TY  - JOUR
AU  - Đukić, Ivana
AU  - Kaličanin, Nevena
AU  - Senćanski, Milan
AU  - Pajović, Snežana B.
AU  - Milićević, Jelena
AU  - Prljić, Jelena
AU  - Paessler, Slobodan
AU  - Prodanović, Radivoje
AU  - Glišić, Sanja
PY  - 2023
UR  - https://cer.ihtm.bg.ac.rs/handle/123456789/5629
AB  - Drug resistance is a critical problem in health care that affects therapy outcomes and requires new approaches to drug
design. SARS-CoV-2 Mpro mutations are of concern as they can potentially reduce therapeutic efficacy. Viral infections are amongst the
many disorders for which nutraceuticals have been employed as an adjunct therapy. The aim of this study was to examine the potential
in vitro activity of L-arginine and vitamin C against SARS-CoV-2 Mpro. Methods: The Mpro inhibition assay was developed by cloning,
expression, purification, and characterization of Mpro. Selected compounds were then screened for protease inhibition. Results: Larginine
was found to be active against SARS-CoV-2 Mpro, while a vitamin C/L-arginine combination had a synergistic antiviral action
against Mpro. These findings confirm the results of our previous in silico repurposing study that showed L-arginine and vitamin C were
potential Mpro inhibitors. Moreover, they suggest a possible molecular mechanism to explain the beneficial effect of arginine in COVID
patients. Conclusions: The findings of the current study are important because they help to identify COVID-19 treatments that are
efficient, inexpensive, and have a favorable safety profile. The results of this study also suggest a possible adjuvant nutritional strategy
for COVID-19 that could be used in conjunction with pharmacological agents.
PB  - IMR Press
T2  - Frontiers in Bioscience Landmark
T1  - Inhibition of SARS-CoV-2 Mpro with Vitamin C, L-Arginine and a Vitamin C/L-Arginine Combination
VL  - 28
IS  - 1
SP  - 8
DO  - 10.31083/j.fbl2801008
ER  - 

@article{
author = "Đukić, Ivana and Kaličanin, Nevena and Senćanski, Milan and Pajović, Snežana B. and Milićević, Jelena and Prljić, Jelena and Paessler, Slobodan and Prodanović, Radivoje and Glišić, Sanja",
year = "2023",
abstract = "Drug resistance is a critical problem in health care that affects therapy outcomes and requires new approaches to drug
design. SARS-CoV-2 Mpro mutations are of concern as they can potentially reduce therapeutic efficacy. Viral infections are amongst the
many disorders for which nutraceuticals have been employed as an adjunct therapy. The aim of this study was to examine the potential
in vitro activity of L-arginine and vitamin C against SARS-CoV-2 Mpro. Methods: The Mpro inhibition assay was developed by cloning,
expression, purification, and characterization of Mpro. Selected compounds were then screened for protease inhibition. Results: Larginine
was found to be active against SARS-CoV-2 Mpro, while a vitamin C/L-arginine combination had a synergistic antiviral action
against Mpro. These findings confirm the results of our previous in silico repurposing study that showed L-arginine and vitamin C were
potential Mpro inhibitors. Moreover, they suggest a possible molecular mechanism to explain the beneficial effect of arginine in COVID
patients. Conclusions: The findings of the current study are important because they help to identify COVID-19 treatments that are
efficient, inexpensive, and have a favorable safety profile. The results of this study also suggest a possible adjuvant nutritional strategy
for COVID-19 that could be used in conjunction with pharmacological agents.",
publisher = "IMR Press",
journal = "Frontiers in Bioscience Landmark",
title = "Inhibition of SARS-CoV-2 Mpro with Vitamin C, L-Arginine and a Vitamin C/L-Arginine Combination",
volume = "28",
number = "1",
pages = "8",
doi = "10.31083/j.fbl2801008"
}

Đukić, I., Kaličanin, N., Senćanski, M., Pajović, S. B., Milićević, J., Prljić, J., Paessler, S., Prodanović, R.,& Glišić, S.. (2023). Inhibition of SARS-CoV-2 Mpro with Vitamin C, L-Arginine and a Vitamin C/L-Arginine Combination. in Frontiers in Bioscience Landmark
IMR Press., 28(1), 8.
https://doi.org/10.31083/j.fbl2801008

Đukić I, Kaličanin N, Senćanski M, Pajović SB, Milićević J, Prljić J, Paessler S, Prodanović R, Glišić S. Inhibition of SARS-CoV-2 Mpro with Vitamin C, L-Arginine and a Vitamin C/L-Arginine Combination. in Frontiers in Bioscience Landmark. 2023;28(1):8.
doi:10.31083/j.fbl2801008 .

Đukić, Ivana, Kaličanin, Nevena, Senćanski, Milan, Pajović, Snežana B., Milićević, Jelena, Prljić, Jelena, Paessler, Slobodan, Prodanović, Radivoje, Glišić, Sanja, "Inhibition of SARS-CoV-2 Mpro with Vitamin C, L-Arginine and a Vitamin C/L-Arginine Combination" in Frontiers in Bioscience Landmark, 28, no. 1 (2023):8,
https://doi.org/10.31083/j.fbl2801008 . .

TY  - JOUR
AU  - Protić, Sara
AU  - Kaličanin, Nevena
AU  - Senćanski, Milan
AU  - Prodanović, Olivera
AU  - Milićević, Jelena
AU  - Perović, Vladimir
AU  - Paessler, Slobodan
AU  - Prodanović, Radivoje
AU  - Glišić, Sanja
PY  - 2023
UR  - https://cer.ihtm.bg.ac.rs/handle/123456789/5652
AB  - Finding an effective drug to prevent or treat COVID-19 is of utmost importance in tcurrent
pandemic. Since developing a new treatment takes a significant amount of time, drug repurposing
can be an effective option for achieving a rapid response. This study used a combined in silico virtual
screening protocol for candidate SARS-CoV-2 PLpro inhibitors. The Drugbank database was searched
first, using the Informational Spectrum Method for Small Molecules, followed by molecular docking.
Gramicidin D was selected as a peptide drug, showing the best in silico interaction profile with PLpro.
After the expression and purification of PLpro, gramicidin D was screened for protease inhibition
in vitro and was found to be active against PLpro. The current study’s findings are significant
because it is critical to identify COVID-19 therapies that are efficient, affordable, and have a favorable
safety profile.
PB  - Switzerland : Multidisciplinary Digital Publishing Institute (MDPI)
T2  - International Journal of Molecular Sciences
T1  - In Silico and In Vitro Inhibition of SARS-CoV-2 PLpro with Gramicidin D
VL  - 24
IS  - 3
SP  - 1955
DO  - 10.3390/ijms24031955
ER  - 

@article{
author = "Protić, Sara and Kaličanin, Nevena and Senćanski, Milan and Prodanović, Olivera and Milićević, Jelena and Perović, Vladimir and Paessler, Slobodan and Prodanović, Radivoje and Glišić, Sanja",
year = "2023",
abstract = "Finding an effective drug to prevent or treat COVID-19 is of utmost importance in tcurrent
pandemic. Since developing a new treatment takes a significant amount of time, drug repurposing
can be an effective option for achieving a rapid response. This study used a combined in silico virtual
screening protocol for candidate SARS-CoV-2 PLpro inhibitors. The Drugbank database was searched
first, using the Informational Spectrum Method for Small Molecules, followed by molecular docking.
Gramicidin D was selected as a peptide drug, showing the best in silico interaction profile with PLpro.
After the expression and purification of PLpro, gramicidin D was screened for protease inhibition
in vitro and was found to be active against PLpro. The current study’s findings are significant
because it is critical to identify COVID-19 therapies that are efficient, affordable, and have a favorable
safety profile.",
publisher = "Switzerland : Multidisciplinary Digital Publishing Institute (MDPI)",
journal = "International Journal of Molecular Sciences",
title = "In Silico and In Vitro Inhibition of SARS-CoV-2 PLpro with Gramicidin D",
volume = "24",
number = "3",
pages = "1955",
doi = "10.3390/ijms24031955"
}

Protić, S., Kaličanin, N., Senćanski, M., Prodanović, O., Milićević, J., Perović, V., Paessler, S., Prodanović, R.,& Glišić, S.. (2023). In Silico and In Vitro Inhibition of SARS-CoV-2 PLpro with Gramicidin D. in International Journal of Molecular Sciences
Switzerland : Multidisciplinary Digital Publishing Institute (MDPI)., 24(3), 1955.
https://doi.org/10.3390/ijms24031955

Protić S, Kaličanin N, Senćanski M, Prodanović O, Milićević J, Perović V, Paessler S, Prodanović R, Glišić S. In Silico and In Vitro Inhibition of SARS-CoV-2 PLpro with Gramicidin D. in International Journal of Molecular Sciences. 2023;24(3):1955.
doi:10.3390/ijms24031955 .

Protić, Sara, Kaličanin, Nevena, Senćanski, Milan, Prodanović, Olivera, Milićević, Jelena, Perović, Vladimir, Paessler, Slobodan, Prodanović, Radivoje, Glišić, Sanja, "In Silico and In Vitro Inhibition of SARS-CoV-2 PLpro with Gramicidin D" in International Journal of Molecular Sciences, 24, no. 3 (2023):1955,
https://doi.org/10.3390/ijms24031955 . .

TY  - JOUR
AU  - Kaličanin, Nevena
AU  - Balaž, Ana Marija
AU  - Prodanović, Olivera
AU  - Prodanović, Radivoje
PY  - 2023
UR  - https://cer.ihtm.bg.ac.rs/handle/123456789/7171
AB  - The aim of this research was to prove the function of the putative opine dehydrogenase from Desulfohalobium retbaense and to characterize the enzyme in terms of functional and kinetic parameters. A putative opine dehydrogenase was identified from a metagenomic library by a sequence-based technique search of the metagenomic library, and afterward was successfully heterologously produced in Escherichia coli. In order to examine its potential for applications in the synthesis of secondary amines, first the substrate specificity of the enzyme towards different amino donors and amino acceptors was determined. The highest affinity was observed towards small amino acids, preferentially L-alanine, and when it comes to α-keto acids, pyruvate proved to be a preferential amino acceptor. The highest activity was observed at pH 6.5 in the absence of salts. The enzyme showed remarkable stability in a wide range of experimental conditions, such as broad pH stability (from 6.0–11.0 after 30 min incubation in buffers at a certain pH), stability in the presence of NaCl up to 3.0 M for 24 h, it retained 80% of the initial activity after 1 h incubation at 45°C, and 65% of the initial activity after 24 h incubation in 30% dimethyl sulfoxide.
PB  - Wiley-VCH GmbH
T2  - ChemBioChem
T1  - Heterologous Expression and Partial Characterization of a Putative Opine Dehydrogenase from a Metagenomic Sequence of Desulfohalobium retbaense
VL  - 24
IS  - 20
IS  - e202300414
DO  - 10.1002/cbic.202300414
ER  - 

@article{
author = "Kaličanin, Nevena and Balaž, Ana Marija and Prodanović, Olivera and Prodanović, Radivoje",
year = "2023",
abstract = "The aim of this research was to prove the function of the putative opine dehydrogenase from Desulfohalobium retbaense and to characterize the enzyme in terms of functional and kinetic parameters. A putative opine dehydrogenase was identified from a metagenomic library by a sequence-based technique search of the metagenomic library, and afterward was successfully heterologously produced in Escherichia coli. In order to examine its potential for applications in the synthesis of secondary amines, first the substrate specificity of the enzyme towards different amino donors and amino acceptors was determined. The highest affinity was observed towards small amino acids, preferentially L-alanine, and when it comes to α-keto acids, pyruvate proved to be a preferential amino acceptor. The highest activity was observed at pH 6.5 in the absence of salts. The enzyme showed remarkable stability in a wide range of experimental conditions, such as broad pH stability (from 6.0–11.0 after 30 min incubation in buffers at a certain pH), stability in the presence of NaCl up to 3.0 M for 24 h, it retained 80% of the initial activity after 1 h incubation at 45°C, and 65% of the initial activity after 24 h incubation in 30% dimethyl sulfoxide.",
publisher = "Wiley-VCH GmbH",
journal = "ChemBioChem",
title = "Heterologous Expression and Partial Characterization of a Putative Opine Dehydrogenase from a Metagenomic Sequence of Desulfohalobium retbaense",
volume = "24",
number = "20, e202300414",
doi = "10.1002/cbic.202300414"
}

Kaličanin, N., Balaž, A. M., Prodanović, O.,& Prodanović, R.. (2023). Heterologous Expression and Partial Characterization of a Putative Opine Dehydrogenase from a Metagenomic Sequence of Desulfohalobium retbaense. in ChemBioChem
Wiley-VCH GmbH., 24(20).
https://doi.org/10.1002/cbic.202300414

Kaličanin N, Balaž AM, Prodanović O, Prodanović R. Heterologous Expression and Partial Characterization of a Putative Opine Dehydrogenase from a Metagenomic Sequence of Desulfohalobium retbaense. in ChemBioChem. 2023;24(20).
doi:10.1002/cbic.202300414 .

Kaličanin, Nevena, Balaž, Ana Marija, Prodanović, Olivera, Prodanović, Radivoje, "Heterologous Expression and Partial Characterization of a Putative Opine Dehydrogenase from a Metagenomic Sequence of Desulfohalobium retbaense" in ChemBioChem, 24, no. 20 (2023),
https://doi.org/10.1002/cbic.202300414 . .

TY  - JOUR
AU  - Pantić, Nevena
AU  - Spasojević, Milica
AU  - Stojanović, Željko
AU  - Veljović, Đorđe
AU  - Krstić, Jugoslav
AU  - Balaž, Ana Marija
AU  - Prodanović, Radivoje
AU  - Prodanović, Olivera
PY  - 2022
UR  - https://cer.ihtm.bg.ac.rs/handle/123456789/7532
AB  - Novel macroporous copolymers of glycidyl methacrylate and ethylene glycol dimethacrylate with mean pore size diameters ranging from 150 to 310 nm were synthesized by dispersion polymerization and modified with ethylenediamine. The glutaraldehyde and periodate method were employed to immobilize horseradish peroxidase (HRP) onto these carriers. The activity of the immobilized enzyme was greatly affected by the pore size of the carrier. The highest specific activities of 9.65 and 8.94 U/g of dry weight were obtained for HRP immobilized by the periodate-route onto poly(GMA‐co‐EGDMA) carriers with pore size diameters of 234 and 297 nm, respectively. Stability studies showed an improved operational stability of immobilized peroxidase at 65 °C and in an organic solvent. HRP immobilized on a copolymer with a pore size of 234 nm, showing the highest specific activity and good stability, had higher activities at almost all pH values than the native enzyme and the increased Km value for pyrogallol oxidation. Immobilized HRP retained 80% of its original activity after five consecutive cycles of the pyrogallol oxidation and 98% of its initial activity in a storage stability study. Enzyme immobilized onto the macroporous copolymer with the pore size diameter of 234 nm showed a substantial degree of phenol removal achieved by immobilized peroxidase.
PB  - Springer
T2  - Journal of Polymers and the Environment
T1  - Immobilization of Horseradish Peroxidase on Macroporous Glycidyl-Based Copolymers with Different Surface Characteristics for the Removal of Phenol
VL  - 30
SP  - 3005
EP  - 3020
DO  - 10.1007/s10924-021-02364-3
ER  - 

@article{
author = "Pantić, Nevena and Spasojević, Milica and Stojanović, Željko and Veljović, Đorđe and Krstić, Jugoslav and Balaž, Ana Marija and Prodanović, Radivoje and Prodanović, Olivera",
year = "2022",
abstract = "Novel macroporous copolymers of glycidyl methacrylate and ethylene glycol dimethacrylate with mean pore size diameters ranging from 150 to 310 nm were synthesized by dispersion polymerization and modified with ethylenediamine. The glutaraldehyde and periodate method were employed to immobilize horseradish peroxidase (HRP) onto these carriers. The activity of the immobilized enzyme was greatly affected by the pore size of the carrier. The highest specific activities of 9.65 and 8.94 U/g of dry weight were obtained for HRP immobilized by the periodate-route onto poly(GMA‐co‐EGDMA) carriers with pore size diameters of 234 and 297 nm, respectively. Stability studies showed an improved operational stability of immobilized peroxidase at 65 °C and in an organic solvent. HRP immobilized on a copolymer with a pore size of 234 nm, showing the highest specific activity and good stability, had higher activities at almost all pH values than the native enzyme and the increased Km value for pyrogallol oxidation. Immobilized HRP retained 80% of its original activity after five consecutive cycles of the pyrogallol oxidation and 98% of its initial activity in a storage stability study. Enzyme immobilized onto the macroporous copolymer with the pore size diameter of 234 nm showed a substantial degree of phenol removal achieved by immobilized peroxidase.",
publisher = "Springer",
journal = "Journal of Polymers and the Environment",
title = "Immobilization of Horseradish Peroxidase on Macroporous Glycidyl-Based Copolymers with Different Surface Characteristics for the Removal of Phenol",
volume = "30",
pages = "3005-3020",
doi = "10.1007/s10924-021-02364-3"
}

Pantić, N., Spasojević, M., Stojanović, Ž., Veljović, Đ., Krstić, J., Balaž, A. M., Prodanović, R.,& Prodanović, O.. (2022). Immobilization of Horseradish Peroxidase on Macroporous Glycidyl-Based Copolymers with Different Surface Characteristics for the Removal of Phenol. in Journal of Polymers and the Environment
Springer., 30, 3005-3020.
https://doi.org/10.1007/s10924-021-02364-3

Pantić N, Spasojević M, Stojanović Ž, Veljović Đ, Krstić J, Balaž AM, Prodanović R, Prodanović O. Immobilization of Horseradish Peroxidase on Macroporous Glycidyl-Based Copolymers with Different Surface Characteristics for the Removal of Phenol. in Journal of Polymers and the Environment. 2022;30:3005-3020.
doi:10.1007/s10924-021-02364-3 .

Pantić, Nevena, Spasojević, Milica, Stojanović, Željko, Veljović, Đorđe, Krstić, Jugoslav, Balaž, Ana Marija, Prodanović, Radivoje, Prodanović, Olivera, "Immobilization of Horseradish Peroxidase on Macroporous Glycidyl-Based Copolymers with Different Surface Characteristics for the Removal of Phenol" in Journal of Polymers and the Environment, 30 (2022):3005-3020,
https://doi.org/10.1007/s10924-021-02364-3 . .

Stanišić, M., Ristić, P., Balaž, A. M., Senćanski, M., Mitić, D., Prodanović, R.,& Todorović, T.. (2022). Efficient enzyme@MOF composites for biocatalysis. in Book of abstracts - EUROBIOTECH 8th Central European Congress of Life Sciences, 20-22 June 2022, Krakow, Poland, 138-138.
https://hdl.handle.net/21.15107/rcub_cherry_5751

Stanišić M, Ristić P, Balaž AM, Senćanski M, Mitić D, Prodanović R, Todorović T. Efficient enzyme@MOF composites for biocatalysis. in Book of abstracts - EUROBIOTECH 8th Central European Congress of Life Sciences, 20-22 June 2022, Krakow, Poland. 2022;:138-138.
https://hdl.handle.net/21.15107/rcub_cherry_5751 .

Stanišić, Marija, Ristić, Predrag, Balaž, Ana Marija, Senćanski, Milan, Mitić, Dragana, Prodanović, Radivoje, Todorović, Tamara, "Efficient enzyme@MOF composites for biocatalysis" in Book of abstracts - EUROBIOTECH 8th Central European Congress of Life Sciences, 20-22 June 2022, Krakow, Poland (2022):138-138,
https://hdl.handle.net/21.15107/rcub_cherry_5751 .

TY  - JOUR
AU  - Kaličanin, Nevena
AU  - Kovačević, Gordana
AU  - Spasojević, Milica
AU  - Prodanović, Olivera
AU  - Jovanović-Šanta, Suzana
AU  - Škorić, Dušan
AU  - Opsenica, Dejan
AU  - Prodanović, Radivoje
PY  - 2022
UR  - https://cer.ihtm.bg.ac.rs/handle/123456789/5370
AB  - The aim of this research was to improve the operational stability and enable the reusability of ω-transaminase for synthesis of new enantiopure chiral amines of steroids. Dihydrotestosterone was used to optimize the synthetic procedure of corresponding amino-steroid on a larger scale. The obtained product 3α-amino-5α-androstan-17β-ol was isolated and characterized. The enzyme was immobilized on a methacrylate-based carrier, giving the specific activity of 1.84 U/g of dry polymer. Higher residual activity of the immobilized enzyme in comparison to the soluble form (100 % versus 35%) after 24 h incubation in 35 % dimethylformamide (DMF) was obtained. The soluble enzyme retained 19 % of the initial activity after 2 h incubation in 35 % DMF at 70 °C, while the activity of the immobilized enzyme decreased only to 75 %. Immobilized retained 85 % of initial activity after ten consecutive cycles of 3α-amino-5α-androstan-17β-ol synthesis. We have tested the specificity of the ArRMut11 variant, further increased its stability by immobilization, and used it in several cycles for the synthesis of 3α-amino-5α-androstan-17β-ol. We showed that the enzyme previously evolved for higher stability as the immobilized variant showed more increased stability and high reusability that can more effectively be applied for the biosynthesis of amino steroids.
PB  - Elsevier
T2  - Process Biochemistry
T1  - Immobilization of ArRMut11 omega-transaminase for increased operational stability and reusability in the synthesis of 3α-amino-5α-androstan-17β-ol
VL  - 121
SP  - 674
EP  - 680
DO  - 10.1016/j.procbio.2022.08.016
ER  - 

@article{
author = "Kaličanin, Nevena and Kovačević, Gordana and Spasojević, Milica and Prodanović, Olivera and Jovanović-Šanta, Suzana and Škorić, Dušan and Opsenica, Dejan and Prodanović, Radivoje",
year = "2022",
abstract = "The aim of this research was to improve the operational stability and enable the reusability of ω-transaminase for synthesis of new enantiopure chiral amines of steroids. Dihydrotestosterone was used to optimize the synthetic procedure of corresponding amino-steroid on a larger scale. The obtained product 3α-amino-5α-androstan-17β-ol was isolated and characterized. The enzyme was immobilized on a methacrylate-based carrier, giving the specific activity of 1.84 U/g of dry polymer. Higher residual activity of the immobilized enzyme in comparison to the soluble form (100 % versus 35%) after 24 h incubation in 35 % dimethylformamide (DMF) was obtained. The soluble enzyme retained 19 % of the initial activity after 2 h incubation in 35 % DMF at 70 °C, while the activity of the immobilized enzyme decreased only to 75 %. Immobilized retained 85 % of initial activity after ten consecutive cycles of 3α-amino-5α-androstan-17β-ol synthesis. We have tested the specificity of the ArRMut11 variant, further increased its stability by immobilization, and used it in several cycles for the synthesis of 3α-amino-5α-androstan-17β-ol. We showed that the enzyme previously evolved for higher stability as the immobilized variant showed more increased stability and high reusability that can more effectively be applied for the biosynthesis of amino steroids.",
publisher = "Elsevier",
journal = "Process Biochemistry",
title = "Immobilization of ArRMut11 omega-transaminase for increased operational stability and reusability in the synthesis of 3α-amino-5α-androstan-17β-ol",
volume = "121",
pages = "674-680",
doi = "10.1016/j.procbio.2022.08.016"
}

Kaličanin, N., Kovačević, G., Spasojević, M., Prodanović, O., Jovanović-Šanta, S., Škorić, D., Opsenica, D.,& Prodanović, R.. (2022). Immobilization of ArRMut11 omega-transaminase for increased operational stability and reusability in the synthesis of 3α-amino-5α-androstan-17β-ol. in Process Biochemistry
Elsevier., 121, 674-680.
https://doi.org/10.1016/j.procbio.2022.08.016

Kaličanin N, Kovačević G, Spasojević M, Prodanović O, Jovanović-Šanta S, Škorić D, Opsenica D, Prodanović R. Immobilization of ArRMut11 omega-transaminase for increased operational stability and reusability in the synthesis of 3α-amino-5α-androstan-17β-ol. in Process Biochemistry. 2022;121:674-680.
doi:10.1016/j.procbio.2022.08.016 .

Kaličanin, Nevena, Kovačević, Gordana, Spasojević, Milica, Prodanović, Olivera, Jovanović-Šanta, Suzana, Škorić, Dušan, Opsenica, Dejan, Prodanović, Radivoje, "Immobilization of ArRMut11 omega-transaminase for increased operational stability and reusability in the synthesis of 3α-amino-5α-androstan-17β-ol" in Process Biochemistry, 121 (2022):674-680,
https://doi.org/10.1016/j.procbio.2022.08.016 . .

TY  - JOUR
AU  - Kaličanin, Nevena
AU  - Kovačević, Gordana
AU  - Spasojević, Milica
AU  - Prodanović, Olivera
AU  - Jovanović-Šanta, Suzana
AU  - Škorić, Dušan
AU  - Opsenica, Dejan
AU  - Prodanović, Radivoje
PY  - 2022
UR  - https://cer.ihtm.bg.ac.rs/handle/123456789/5545
AB  - The aim of this research was to improve the operational stability and enable the reusability of ω-transaminase for synthesis of new enantiopure chiral amines of steroids. Dihydrotestosterone was used to optimize the synthetic procedure of corresponding amino-steroid on a larger scale. The obtained product 3α-amino-5α-androstan-17β-ol was isolated and characterized. The enzyme was immobilized on a methacrylate-based carrier, giving the specific activity of 1.84 U/g of dry polymer. Higher residual activity of the immobilized enzyme in comparison to the soluble form (100 % versus 35%) after 24 h incubation in 35 % dimethylformamide (DMF) was obtained. The soluble enzyme retained 19 % of the initial activity after 2 h incubation in 35 % DMF at 70 °C, while the activity of the immobilized enzyme decreased only to 75 %. Immobilized retained 85 % of initial activity after ten consecutive cycles of 3α-amino-5α-androstan-17β-ol synthesis. We have tested the specificity of the ArRMut11 variant, further increased its stability by immobilization, and used it in several cycles for the synthesis of 3α-amino-5α-androstan-17β-ol. We showed that the enzyme previously evolved for higher stability as the immobilized variant showed more increased stability and high reusability that can more effectively be applied for the biosynthesis of amino steroids.
PB  - Elsevier
T2  - Process Biochemistry
T1  - Immobilization of ArRMut11 omega-transaminase for increased operational stability and reusability in the synthesis of 3α-amino-5α-androstan-17β-ol
VL  - 121
SP  - 674
EP  - 680
DO  - 10.1016/j.procbio.2022.08.016
ER  - 

@article{
author = "Kaličanin, Nevena and Kovačević, Gordana and Spasojević, Milica and Prodanović, Olivera and Jovanović-Šanta, Suzana and Škorić, Dušan and Opsenica, Dejan and Prodanović, Radivoje",
year = "2022",
abstract = "The aim of this research was to improve the operational stability and enable the reusability of ω-transaminase for synthesis of new enantiopure chiral amines of steroids. Dihydrotestosterone was used to optimize the synthetic procedure of corresponding amino-steroid on a larger scale. The obtained product 3α-amino-5α-androstan-17β-ol was isolated and characterized. The enzyme was immobilized on a methacrylate-based carrier, giving the specific activity of 1.84 U/g of dry polymer. Higher residual activity of the immobilized enzyme in comparison to the soluble form (100 % versus 35%) after 24 h incubation in 35 % dimethylformamide (DMF) was obtained. The soluble enzyme retained 19 % of the initial activity after 2 h incubation in 35 % DMF at 70 °C, while the activity of the immobilized enzyme decreased only to 75 %. Immobilized retained 85 % of initial activity after ten consecutive cycles of 3α-amino-5α-androstan-17β-ol synthesis. We have tested the specificity of the ArRMut11 variant, further increased its stability by immobilization, and used it in several cycles for the synthesis of 3α-amino-5α-androstan-17β-ol. We showed that the enzyme previously evolved for higher stability as the immobilized variant showed more increased stability and high reusability that can more effectively be applied for the biosynthesis of amino steroids.",
publisher = "Elsevier",
journal = "Process Biochemistry",
title = "Immobilization of ArRMut11 omega-transaminase for increased operational stability and reusability in the synthesis of 3α-amino-5α-androstan-17β-ol",
volume = "121",
pages = "674-680",
doi = "10.1016/j.procbio.2022.08.016"
}

Kaličanin, N., Kovačević, G., Spasojević, M., Prodanović, O., Jovanović-Šanta, S., Škorić, D., Opsenica, D.,& Prodanović, R.. (2022). Immobilization of ArRMut11 omega-transaminase for increased operational stability and reusability in the synthesis of 3α-amino-5α-androstan-17β-ol. in Process Biochemistry
Elsevier., 121, 674-680.
https://doi.org/10.1016/j.procbio.2022.08.016

Kaličanin N, Kovačević G, Spasojević M, Prodanović O, Jovanović-Šanta S, Škorić D, Opsenica D, Prodanović R. Immobilization of ArRMut11 omega-transaminase for increased operational stability and reusability in the synthesis of 3α-amino-5α-androstan-17β-ol. in Process Biochemistry. 2022;121:674-680.
doi:10.1016/j.procbio.2022.08.016 .

Kaličanin, Nevena, Kovačević, Gordana, Spasojević, Milica, Prodanović, Olivera, Jovanović-Šanta, Suzana, Škorić, Dušan, Opsenica, Dejan, Prodanović, Radivoje, "Immobilization of ArRMut11 omega-transaminase for increased operational stability and reusability in the synthesis of 3α-amino-5α-androstan-17β-ol" in Process Biochemistry, 121 (2022):674-680,
https://doi.org/10.1016/j.procbio.2022.08.016 . .

TY  - CONF
AU  - Kaličanin, Nevena
AU  - Balaž, Ana Marija
AU  - Prodanović, Olivera
AU  - Prodanović, Radivoje
PY  - 2022
UR  - https://cer.ihtm.bg.ac.rs/handle/123456789/7099
AB  - Opine dehydrogenases are a family of NAD(P)H dependent oxidoreductases, whichcatalyze the reductive condensation of an α amino group from an amino acid with an αketo acid during anaerobic glycolysis by regenerating NAD. They are widespread incephalopods and mollusks. Opines are associated with crown gall tumor pathogenesiscaused by A. tumefaciens providing nutrients to the pathogen, and novel opine compoundsacting as metallophores have been identified. Besides, opine-type secondary aminedicarboxylic acids are chiral intermediates of angiotensin-converting enzyme inhibitors. Anovel enzyme originating from an extremophile bacterium, with assumed opinedehydrogenase function was successfully expressed in Escherichia coli STAR cells andpurified by affinity chromatography. Molecular mass determined by SDS-PAGE wasapproximately 40 kDa. The activity was measured by using pyruvate and alanine assubstrates, by which proved that it has opine dehydrogenase activity.
PB  - Faculty of Chemistry
PB  - Serbian Biochemical Society
C3  - Proceedings - Eleventh Conference, Scientific meeting of an international character "Amazing Biochemistry", September 22nd and 23rd, 2022, Novi Sad, Serbia
T1  - Production of a novel opine dehydrogenase
SP  - 79
EP  - 79
UR  - https://hdl.handle.net/21.15107/rcub_cer_7099
ER  - 

@conference{
author = "Kaličanin, Nevena and Balaž, Ana Marija and Prodanović, Olivera and Prodanović, Radivoje",
year = "2022",
abstract = "Opine dehydrogenases are a family of NAD(P)H dependent oxidoreductases, whichcatalyze the reductive condensation of an α amino group from an amino acid with an αketo acid during anaerobic glycolysis by regenerating NAD. They are widespread incephalopods and mollusks. Opines are associated with crown gall tumor pathogenesiscaused by A. tumefaciens providing nutrients to the pathogen, and novel opine compoundsacting as metallophores have been identified. Besides, opine-type secondary aminedicarboxylic acids are chiral intermediates of angiotensin-converting enzyme inhibitors. Anovel enzyme originating from an extremophile bacterium, with assumed opinedehydrogenase function was successfully expressed in Escherichia coli STAR cells andpurified by affinity chromatography. Molecular mass determined by SDS-PAGE wasapproximately 40 kDa. The activity was measured by using pyruvate and alanine assubstrates, by which proved that it has opine dehydrogenase activity.",
publisher = "Faculty of Chemistry, Serbian Biochemical Society",
journal = "Proceedings - Eleventh Conference, Scientific meeting of an international character "Amazing Biochemistry", September 22nd and 23rd, 2022, Novi Sad, Serbia",
title = "Production of a novel opine dehydrogenase",
pages = "79-79",
url = "https://hdl.handle.net/21.15107/rcub_cer_7099"
}

Kaličanin, N., Balaž, A. M., Prodanović, O.,& Prodanović, R.. (2022). Production of a novel opine dehydrogenase. in Proceedings - Eleventh Conference, Scientific meeting of an international character "Amazing Biochemistry", September 22nd and 23rd, 2022, Novi Sad, Serbia
Faculty of Chemistry., 79-79.
https://hdl.handle.net/21.15107/rcub_cer_7099

Kaličanin N, Balaž AM, Prodanović O, Prodanović R. Production of a novel opine dehydrogenase. in Proceedings - Eleventh Conference, Scientific meeting of an international character "Amazing Biochemistry", September 22nd and 23rd, 2022, Novi Sad, Serbia. 2022;:79-79.
https://hdl.handle.net/21.15107/rcub_cer_7099 .

Kaličanin, Nevena, Balaž, Ana Marija, Prodanović, Olivera, Prodanović, Radivoje, "Production of a novel opine dehydrogenase" in Proceedings - Eleventh Conference, Scientific meeting of an international character "Amazing Biochemistry", September 22nd and 23rd, 2022, Novi Sad, Serbia (2022):79-79,
https://hdl.handle.net/21.15107/rcub_cer_7099 .

TY  - JOUR
AU  - Stanišić, Marija D.
AU  - Popović Kokar, Nikolina
AU  - Ristić, Predrag
AU  - Balaž, Ana Marija
AU  - Ognjanović, Miloš
AU  - Đokić, Veljko R.
AU  - Prodanović, Radivoje
AU  - Todorović, Tamara
PY  - 2022
UR  - https://cer.ihtm.bg.ac.rs/handle/123456789/5561
AB  - Many articles in the literature deal with horseradish peroxidase (HRP) biomineralization,but none pay attention to the isoenzyme composition of commercial HRP or the influence of thecarbohydrate component of the protein molecule on the biomineralization process. To study theimpact of these factors, we performed periodate oxidation of commercial HRP and a purified HRP-Cisoform for biomineralization within ZIF-8. With purified HRP, enzyme@ZIF-8 biocomposites withhigher activity were obtained, while periodate oxidation of the carbohydrate component of bothcommercial HRP and purified HRP-C yields biocomposites with very high activity in acetate bufferthat does not degrade the ZIF-8 structure. Using acetate instead of phosphate buffer can prevent thefalse high activity of HRP@ZIF-8 biocomposites caused by the degradation of ZIF-8 coating. At thesame time, purification and especially oxidation of the carbohydrate component of enzymes prior tobiomineralization lead to significantly improved activity of the biocomposites.
PB  - MDPI
T2  - Polymers
T1  - The influence of isoenzymes composition and chemical modification on horseradish peroxidase@ZIF-8 bio-composite performance
VL  - 14
IS  - 22
SP  - 4834
DO  - 10.3390/polym14224834
ER  - 

@article{
author = "Stanišić, Marija D. and Popović Kokar, Nikolina and Ristić, Predrag and Balaž, Ana Marija and Ognjanović, Miloš and Đokić, Veljko R. and Prodanović, Radivoje and Todorović, Tamara",
year = "2022",
abstract = "Many articles in the literature deal with horseradish peroxidase (HRP) biomineralization,but none pay attention to the isoenzyme composition of commercial HRP or the influence of thecarbohydrate component of the protein molecule on the biomineralization process. To study theimpact of these factors, we performed periodate oxidation of commercial HRP and a purified HRP-Cisoform for biomineralization within ZIF-8. With purified HRP, enzyme@ZIF-8 biocomposites withhigher activity were obtained, while periodate oxidation of the carbohydrate component of bothcommercial HRP and purified HRP-C yields biocomposites with very high activity in acetate bufferthat does not degrade the ZIF-8 structure. Using acetate instead of phosphate buffer can prevent thefalse high activity of HRP@ZIF-8 biocomposites caused by the degradation of ZIF-8 coating. At thesame time, purification and especially oxidation of the carbohydrate component of enzymes prior tobiomineralization lead to significantly improved activity of the biocomposites.",
publisher = "MDPI",
journal = "Polymers",
title = "The influence of isoenzymes composition and chemical modification on horseradish peroxidase@ZIF-8 bio-composite performance",
volume = "14",
number = "22",
pages = "4834",
doi = "10.3390/polym14224834"
}

Stanišić, M. D., Popović Kokar, N., Ristić, P., Balaž, A. M., Ognjanović, M., Đokić, V. R., Prodanović, R.,& Todorović, T.. (2022). The influence of isoenzymes composition and chemical modification on horseradish peroxidase@ZIF-8 bio-composite performance. in Polymers
MDPI., 14(22), 4834.
https://doi.org/10.3390/polym14224834

Stanišić MD, Popović Kokar N, Ristić P, Balaž AM, Ognjanović M, Đokić VR, Prodanović R, Todorović T. The influence of isoenzymes composition and chemical modification on horseradish peroxidase@ZIF-8 bio-composite performance. in Polymers. 2022;14(22):4834.
doi:10.3390/polym14224834 .

Stanišić, Marija D., Popović Kokar, Nikolina, Ristić, Predrag, Balaž, Ana Marija, Ognjanović, Miloš, Đokić, Veljko R., Prodanović, Radivoje, Todorović, Tamara, "The influence of isoenzymes composition and chemical modification on horseradish peroxidase@ZIF-8 bio-composite performance" in Polymers, 14, no. 22 (2022):4834,
https://doi.org/10.3390/polym14224834 . .

TY  - CONF
AU  - Ristić, Predrag
AU  - Stanišić, Marija
AU  - Đokić, Veljko
AU  - Balaž, Ana Marija
AU  - Mitić, Dragana
AU  - Prodanović, Radivoje
AU  - Todorović, Tamara
PY  - 2022
UR  - https://cer.ihtm.bg.ac.rs/handle/123456789/5563
AB  - The durability of enzymes in harsh conditions can be enhanced by immobilization within metal-organic frameworks(MOFs) via a process called biomimetic mineralisation. Zeolitic imidazolate framework-8 (ZIF-8) is widely used as aprotective coating to encapsulate proteins. The formation of nucleation centres and further biocomposite particlegrowth is entirely governed by the pure electrostatic interactions between the protein’s surface and positively chargedZn(II) metal ions. It was previously shown that enhancing these electrostatic interactions by a chemical modificationof surface amino acid residues can lead to a rapid biocomposite formation. However, a chemical modification ofcarbohydrate components by periodate oxidation for glycoproteins can serve as an alternative strategy. In the presentstudy, an industrially important enzyme glucose oxidase (GOx) was selected as a model system. Periodate oxidationof GOx by 2.5 mM sodium periodate increased negative charge on the enzyme molecule. Biomineralizationexperiments with oxidized GOx resulted in higher specific activity, effectiveness factor, and higher thermostability ofthe ZIF-8 biocomposites.
C3  - Book of abstracts - NANOTEXNOLOGY NN22, 19th International Conference on Nanosciences & Nanotechnologies, 5-8 July 2022, Thessaloniki, Greece
T1  - Periodate oxidized glucose oxidase@ZIF-8 nanocomposite
SP  - 138
EP  - 138
UR  - https://hdl.handle.net/21.15107/rcub_cherry_5753
ER  - 

@conference{
author = "Ristić, Predrag and Stanišić, Marija and Đokić, Veljko and Balaž, Ana Marija and Mitić, Dragana and Prodanović, Radivoje and Todorović, Tamara",
year = "2022",
abstract = "The durability of enzymes in harsh conditions can be enhanced by immobilization within metal-organic frameworks(MOFs) via a process called biomimetic mineralisation. Zeolitic imidazolate framework-8 (ZIF-8) is widely used as aprotective coating to encapsulate proteins. The formation of nucleation centres and further biocomposite particlegrowth is entirely governed by the pure electrostatic interactions between the protein’s surface and positively chargedZn(II) metal ions. It was previously shown that enhancing these electrostatic interactions by a chemical modificationof surface amino acid residues can lead to a rapid biocomposite formation. However, a chemical modification ofcarbohydrate components by periodate oxidation for glycoproteins can serve as an alternative strategy. In the presentstudy, an industrially important enzyme glucose oxidase (GOx) was selected as a model system. Periodate oxidationof GOx by 2.5 mM sodium periodate increased negative charge on the enzyme molecule. Biomineralizationexperiments with oxidized GOx resulted in higher specific activity, effectiveness factor, and higher thermostability ofthe ZIF-8 biocomposites.",
journal = "Book of abstracts - NANOTEXNOLOGY NN22, 19th International Conference on Nanosciences & Nanotechnologies, 5-8 July 2022, Thessaloniki, Greece",
title = "Periodate oxidized glucose oxidase@ZIF-8 nanocomposite",
pages = "138-138",
url = "https://hdl.handle.net/21.15107/rcub_cherry_5753"
}

Ristić, P., Stanišić, M., Đokić, V., Balaž, A. M., Mitić, D., Prodanović, R.,& Todorović, T.. (2022). Periodate oxidized glucose oxidase@ZIF-8 nanocomposite. in Book of abstracts - NANOTEXNOLOGY NN22, 19th International Conference on Nanosciences & Nanotechnologies, 5-8 July 2022, Thessaloniki, Greece, 138-138.
https://hdl.handle.net/21.15107/rcub_cherry_5753

Ristić P, Stanišić M, Đokić V, Balaž AM, Mitić D, Prodanović R, Todorović T. Periodate oxidized glucose oxidase@ZIF-8 nanocomposite. in Book of abstracts - NANOTEXNOLOGY NN22, 19th International Conference on Nanosciences & Nanotechnologies, 5-8 July 2022, Thessaloniki, Greece. 2022;:138-138.
https://hdl.handle.net/21.15107/rcub_cherry_5753 .

Ristić, Predrag, Stanišić, Marija, Đokić, Veljko, Balaž, Ana Marija, Mitić, Dragana, Prodanović, Radivoje, Todorović, Tamara, "Periodate oxidized glucose oxidase@ZIF-8 nanocomposite" in Book of abstracts - NANOTEXNOLOGY NN22, 19th International Conference on Nanosciences & Nanotechnologies, 5-8 July 2022, Thessaloniki, Greece (2022):138-138,
https://hdl.handle.net/21.15107/rcub_cherry_5753 .

TY  - CONF
AU  - Stanišić, Marija
AU  - Ristić, Predrag
AU  - Đokić, Veljko
AU  - Balaž, Ana Marija
AU  - Prodanović, Radivoje
AU  - Todorović, Tamara
PY  - 2022
UR  - https://cer.ihtm.bg.ac.rs/handle/123456789/5562
AB  - Metal-organic frameworks (MOFs) are a class of materials well-known for their high degree of crystallinity andultrahigh porosity. Modular synthesis from organic linkers and metal nodes allows for precise control of structure,pore size and chemical functionality of MOFs. Recently, MOFs have been explored for their potential to form novelbiocomposites with proteins by a process termed biomimetic mineralization. These novel MOF biocomposites showgreat promise for application to industrial biocatalysis where strategies for enhancing enzyme stability are ofsignificant interest. The protective capacity and applications of biomimetically mineralized biomacromolecule zeoliticimidazolate framework (ZIF-8) composites are likely dependent on the charge of the biomolecule and the topologyof the mineralized ZIF-8 coating. Herein, we identify conditions to reliably yield the porous periodate oxidizedhorseradish peroxidase@ZIF-8 sodalite topology biocomposite in preference to other more dense phases.
C3  - NANOTEXNOLOGY NN22, 19th International Conference on Nanosciences & Nanotechnologies, 5-8 July 2022, Thessaloniki, Greece
T1  - Periodate oxidized horseradish peroxidase@ZIF-8 nanocomposite
SP  - 138
EP  - 138
UR  - https://hdl.handle.net/21.15107/rcub_cherry_5752
ER  - 

@conference{
author = "Stanišić, Marija and Ristić, Predrag and Đokić, Veljko and Balaž, Ana Marija and Prodanović, Radivoje and Todorović, Tamara",
year = "2022",
abstract = "Metal-organic frameworks (MOFs) are a class of materials well-known for their high degree of crystallinity andultrahigh porosity. Modular synthesis from organic linkers and metal nodes allows for precise control of structure,pore size and chemical functionality of MOFs. Recently, MOFs have been explored for their potential to form novelbiocomposites with proteins by a process termed biomimetic mineralization. These novel MOF biocomposites showgreat promise for application to industrial biocatalysis where strategies for enhancing enzyme stability are ofsignificant interest. The protective capacity and applications of biomimetically mineralized biomacromolecule zeoliticimidazolate framework (ZIF-8) composites are likely dependent on the charge of the biomolecule and the topologyof the mineralized ZIF-8 coating. Herein, we identify conditions to reliably yield the porous periodate oxidizedhorseradish peroxidase@ZIF-8 sodalite topology biocomposite in preference to other more dense phases.",
journal = "NANOTEXNOLOGY NN22, 19th International Conference on Nanosciences & Nanotechnologies, 5-8 July 2022, Thessaloniki, Greece",
title = "Periodate oxidized horseradish peroxidase@ZIF-8 nanocomposite",
pages = "138-138",
url = "https://hdl.handle.net/21.15107/rcub_cherry_5752"
}

Stanišić, M., Ristić, P., Đokić, V., Balaž, A. M., Prodanović, R.,& Todorović, T.. (2022). Periodate oxidized horseradish peroxidase@ZIF-8 nanocomposite. in NANOTEXNOLOGY NN22, 19th International Conference on Nanosciences & Nanotechnologies, 5-8 July 2022, Thessaloniki, Greece, 138-138.
https://hdl.handle.net/21.15107/rcub_cherry_5752

Stanišić M, Ristić P, Đokić V, Balaž AM, Prodanović R, Todorović T. Periodate oxidized horseradish peroxidase@ZIF-8 nanocomposite. in NANOTEXNOLOGY NN22, 19th International Conference on Nanosciences & Nanotechnologies, 5-8 July 2022, Thessaloniki, Greece. 2022;:138-138.
https://hdl.handle.net/21.15107/rcub_cherry_5752 .

Stanišić, Marija, Ristić, Predrag, Đokić, Veljko, Balaž, Ana Marija, Prodanović, Radivoje, Todorović, Tamara, "Periodate oxidized horseradish peroxidase@ZIF-8 nanocomposite" in NANOTEXNOLOGY NN22, 19th International Conference on Nanosciences & Nanotechnologies, 5-8 July 2022, Thessaloniki, Greece (2022):138-138,
https://hdl.handle.net/21.15107/rcub_cherry_5752 .

TY  - JOUR
AU  - Kovačević, Gordana
AU  - Elgahwash, Reyadh Gomah Amar
AU  - Blažić, Marija
AU  - Pantić, Nevena
AU  - Prodanović, Olivera
AU  - Balaž, Ana Marija
AU  - Prodanović, Radivoje
PY  - 2022
UR  - https://cer.ihtm.bg.ac.rs/handle/123456789/5472
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
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
IS  - 112215
DO  - 10.1016/j.mcat.2022.112215
ER  - 

@article{
author = "Kovačević, Gordana and Elgahwash, Reyadh Gomah Amar and Blažić, Marija 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",
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",
number = "112215",
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., 522(112215).
https://doi.org/10.1016/j.mcat.2022.112215

Kovačević G, Elgahwash RGA, 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(112215).
doi:10.1016/j.mcat.2022.112215 .

Kovačević, Gordana, Elgahwash, Reyadh Gomah Amar, Blažić, Marija, 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, no. 112215 (2022),
https://doi.org/10.1016/j.mcat.2022.112215 . .

TY  - JOUR
AU  - Stanišić, Marija D.
AU  - Popović Kokar, Nikolina
AU  - Ristić, Predrag
AU  - Balaž, Ana Marija
AU  - Senćanski, Milan
AU  - Ognanović, Miloš
AU  - Đokić, Veljko R.
AU  - Prodanović, Radivoje
AU  - Todorović, Tamara
PY  - 2021
UR  - https://cer.ihtm.bg.ac.rs/handle/123456789/4895
AB  - Zeolitic imidazolate framework-8 (ZIF-8) is widely used as a protective coating to en-capsulate proteins via biomimetic mineralization. The formation of nucleation centers and further biocomposite crystal growth is entirely governed by the pure electrostatic interactions between the protein’s surface and the positively charged Zn(II) metal ions. It was previously shown that enhancing these electrostatic interactions by a chemical modification of surface amino acid residues can lead to a rapid biocomposite crystal formation. However, a chemical modification of carbohydrate components by periodate oxidation for glycoproteins can serve as an alternative strategy. In the present study, an industrially important enzyme glucose oxidase (GOx) was selected as a model system. Periodate oxidation of GOx by 2.5 mM sodium periodate increased negative charge on the enzyme molecule, from −10.2 to −36.9 mV, as shown by zeta potential measurements and native PAGE electrophoresis. Biomineralization experiments with oxidized GOx resulted in higher specific activity, effectiveness factor, and higher thermostability of the ZIF-8 biocomposites. Periodate oxidation of carbohydrate components for glycoproteins can serve as a facile and general method for facilitating the biomimetic mineralization of other industrially relevant glycoproteins.
PB  - MDPI
T2  - Polymers
T1  - Chemical modification of glycoproteins’ carbohydrate moiety as a general strategy for the synthesis of efficient biocatalysts by biomimetic mineralization: The case of glucose oxidase
VL  - 13
IS  - 22
SP  - 2
EP  - 12
DO  - 10.3390/polym13223875
ER  - 

@article{
author = "Stanišić, Marija D. and Popović Kokar, Nikolina and Ristić, Predrag and Balaž, Ana Marija and Senćanski, Milan and Ognanović, Miloš and Đokić, Veljko R. and Prodanović, Radivoje and Todorović, Tamara",
year = "2021",
abstract = "Zeolitic imidazolate framework-8 (ZIF-8) is widely used as a protective coating to en-capsulate proteins via biomimetic mineralization. The formation of nucleation centers and further biocomposite crystal growth is entirely governed by the pure electrostatic interactions between the protein’s surface and the positively charged Zn(II) metal ions. It was previously shown that enhancing these electrostatic interactions by a chemical modification of surface amino acid residues can lead to a rapid biocomposite crystal formation. However, a chemical modification of carbohydrate components by periodate oxidation for glycoproteins can serve as an alternative strategy. In the present study, an industrially important enzyme glucose oxidase (GOx) was selected as a model system. Periodate oxidation of GOx by 2.5 mM sodium periodate increased negative charge on the enzyme molecule, from −10.2 to −36.9 mV, as shown by zeta potential measurements and native PAGE electrophoresis. Biomineralization experiments with oxidized GOx resulted in higher specific activity, effectiveness factor, and higher thermostability of the ZIF-8 biocomposites. Periodate oxidation of carbohydrate components for glycoproteins can serve as a facile and general method for facilitating the biomimetic mineralization of other industrially relevant glycoproteins.",
publisher = "MDPI",
journal = "Polymers",
title = "Chemical modification of glycoproteins’ carbohydrate moiety as a general strategy for the synthesis of efficient biocatalysts by biomimetic mineralization: The case of glucose oxidase",
volume = "13",
number = "22",
pages = "2-12",
doi = "10.3390/polym13223875"
}

Stanišić, M. D., Popović Kokar, N., Ristić, P., Balaž, A. M., Senćanski, M., Ognanović, M., Đokić, V. R., Prodanović, R.,& Todorović, T.. (2021). Chemical modification of glycoproteins’ carbohydrate moiety as a general strategy for the synthesis of efficient biocatalysts by biomimetic mineralization: The case of glucose oxidase. in Polymers
MDPI., 13(22), 2-12.
https://doi.org/10.3390/polym13223875

Stanišić MD, Popović Kokar N, Ristić P, Balaž AM, Senćanski M, Ognanović M, Đokić VR, Prodanović R, Todorović T. Chemical modification of glycoproteins’ carbohydrate moiety as a general strategy for the synthesis of efficient biocatalysts by biomimetic mineralization: The case of glucose oxidase. in Polymers. 2021;13(22):2-12.
doi:10.3390/polym13223875 .

Stanišić, Marija D., Popović Kokar, Nikolina, Ristić, Predrag, Balaž, Ana Marija, Senćanski, Milan, Ognanović, Miloš, Đokić, Veljko R., Prodanović, Radivoje, Todorović, Tamara, "Chemical modification of glycoproteins’ carbohydrate moiety as a general strategy for the synthesis of efficient biocatalysts by biomimetic mineralization: The case of glucose oxidase" in Polymers, 13, no. 22 (2021):2-12,
https://doi.org/10.3390/polym13223875 . .

TY  - CONF
AU  - Stanišić, Marija
AU  - Popović, Nikolina
AU  - Ristić, Predrag
AU  - Balaž, Ana Marija
AU  - Senćanski, Milan
AU  - Prodanović, Radivoje
AU  - Todorović, Tamara
PY  - 2021
UR  - https://cer.ihtm.bg.ac.rs/handle/123456789/5564
AB  - Glucose oxidase (GOx) is an enzyme that belongs to a group of oxidoreductases. This enzyme catalyzes the oxidation of glucose to gluconic acid using molecular oxygen as an electron acceptor. Glucose oxidase contains carbohydrates in its structure, most often mannose and glucose (11-13%) 1. Durability of GOx in harsh conditions can be enhanced by encapsulation within metal–organic frameworks via a process called biomimetic mineralisation. We demonstrate that chemical modification of carbohydrate parts on the protein surface by periodate oxidation is an effective method for control of biomimetic mineralisation by zeolitic imidazolate framework-8 (ZIF-8). Obtained GOx-ZIF-8 biocomposite had the higher half-life at 65oC, and higher specific activity than native GOx.
PB  - Beograd : Biohemijsko društvo Srbije
C3  - Tenth Conference of Serbian Biochemical Society, 24 September 2021, Kragujevac, Book of Abstracts
T1  - Biomimetic mineralisation of periodate oxidized glucose oxidase
SP  - 148
EP  - 148
UR  - https://hdl.handle.net/21.15107/rcub_cherry_5748
ER  - 

@conference{
author = "Stanišić, Marija and Popović, Nikolina and Ristić, Predrag and Balaž, Ana Marija and Senćanski, Milan and Prodanović, Radivoje and Todorović, Tamara",
year = "2021",
abstract = "Glucose oxidase (GOx) is an enzyme that belongs to a group of oxidoreductases. This enzyme catalyzes the oxidation of glucose to gluconic acid using molecular oxygen as an electron acceptor. Glucose oxidase contains carbohydrates in its structure, most often mannose and glucose (11-13%) 1. Durability of GOx in harsh conditions can be enhanced by encapsulation within metal–organic frameworks via a process called biomimetic mineralisation. We demonstrate that chemical modification of carbohydrate parts on the protein surface by periodate oxidation is an effective method for control of biomimetic mineralisation by zeolitic imidazolate framework-8 (ZIF-8). Obtained GOx-ZIF-8 biocomposite had the higher half-life at 65oC, and higher specific activity than native GOx.",
publisher = "Beograd : Biohemijsko društvo Srbije",
journal = "Tenth Conference of Serbian Biochemical Society, 24 September 2021, Kragujevac, Book of Abstracts",
title = "Biomimetic mineralisation of periodate oxidized glucose oxidase",
pages = "148-148",
url = "https://hdl.handle.net/21.15107/rcub_cherry_5748"
}

Stanišić, M., Popović, N., Ristić, P., Balaž, A. M., Senćanski, M., Prodanović, R.,& Todorović, T.. (2021). Biomimetic mineralisation of periodate oxidized glucose oxidase. in Tenth Conference of Serbian Biochemical Society, 24 September 2021, Kragujevac, Book of Abstracts
Beograd : Biohemijsko društvo Srbije., 148-148.
https://hdl.handle.net/21.15107/rcub_cherry_5748

Stanišić M, Popović N, Ristić P, Balaž AM, Senćanski M, Prodanović R, Todorović T. Biomimetic mineralisation of periodate oxidized glucose oxidase. in Tenth Conference of Serbian Biochemical Society, 24 September 2021, Kragujevac, Book of Abstracts. 2021;:148-148.
https://hdl.handle.net/21.15107/rcub_cherry_5748 .

Stanišić, Marija, Popović, Nikolina, Ristić, Predrag, Balaž, Ana Marija, Senćanski, Milan, Prodanović, Radivoje, Todorović, Tamara, "Biomimetic mineralisation of periodate oxidized glucose oxidase" in Tenth Conference of Serbian Biochemical Society, 24 September 2021, Kragujevac, Book of Abstracts (2021):148-148,
https://hdl.handle.net/21.15107/rcub_cherry_5748 .

TY  - CONF
AU  - Balaž, Ana Marija
AU  - Crnoglavac Popović, Milica
AU  - Stanišić, Marija
AU  - Ristić, Predrag
AU  - Senćanski, Milan
AU  - Todorović, Tamara
AU  - Prodanović, Radivoje
PY  - 2021
UR  - https://cer.ihtm.bg.ac.rs/handle/123456789/5566
AB  - Enzyme immobilization enables maintenance of enzyme activity and structural stability even in adverse conditions 1. Structural changes in enzymes that can occur due to the action of organic solvents, inhibitors or increased temperature can be prevented by immobilization of the enzymes in metal–organic frameworks (MOFs). It is reported that several enzymes, such as cytochrome c and horseradish peroxidase (HRP) have been successfully incorporated into MOFs 2. The aim of this work is to produce wild type horseradish peroxidase, isoform C1A, and several mutants specially designed to increase the activity and stability of HRP while immobilized within selected MOFs. Wild type and its variants were produced in metalotrophic yeast, Pichia pastoris KM71H strain, their activity and basic kinetic parameters were determined and compared prior imobilization.
C3  - Tenth Conference of Serbian Biochemical Society, 24 September 2021, Kragujevac
T1  - Horseradish peroxidase C1A wild type gene and its variants expressed in Pichia pastoris KM71H strain
SP  - 49
EP  - 49
UR  - https://hdl.handle.net/21.15107/rcub_cherry_5747
ER  - 

@conference{
author = "Balaž, Ana Marija and Crnoglavac Popović, Milica and Stanišić, Marija and Ristić, Predrag and Senćanski, Milan and Todorović, Tamara and Prodanović, Radivoje",
year = "2021",
abstract = "Enzyme immobilization enables maintenance of enzyme activity and structural stability even in adverse conditions 1. Structural changes in enzymes that can occur due to the action of organic solvents, inhibitors or increased temperature can be prevented by immobilization of the enzymes in metal–organic frameworks (MOFs). It is reported that several enzymes, such as cytochrome c and horseradish peroxidase (HRP) have been successfully incorporated into MOFs 2. The aim of this work is to produce wild type horseradish peroxidase, isoform C1A, and several mutants specially designed to increase the activity and stability of HRP while immobilized within selected MOFs. Wild type and its variants were produced in metalotrophic yeast, Pichia pastoris KM71H strain, their activity and basic kinetic parameters were determined and compared prior imobilization.",
journal = "Tenth Conference of Serbian Biochemical Society, 24 September 2021, Kragujevac",
title = "Horseradish peroxidase C1A wild type gene and its variants expressed in Pichia pastoris KM71H strain",
pages = "49-49",
url = "https://hdl.handle.net/21.15107/rcub_cherry_5747"
}

Balaž, A. M., Crnoglavac Popović, M., Stanišić, M., Ristić, P., Senćanski, M., Todorović, T.,& Prodanović, R.. (2021). Horseradish peroxidase C1A wild type gene and its variants expressed in Pichia pastoris KM71H strain. in Tenth Conference of Serbian Biochemical Society, 24 September 2021, Kragujevac, 49-49.
https://hdl.handle.net/21.15107/rcub_cherry_5747

Balaž AM, Crnoglavac Popović M, Stanišić M, Ristić P, Senćanski M, Todorović T, Prodanović R. Horseradish peroxidase C1A wild type gene and its variants expressed in Pichia pastoris KM71H strain. in Tenth Conference of Serbian Biochemical Society, 24 September 2021, Kragujevac. 2021;:49-49.
https://hdl.handle.net/21.15107/rcub_cherry_5747 .

Balaž, Ana Marija, Crnoglavac Popović, Milica, Stanišić, Marija, Ristić, Predrag, Senćanski, Milan, Todorović, Tamara, Prodanović, Radivoje, "Horseradish peroxidase C1A wild type gene and its variants expressed in Pichia pastoris KM71H strain" in Tenth Conference of Serbian Biochemical Society, 24 September 2021, Kragujevac (2021):49-49,
https://hdl.handle.net/21.15107/rcub_cherry_5747 .

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  - Spasojević, Dragica
AU  - Prokopijević, Miloš
AU  - Prodanović, Olivera
AU  - Zelenović, Nevena
AU  - Polović, Natalija
AU  - Radotić, Ksenija
AU  - Prodanović, Radivoje
PY  - 2019
UR  - https://cer.ihtm.bg.ac.rs/handle/123456789/3028
AB  - Derivatives of xylans were synthesized from corncob xylan by carboxymethylation, oxidization with different molar ratios of periodate (5, 10 15 and 20 mol%) and by reductive amination with tyramine. Modifications of tyramine carboxymethyl xylans (Tyr-CMX) were confirmed by FTIR, UV and NMR spectra. Concentration of ionizable groups increased from 1.5 mmol/g for carboxymethyl xylan (CMX) to 5.4 mmol/g for Tyr-CMX oxidized with 20 mol% of periodate. All Tyr-CMXs were able to form hydrogels the cross-linking reaction with horseradish peroxidase and peroxide. Tyr-CMXs were tested for amyloglucosidase (AG) encapsulation within hydrogel microbeads obtained in a reaction of emulsion polymerization with peroxidase. Average diameter of Tyr-CMX hydrogel microbeads was 52±25 µm and after encapsulation optimization with respect to the extent of CMX modification with tyramine, the concentration of Tyr-CMX, and the amount of added AG, microbeads with AG specific activity of 2 U/mL and 20% yield of immobilization were obtained. The optimum pH of the immobilized AG was not changed compared to the soluble one, while half-life at 60 °C was increased around 10 times. The Michaelis-Menten constant for the immobilized enzyme, 1.03 mM, was significantly lower than that for the soluble one, 1.54 mM. After 5 cycles of repetitive use in batch reactor, the immobilized AG retained 68% of initial activity.
PB  - Springer
T2  - Macromolecular Research
T1  - Peroxidase-Sensitive Tyramine Carboxymethyl Xylan Hydrogels for Enzyme Encapsulation
VL  - 27
IS  - 8
SP  - 764
EP  - 771
DO  - 10.1007/s13233-019-7111-7
ER  - 

@article{
author = "Spasojević, Dragica and Prokopijević, Miloš and Prodanović, Olivera and Zelenović, Nevena and Polović, Natalija and Radotić, Ksenija and Prodanović, Radivoje",
year = "2019",
abstract = "Derivatives of xylans were synthesized from corncob xylan by carboxymethylation, oxidization with different molar ratios of periodate (5, 10 15 and 20 mol%) and by reductive amination with tyramine. Modifications of tyramine carboxymethyl xylans (Tyr-CMX) were confirmed by FTIR, UV and NMR spectra. Concentration of ionizable groups increased from 1.5 mmol/g for carboxymethyl xylan (CMX) to 5.4 mmol/g for Tyr-CMX oxidized with 20 mol% of periodate. All Tyr-CMXs were able to form hydrogels the cross-linking reaction with horseradish peroxidase and peroxide. Tyr-CMXs were tested for amyloglucosidase (AG) encapsulation within hydrogel microbeads obtained in a reaction of emulsion polymerization with peroxidase. Average diameter of Tyr-CMX hydrogel microbeads was 52±25 µm and after encapsulation optimization with respect to the extent of CMX modification with tyramine, the concentration of Tyr-CMX, and the amount of added AG, microbeads with AG specific activity of 2 U/mL and 20% yield of immobilization were obtained. The optimum pH of the immobilized AG was not changed compared to the soluble one, while half-life at 60 °C was increased around 10 times. The Michaelis-Menten constant for the immobilized enzyme, 1.03 mM, was significantly lower than that for the soluble one, 1.54 mM. After 5 cycles of repetitive use in batch reactor, the immobilized AG retained 68% of initial activity.",
publisher = "Springer",
journal = "Macromolecular Research",
title = "Peroxidase-Sensitive Tyramine Carboxymethyl Xylan Hydrogels for Enzyme Encapsulation",
volume = "27",
number = "8",
pages = "764-771",
doi = "10.1007/s13233-019-7111-7"
}

Spasojević, D., Prokopijević, M., Prodanović, O., Zelenović, N., Polović, N., Radotić, K.,& Prodanović, R.. (2019). Peroxidase-Sensitive Tyramine Carboxymethyl Xylan Hydrogels for Enzyme Encapsulation. in Macromolecular Research
Springer., 27(8), 764-771.
https://doi.org/10.1007/s13233-019-7111-7

Spasojević D, Prokopijević M, Prodanović O, Zelenović N, Polović N, Radotić K, Prodanović R. Peroxidase-Sensitive Tyramine Carboxymethyl Xylan Hydrogels for Enzyme Encapsulation. in Macromolecular Research. 2019;27(8):764-771.
doi:10.1007/s13233-019-7111-7 .

Spasojević, Dragica, Prokopijević, Miloš, Prodanović, Olivera, Zelenović, Nevena, Polović, Natalija, Radotić, Ksenija, Prodanović, Radivoje, "Peroxidase-Sensitive Tyramine Carboxymethyl Xylan Hydrogels for Enzyme Encapsulation" in Macromolecular Research, 27, no. 8 (2019):764-771,
https://doi.org/10.1007/s13233-019-7111-7 . .

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  - Blažić, Marija
AU  - Prodanović, Radivoje
PY  - 2017
UR  - https://cer.ihtm.bg.ac.rs/handle/123456789/2168
UR  - https://doi.org/10.1111/febs.14174
PB  - Wiley, Hoboken
C3  - FEBS Journal
T1  - Directed evolution of cellobiose dehydrogenase from Phanerochaete chrysosporium in yeast Saccharomyces cerevisiae for increased activity
VL  - 284
IS  - S1
SP  - 104
EP  - 104
UR  - https://hdl.handle.net/21.15107/rcub_cherry_2406
ER  - 

@conference{
author = "Blažić, Marija and Prodanović, Radivoje",
year = "2017",
publisher = "Wiley, Hoboken",
journal = "FEBS Journal",
title = "Directed evolution of cellobiose dehydrogenase from Phanerochaete chrysosporium in yeast Saccharomyces cerevisiae for increased activity",
volume = "284",
number = "S1",
pages = "104-104",
url = "https://hdl.handle.net/21.15107/rcub_cherry_2406"
}

Blažić, M.,& Prodanović, R.. (2017). Directed evolution of cellobiose dehydrogenase from Phanerochaete chrysosporium in yeast Saccharomyces cerevisiae for increased activity. in FEBS Journal
Wiley, Hoboken., 284(S1), 104-104.
https://hdl.handle.net/21.15107/rcub_cherry_2406

Blažić M, Prodanović R. Directed evolution of cellobiose dehydrogenase from Phanerochaete chrysosporium in yeast Saccharomyces cerevisiae for increased activity. in FEBS Journal. 2017;284(S1):104-104.
https://hdl.handle.net/21.15107/rcub_cherry_2406 .

Blažić, Marija, Prodanović, Radivoje, "Directed evolution of cellobiose dehydrogenase from Phanerochaete chrysosporium in yeast Saccharomyces cerevisiae for increased activity" in FEBS Journal, 284, no. S1 (2017):104-104,
https://hdl.handle.net/21.15107/rcub_cherry_2406 .

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 .