 hour incubation in the hydrogen peroxide were over 50%, whereas wild-type has 30%.
 Selected mutants were expressed in S. cerevisiae and purified on DEAE column. Purity
 and activity of the enzymes were detected on the electrophoresis gel, oxidative stability of
 purified mutants was measured once again and characterization of these mutants was done. Mutants showing increased oxidative stability were sequenced and we have decided to
 combine these mutations with each other in order to make combined mutants that will be
 tested for oxidative stability. Screening library mutants for improved features in
 microtitatar plates is a long time process, in order to shorten the time necessary for
 screening libraries with 106 mutants we are developing fluorescent assay for flou
 cytometry.",
publisher = "Faculty of Chemistry, Serbian Biochemical Society",
journal = "Serbian Biochemical Society Seventh Conference "Biochemistry of Control in Life and Technology" - Proceedings",
title = "Semi - rational design of cellobiose dehydrogenase from Phanerochaete chrysosporium for increased oxidative stability and high-throughput screening of library mutants",
url = "https://hdl.handle.net/21.15107/rcub_cer_3279"
}

Balaž, A. M., Ostafe, R., Fischer, R.,& Prodanović, R.. (2017). Semi - rational design of cellobiose dehydrogenase from Phanerochaete chrysosporium for increased oxidative stability and high-throughput screening of library mutants. in Serbian Biochemical Society Seventh Conference "Biochemistry of Control in Life and Technology" - Proceedings
Faculty of Chemistry, Serbian Biochemical Society..
https://hdl.handle.net/21.15107/rcub_cer_3279

Balaž AM, Ostafe R, Fischer R, Prodanović R. Semi - rational design of cellobiose dehydrogenase from Phanerochaete chrysosporium for increased oxidative stability and high-throughput screening of library mutants. in Serbian Biochemical Society Seventh Conference "Biochemistry of Control in Life and Technology" - Proceedings. 2017;.
https://hdl.handle.net/21.15107/rcub_cer_3279 .

Balaž, Ana Marija, Ostafe, Raluca, Fischer, Rainer, Prodanović, Radivoje, "Semi - rational design of cellobiose dehydrogenase from Phanerochaete chrysosporium for increased oxidative stability and high-throughput screening of library mutants" in Serbian Biochemical Society Seventh Conference "Biochemistry of Control in Life and Technology" - Proceedings (2017),
https://hdl.handle.net/21.15107/rcub_cer_3279 .

TY  - CONF
AU  - Zelenović, Jelena
AU  - Ostafe, Raluca
AU  - Fischer, Rainer
AU  - Prodanović, Radivoje
PY  - 2017
UR  - http://www.bds.org.rs/en/conferences.php
UR  - https://cer.ihtm.bg.ac.rs/handle/123456789/3282
AB  - Cellulase (EC 3.2.1.4) are important enzymes in food, paper, textile, detergent and biofuel industries. Most cellulases have low activity and stability. Improving these properties would have substantial impact on numerous industrial processes. Enzymatic properties can be improved by directed evolution, but the screening process is the limiting step. Coupled cellulase assay has been developed in order to improve the screening process. This method does not require boiling samples and allows rapid screening of mutants in a microtiter plate. The aim of this study was to establish enzyme coupled assay where cellulase first hydrolyzes carboxymethylcellulose (CMC), and cellobioses dehydrogenase (CBDH) and dichlorophenolindophenol (DCPIP) is used subsequently for detection of reducing ends.
PB  - Faculty of Chemistry, Serbian Biochemical Society
C3  - Serbian Biochemical Society Seventh Conference "Biochemistry of Control in Life and Technology" - Proceedings
T1  - Directed evolution of cellulase from Trichoderma reesei for higher activity and development of microtiter plate assay based on cellobiose dehydrogenase
UR  - https://hdl.handle.net/21.15107/rcub_cer_3282
ER  - 

@conference{
author = "Zelenović, Jelena and Ostafe, Raluca and Fischer, Rainer and Prodanović, Radivoje",
year = "2017",
abstract = "Cellulase (EC 3.2.1.4) are important enzymes in food, paper, textile, detergent and biofuel industries. Most cellulases have low activity and stability. Improving these properties would have substantial impact on numerous industrial processes. Enzymatic properties can be improved by directed evolution, but the screening process is the limiting step. Coupled cellulase assay has been developed in order to improve the screening process. This method does not require boiling samples and allows rapid screening of mutants in a microtiter plate. The aim of this study was to establish enzyme coupled assay where cellulase first hydrolyzes carboxymethylcellulose (CMC), and cellobioses dehydrogenase (CBDH) and dichlorophenolindophenol (DCPIP) is used subsequently for detection of reducing ends.",
publisher = "Faculty of Chemistry, Serbian Biochemical Society",
journal = "Serbian Biochemical Society Seventh Conference "Biochemistry of Control in Life and Technology" - Proceedings",
title = "Directed evolution of cellulase from Trichoderma reesei for higher activity and development of microtiter plate assay based on cellobiose dehydrogenase",
url = "https://hdl.handle.net/21.15107/rcub_cer_3282"
}

Zelenović, J., Ostafe, R., Fischer, R.,& Prodanović, R.. (2017). Directed evolution of cellulase from Trichoderma reesei for higher activity and development of microtiter plate assay based on cellobiose dehydrogenase. in Serbian Biochemical Society Seventh Conference "Biochemistry of Control in Life and Technology" - Proceedings
Faculty of Chemistry, Serbian Biochemical Society..
https://hdl.handle.net/21.15107/rcub_cer_3282

Zelenović J, Ostafe R, Fischer R, Prodanović R. Directed evolution of cellulase from Trichoderma reesei for higher activity and development of microtiter plate assay based on cellobiose dehydrogenase. in Serbian Biochemical Society Seventh Conference "Biochemistry of Control in Life and Technology" - Proceedings. 2017;.
https://hdl.handle.net/21.15107/rcub_cer_3282 .

Zelenović, Jelena, Ostafe, Raluca, Fischer, Rainer, Prodanović, Radivoje, "Directed evolution of cellulase from Trichoderma reesei for higher activity and development of microtiter plate assay based on cellobiose dehydrogenase" in Serbian Biochemical Society Seventh Conference "Biochemistry of Control in Life and Technology" - Proceedings (2017),
https://hdl.handle.net/21.15107/rcub_cer_3282 .

TY  - CONF
AU  - Balaž, Ana Marija
AU  - Ostafe, Raluca
AU  - Prodanović, Radivoje
PY  - 2016
UR  - https://cer.ihtm.bg.ac.rs/handle/123456789/3548
PB  - Serbian Biochemical Society (Belgrade)
C3  - Serbian Biochemical Society Sixth Conference, “Biochemistry and Interdisciplinarity: Transcending the Limits of Field”, Faculty of Chemistry, University of Belgrade, 18.11.2016. Belgrade, Serbia
T1  - Semi rational design of cellobiose dehydrogenaze from Phanerochaete chrysosporium for increase oxidative stability
SP  - 113
EP  - 114
UR  - https://hdl.handle.net/21.15107/rcub_cer_3548
ER  - 

@conference{
author = "Balaž, Ana Marija and Ostafe, Raluca and Prodanović, Radivoje",
year = "2016",
publisher = "Serbian Biochemical Society (Belgrade)",
journal = "Serbian Biochemical Society Sixth Conference, “Biochemistry and Interdisciplinarity: Transcending the Limits of Field”, Faculty of Chemistry, University of Belgrade, 18.11.2016. Belgrade, Serbia",
title = "Semi rational design of cellobiose dehydrogenaze from Phanerochaete chrysosporium for increase oxidative stability",
pages = "113-114",
url = "https://hdl.handle.net/21.15107/rcub_cer_3548"
}

Balaž, A. M., Ostafe, R.,& Prodanović, R.. (2016). Semi rational design of cellobiose dehydrogenaze from Phanerochaete chrysosporium for increase oxidative stability. in Serbian Biochemical Society Sixth Conference, “Biochemistry and Interdisciplinarity: Transcending the Limits of Field”, Faculty of Chemistry, University of Belgrade, 18.11.2016. Belgrade, Serbia
Serbian Biochemical Society (Belgrade)., 113-114.
https://hdl.handle.net/21.15107/rcub_cer_3548

Balaž AM, Ostafe R, Prodanović R. Semi rational design of cellobiose dehydrogenaze from Phanerochaete chrysosporium for increase oxidative stability. in Serbian Biochemical Society Sixth Conference, “Biochemistry and Interdisciplinarity: Transcending the Limits of Field”, Faculty of Chemistry, University of Belgrade, 18.11.2016. Belgrade, Serbia. 2016;:113-114.
https://hdl.handle.net/21.15107/rcub_cer_3548 .

Balaž, Ana Marija, Ostafe, Raluca, Prodanović, Radivoje, "Semi rational design of cellobiose dehydrogenaze from Phanerochaete chrysosporium for increase oxidative stability" in Serbian Biochemical Society Sixth Conference, “Biochemistry and Interdisciplinarity: Transcending the Limits of Field”, Faculty of Chemistry, University of Belgrade, 18.11.2016. Belgrade, Serbia (2016):113-114,
https://hdl.handle.net/21.15107/rcub_cer_3548 .

TY  - JOUR
AU  - Kovačević, Gordana
AU  - Blažić, Marija
AU  - Draganic, Bojana
AU  - Ostafe, Raluca
AU  - Gavrović-Jankulović, Marija
AU  - Fischer, Rainer
AU  - Prodanović, Radivoje
PY  - 2014
UR  - https://cer.ihtm.bg.ac.rs/handle/123456789/1506
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
ER  - 

@article{
author = "Kovačević, Gordana and Blažić, Marija and Draganic, 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"
}

Kovačević, G., Blažić, M., Draganic, 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

Kovačević G, Blažić M, Draganic 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 .

Kovačević, Gordana, Blažić, Marija, Draganic, 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 . .

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  - https://cer.ihtm.bg.ac.rs/handle/123456789/1331
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
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"
}

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

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 .

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 . .