TY  - JOUR
AU  - Kovačević, G.
AU  - Elgahwash, R.G.A.
AU  - Blažić, M.
AU  - Pantić, Nevena
AU  - Prodanović, Olivera
AU  - Balaž, Ana Marija
AU  - Prodanović, Radivoje
PY  - 2022
UR  - http://rimsi.imsi.bg.ac.rs/handle/123456789/1543
AB  - Saccharomyces cerevisiae, known as bakers’ yeast, is one of the most utilized yeasts in industry. Several enzymes that are naturally produced by yeast, such as invertase and catalase, combined with heterologously expressed glucose oxidase (GOx), represent the enzyme machinery for fructose and gluconic acid production. Therefore, we have used yeast cell walls with expressed glucose oxidase as a platform for crosslinking with invertase and catalase to create biocatalyst cells for the high yield sucrose conversion. Using 5% (w/v) suspension of cross-linked yeast cell walls in 0.15 M sucrose solution, 1.86 g L−1 h−1 of gluconic acid has been obtained using wt-GOx, while mutant A2-GOx produced 2.91 g L−1 h−1 of gluconic acid. Increasing the concentration of modified yeast cells walls to 10% (w/v) we were able to obtain almost 100% conversion of glucose to gluconic acid using A2-GOx in the first cycle. Reusing the modified cells walls in three consecutive cycles, conversion dropped to approximately 70% using A2-GOx and 40% using wt-GOx.
PB  - Elsevier B.V.
T2  - Molecular Catalysis
T1  - Production of fructose and gluconic acid from sucrose with cross-linked yeast cell walls expressing glucose oxidase on the surface
VL  - 522
DO  - 10.1016/j.mcat.2022.112215
ER  - 

@article{
author = "Kovačević, G. and Elgahwash, R.G.A. and Blažić, M. and Pantić, Nevena and Prodanović, Olivera and Balaž, Ana Marija and Prodanović, Radivoje",
year = "2022",
abstract = "Saccharomyces cerevisiae, known as bakers’ yeast, is one of the most utilized yeasts in industry. Several enzymes that are naturally produced by yeast, such as invertase and catalase, combined with heterologously expressed glucose oxidase (GOx), represent the enzyme machinery for fructose and gluconic acid production. Therefore, we have used yeast cell walls with expressed glucose oxidase as a platform for crosslinking with invertase and catalase to create biocatalyst cells for the high yield sucrose conversion. Using 5% (w/v) suspension of cross-linked yeast cell walls in 0.15 M sucrose solution, 1.86 g L−1 h−1 of gluconic acid has been obtained using wt-GOx, while mutant A2-GOx produced 2.91 g L−1 h−1 of gluconic acid. Increasing the concentration of modified yeast cells walls to 10% (w/v) we were able to obtain almost 100% conversion of glucose to gluconic acid using A2-GOx in the first cycle. Reusing the modified cells walls in three consecutive cycles, conversion dropped to approximately 70% using A2-GOx and 40% using wt-GOx.",
publisher = "Elsevier B.V.",
journal = "Molecular Catalysis",
title = "Production of fructose and gluconic acid from sucrose with cross-linked yeast cell walls expressing glucose oxidase on the surface",
volume = "522",
doi = "10.1016/j.mcat.2022.112215"
}

Kovačević, G., Elgahwash, R.G.A., Blažić, M., Pantić, N., Prodanović, O., Balaž, A. M.,& Prodanović, R.. (2022). Production of fructose and gluconic acid from sucrose with cross-linked yeast cell walls expressing glucose oxidase on the surface. in Molecular Catalysis
Elsevier B.V.., 522.
https://doi.org/10.1016/j.mcat.2022.112215

Kovačević G, Elgahwash R, Blažić M, Pantić N, Prodanović O, Balaž AM, Prodanović R. Production of fructose and gluconic acid from sucrose with cross-linked yeast cell walls expressing glucose oxidase on the surface. in Molecular Catalysis. 2022;522.
doi:10.1016/j.mcat.2022.112215 .

Kovačević, G., Elgahwash, R.G.A., Blažić, M., Pantić, Nevena, Prodanović, Olivera, Balaž, Ana Marija, Prodanović, Radivoje, "Production of fructose and gluconic acid from sucrose with cross-linked yeast cell walls expressing glucose oxidase on the surface" in Molecular Catalysis, 522 (2022),
https://doi.org/10.1016/j.mcat.2022.112215 . .

TY  - JOUR
AU  - Balaž, Ana Marija
AU  - BLazic, Marija B.
AU  - Popović, Nikolina
AU  - Prodanović, Olivera
AU  - Ostafe, Raluca
AU  - Fischer, Rainer
AU  - Prodanović, Radivoje
PY  - 2020
UR  - http://rimsi.imsi.bg.ac.rs/handle/123456789/1297
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 pPICZ alpha 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.
PB  - Srpsko hemijsko društvo, Beograd
T2  - Journal of the Serbian Chemical Society
T1  - Expression, purification and characterization of cellobiose dehydrogenase mutants from Phanerochaete chrysosporium in Pichia pastoris KM71H strain
EP  - 35
IS  - 1
SP  - 25
VL  - 85
DO  - 10.2298/JSC190320058B
ER  - 

@article{
author = "Balaž, Ana Marija and BLazic, Marija B. 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 pPICZ alpha 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.",
publisher = "Srpsko hemijsko društvo, Beograd",
journal = "Journal of the Serbian Chemical Society",
title = "Expression, purification and characterization of cellobiose dehydrogenase mutants from Phanerochaete chrysosporium in Pichia pastoris KM71H strain",
pages = "35-25",
number = "1",
volume = "85",
doi = "10.2298/JSC190320058B"
}

Balaž, A. M., BLazic, M. B., 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
Srpsko hemijsko društvo, Beograd., 85(1), 25-35.
https://doi.org/10.2298/JSC190320058B

Balaž AM, BLazic MB, 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, BLazic, Marija B., 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  - Blazic, Marija
AU  - Balaž, Ana Marija
AU  - Prodanović, Olivera
AU  - Popović, Nikolina
AU  - Ostafe, Raluca
AU  - Fischer, Rainer
AU  - Prodanović, Radivoje
PY  - 2019
UR  - http://rimsi.imsi.bg.ac.rs/handle/123456789/1240
AB  - Featured Application Developed fluorescent assay and expression system can be used for obtaining improved cellobiose dehydrogenase whole cell biocatalysts for lactobionic acid production and building of biosensors and biofuel cells. 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 (10 mM lactose and 0.08 mM cellobiose), 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, Basel
T2  - Applied Sciences-Basel
T1  - Directed Evolution of Cellobiose Dehydrogenase on the Surface of Yeast Cells Using Resazurin-Based Fluorescent Assay
IS  - 7
VL  - 9
DO  - 10.3390/app9071413
ER  - 

@article{
author = "Blazic, Marija and Balaž, Ana Marija and Prodanović, Olivera and Popović, Nikolina and Ostafe, Raluca and Fischer, Rainer and Prodanović, Radivoje",
year = "2019",
abstract = "Featured Application Developed fluorescent assay and expression system can be used for obtaining improved cellobiose dehydrogenase whole cell biocatalysts for lactobionic acid production and building of biosensors and biofuel cells. 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 (10 mM lactose and 0.08 mM cellobiose), 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, Basel",
journal = "Applied Sciences-Basel",
title = "Directed Evolution of Cellobiose Dehydrogenase on the Surface of Yeast Cells Using Resazurin-Based Fluorescent Assay",
number = "7",
volume = "9",
doi = "10.3390/app9071413"
}

Blazic, 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-Basel
MDPI, Basel., 9(7).
https://doi.org/10.3390/app9071413

Blazic 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-Basel. 2019;9(7).
doi:10.3390/app9071413 .

Blazic, 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-Basel, 9, no. 7 (2019),
https://doi.org/10.3390/app9071413 . .

TY  - JOUR
AU  - Prodanović, Olivera
AU  - Spasojević, Dragica
AU  - Prokopijević, Miloš
AU  - Radotić, Ksenija
AU  - Marković, Nevena
AU  - Blazic, Marija
AU  - Prodanović, Radivoje
PY  - 2015
UR  - http://rimsi.imsi.bg.ac.rs/handle/123456789/841
AB  - Phenol and amino groups were introduced into alginate to different degrees via oxidation with 2.5, 5, 10, 15 and 20 mol% of periodate and reductive amination by tyramine. Modification of alginate with tyramine was confirmed by FTIR spectroscopy and UV-VIS spectroscopy, while concentration of phenol and ionizable groups was determined using absorbance at 275 nm and acid-base titration. All tyramine-alginates were able to form hydrogels after cross-linking with horse radish peroxidase (HRP) and hydrogen peroxide. Tyramine-alginates oxidized with up to 10 mol% of periodate were also capable of forming hydrogels with calcium ions. Tyramine-alginates were tested for HRP immobilization within micro-beads obtained by peroxidase catalyzed droplet polymerization using internal delivery of hydrogen peroxide via glucose oxidase and glucose. Highest activity of immobilized peroxidase was obtained with 20% (w/v) tyramine-alginate obtained via 20 mol% periodate oxidation. Immobilized enzyme was not leaking from the micro-beads and was further kinetically characterized for pyrogallol oxidation. Km for pyrogallol was increased after immobilization from 1.93 mM for soluble HRP to 734 mM for immobilized HRP. The optimum pH was also increased from pH 7.0 to 8.0. Temperature and organic solvent stability improved significantly after immobilization, so that half-life at 70 degrees C increased around four times, while half-life in 80% (v/v) dioxane increased 22 times. After repeated use of 6 times in batch reactor for pyrogallol oxidation immobilized HRP retained 45% of original activity.
PB  - Elsevier Science Bv, Amsterdam
T2  - Reactive & Functional Polymers
T1  - Tyramine modified alginates via periodate oxidation for peroxidase induced hydrogel formation and immobilization
EP  - 83
SP  - 77
VL  - 93
DO  - 10.1016/j.reactfunctpolym.2015.06.004
ER  - 

@article{
author = "Prodanović, Olivera and Spasojević, Dragica and Prokopijević, Miloš and Radotić, Ksenija and Marković, Nevena and Blazic, Marija and Prodanović, Radivoje",
year = "2015",
abstract = "Phenol and amino groups were introduced into alginate to different degrees via oxidation with 2.5, 5, 10, 15 and 20 mol% of periodate and reductive amination by tyramine. Modification of alginate with tyramine was confirmed by FTIR spectroscopy and UV-VIS spectroscopy, while concentration of phenol and ionizable groups was determined using absorbance at 275 nm and acid-base titration. All tyramine-alginates were able to form hydrogels after cross-linking with horse radish peroxidase (HRP) and hydrogen peroxide. Tyramine-alginates oxidized with up to 10 mol% of periodate were also capable of forming hydrogels with calcium ions. Tyramine-alginates were tested for HRP immobilization within micro-beads obtained by peroxidase catalyzed droplet polymerization using internal delivery of hydrogen peroxide via glucose oxidase and glucose. Highest activity of immobilized peroxidase was obtained with 20% (w/v) tyramine-alginate obtained via 20 mol% periodate oxidation. Immobilized enzyme was not leaking from the micro-beads and was further kinetically characterized for pyrogallol oxidation. Km for pyrogallol was increased after immobilization from 1.93 mM for soluble HRP to 734 mM for immobilized HRP. The optimum pH was also increased from pH 7.0 to 8.0. Temperature and organic solvent stability improved significantly after immobilization, so that half-life at 70 degrees C increased around four times, while half-life in 80% (v/v) dioxane increased 22 times. After repeated use of 6 times in batch reactor for pyrogallol oxidation immobilized HRP retained 45% of original activity.",
publisher = "Elsevier Science Bv, Amsterdam",
journal = "Reactive & Functional Polymers",
title = "Tyramine modified alginates via periodate oxidation for peroxidase induced hydrogel formation and immobilization",
pages = "83-77",
volume = "93",
doi = "10.1016/j.reactfunctpolym.2015.06.004"
}

Prodanović, O., Spasojević, D., Prokopijević, M., Radotić, K., Marković, N., Blazic, M.,& Prodanović, R.. (2015). Tyramine modified alginates via periodate oxidation for peroxidase induced hydrogel formation and immobilization. in Reactive & Functional Polymers
Elsevier Science Bv, Amsterdam., 93, 77-83.
https://doi.org/10.1016/j.reactfunctpolym.2015.06.004

Prodanović O, Spasojević D, Prokopijević M, Radotić K, Marković N, Blazic M, Prodanović R. Tyramine modified alginates via periodate oxidation for peroxidase induced hydrogel formation and immobilization. in Reactive & Functional Polymers. 2015;93:77-83.
doi:10.1016/j.reactfunctpolym.2015.06.004 .

Prodanović, Olivera, Spasojević, Dragica, Prokopijević, Miloš, Radotić, Ksenija, Marković, Nevena, Blazic, Marija, Prodanović, Radivoje, "Tyramine modified alginates via periodate oxidation for peroxidase induced hydrogel formation and immobilization" in Reactive & Functional Polymers, 93 (2015):77-83,
https://doi.org/10.1016/j.reactfunctpolym.2015.06.004 . .

TY  - JOUR
AU  - Blazic, Marija B
AU  - Kovacević, Gordana
AU  - Prodanović, Olivera
AU  - Ostafe, Raluca
AU  - Gavrovic-Jankulović, Marija D
AU  - Fischer, Rainer
AU  - Prodanović, Radivoje
PY  - 2013
UR  - http://rimsi.imsi.bg.ac.rs/handle/123456789/705
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
EP  - 180
IS  - 2
SP  - 175
VL  - 89
DO  - 10.1016/j.pep.2013.03.014
ER  - 

@article{
author = "Blazic, Marija B and Kovacević, Gordana and Prodanović, Olivera and Ostafe, Raluca and Gavrovic-Jankulović, Marija D 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",
pages = "180-175",
number = "2",
volume = "89",
doi = "10.1016/j.pep.2013.03.014"
}

Blazic, M. B., Kovacević, G., Prodanović, O., Ostafe, R., Gavrovic-Jankulović, M. D., 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

Blazic MB, Kovacević G, Prodanović O, Ostafe R, Gavrovic-Jankulović MD, 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 .

Blazic, Marija B, Kovacević, Gordana, Prodanović, Olivera, Ostafe, Raluca, Gavrovic-Jankulović, Marija D, 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 . .