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  - 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  - 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  - Kovačević, Gordana
AU  - Blažić, Marija
AU  - Draganic, Bojana
AU  - Ostafe, Raluca
AU  - Gavrović-Jankulović, Marija
AU  - Fischer, Rainer
AU  - Prodanović, Radivoje
PY  - 2014
UR  - https://cer.ihtm.bg.ac.rs/handle/123456789/1506
AB  - Aspergillus niger glucose oxidase (GOx) genes for wild-type (GenBank accession no. X16061, swiss-Prot; P13006) and M12 mutant (N2Y, K13E, T30 V, I94 V, K152R) were cloned into pPICZ alpha A vector for expression in Pichia pastoris KM71H strain. The highest expression level of 17.5 U/mL of fermentation media was obtained in 0.5 % (v/v) methanol after 9 days of fermentation. The recombinant GOx was purified by cross-flow ultrafiltration using membranes of 30 kDa molecular cutoff and DEAE ion-exchange chromatography at pH 6.0. Purified wt GOx had k (cat) of 189.4 s(-1) and K (m) of 28.26 mM while M12 GOx had k (cat) of 352.0 s(-1) and K (m) of 13.33 mM for glucose at pH 5.5. Specificity constants k (cat)/K (m) of wt (6.70 mM(-1) s(-1)) and M12 GOx (26.7 mM(-1) s(-1)) expressed in P. pastoris KM71H were around three times higher than for the same enzymes previously expressed in Saccharomyces cerevisiae InvSc1 strain. The pH optimum and sugar specificity of M12 mutant of GOx remained similar to the wild-type form of the enzyme, while thermostability was slightly decreased. M12 GOx expressed in P. pastoris showed three times higher activity compared to the wt GOx toward redox mediators like N,N-dimethyl-nitroso-aniline used for glucose strips manufacturing. M12 mutant of GOx produced in P. pastoris KM71H could be useful for manufacturing of glucose biosensors and biofuel cells.
PB  - Humana Press Inc, Totowa
T2  - Molecular Biotechnology
T1  - Cloning, Heterologous Expression, Purification and Characterization of M12 Mutant of Aspergillus niger Glucose Oxidase in Yeast Pichia pastoris KM71H
VL  - 56
IS  - 4
SP  - 305
EP  - 311
DO  - 10.1007/s12033-013-9709-x
ER  - 

@article{
author = "Kovačević, Gordana and Blažić, Marija and Draganic, Bojana and Ostafe, Raluca and Gavrović-Jankulović, Marija and Fischer, Rainer and Prodanović, Radivoje",
year = "2014",
abstract = "Aspergillus niger glucose oxidase (GOx) genes for wild-type (GenBank accession no. X16061, swiss-Prot; P13006) and M12 mutant (N2Y, K13E, T30 V, I94 V, K152R) were cloned into pPICZ alpha A vector for expression in Pichia pastoris KM71H strain. The highest expression level of 17.5 U/mL of fermentation media was obtained in 0.5 % (v/v) methanol after 9 days of fermentation. The recombinant GOx was purified by cross-flow ultrafiltration using membranes of 30 kDa molecular cutoff and DEAE ion-exchange chromatography at pH 6.0. Purified wt GOx had k (cat) of 189.4 s(-1) and K (m) of 28.26 mM while M12 GOx had k (cat) of 352.0 s(-1) and K (m) of 13.33 mM for glucose at pH 5.5. Specificity constants k (cat)/K (m) of wt (6.70 mM(-1) s(-1)) and M12 GOx (26.7 mM(-1) s(-1)) expressed in P. pastoris KM71H were around three times higher than for the same enzymes previously expressed in Saccharomyces cerevisiae InvSc1 strain. The pH optimum and sugar specificity of M12 mutant of GOx remained similar to the wild-type form of the enzyme, while thermostability was slightly decreased. M12 GOx expressed in P. pastoris showed three times higher activity compared to the wt GOx toward redox mediators like N,N-dimethyl-nitroso-aniline used for glucose strips manufacturing. M12 mutant of GOx produced in P. pastoris KM71H could be useful for manufacturing of glucose biosensors and biofuel cells.",
publisher = "Humana Press Inc, Totowa",
journal = "Molecular Biotechnology",
title = "Cloning, Heterologous Expression, Purification and Characterization of M12 Mutant of Aspergillus niger Glucose Oxidase in Yeast Pichia pastoris KM71H",
volume = "56",
number = "4",
pages = "305-311",
doi = "10.1007/s12033-013-9709-x"
}

Kovačević, G., Blažić, M., Draganic, B., Ostafe, R., Gavrović-Jankulović, M., Fischer, R.,& Prodanović, R.. (2014). Cloning, Heterologous Expression, Purification and Characterization of M12 Mutant of Aspergillus niger Glucose Oxidase in Yeast Pichia pastoris KM71H. in Molecular Biotechnology
Humana Press Inc, Totowa., 56(4), 305-311.
https://doi.org/10.1007/s12033-013-9709-x

Kovačević G, Blažić M, Draganic B, Ostafe R, Gavrović-Jankulović M, Fischer R, Prodanović R. Cloning, Heterologous Expression, Purification and Characterization of M12 Mutant of Aspergillus niger Glucose Oxidase in Yeast Pichia pastoris KM71H. in Molecular Biotechnology. 2014;56(4):305-311.
doi:10.1007/s12033-013-9709-x .

Kovačević, Gordana, Blažić, Marija, Draganic, Bojana, Ostafe, Raluca, Gavrović-Jankulović, Marija, Fischer, Rainer, Prodanović, Radivoje, "Cloning, Heterologous Expression, Purification and Characterization of M12 Mutant of Aspergillus niger Glucose Oxidase in Yeast Pichia pastoris KM71H" in Molecular Biotechnology, 56, no. 4 (2014):305-311,
https://doi.org/10.1007/s12033-013-9709-x . .

TY  - JOUR
AU  - Blažić, Marija
AU  - Kovačević, Gordana
AU  - Prodanović, Olivera
AU  - Ostafe, Raluca
AU  - Gavrović-Jankulović, Marija
AU  - Fischer, Rainer
AU  - Prodanović, Radivoje
PY  - 2013
UR  - https://cer.ihtm.bg.ac.rs/handle/123456789/1331
AB  - Glucose oxidase (GOx) catalyzes the oxidation of glucose to form gluconic acid and hydrogen peroxide, a reaction with important applications in food preservation, the manufacture of cosmetics and pharmaceuticals, and the development of glucose monitoring devices and biofuel cells. We expressed Aspergillus niger wild type GOx and the B11 mutant, which has twice the activity of the wild type enzyme at pH 5.5, as C-terminal fusions with the Saccharomyces cerevisiae Aga2 protein, allowing the fusion proteins to be displayed on the surface of yeast EBY100 cells. After expression, we extracted the proteins from the yeast cell wall and purified them by ion-exchange chromatography and ultrafiltration. This produced a broad 100-140 kDa band by denaturing SDS-PAGE and a high-molecular-weight band by native PAGE corresponding to the activity band revealed by zymography. The wild type and B11 fusion proteins had k(cat) values of 33.3 and 61.3 s(-1) and K-m values for glucose of 33.4 and 27.9 mM, respectively. The pH optimum for both enzymes was 5.0. The kinetic properties of the fusion proteins displayed the same ratio as their native counterparts, confirming that yeast surface display is suitable for the high-throughput directed evolution of GOx using flow cytometry for selection. Aga2-GOx fusion proteins in the yeast cell wall could also be used as immobilized catalysts for the production of gluconic acid.
PB  - Academic Press Inc Elsevier Science, San Diego
T2  - Protein Expression and Purification
T1  - Yeast surface display for the expression, purification and characterization of wild-type and B11 mutant glucose oxidases
VL  - 89
IS  - 2
SP  - 175
EP  - 180
DO  - 10.1016/j.pep.2013.03.014
ER  - 

@article{
author = "Blažić, Marija and Kovačević, Gordana and Prodanović, Olivera and Ostafe, Raluca and Gavrović-Jankulović, Marija and Fischer, Rainer and Prodanović, Radivoje",
year = "2013",
abstract = "Glucose oxidase (GOx) catalyzes the oxidation of glucose to form gluconic acid and hydrogen peroxide, a reaction with important applications in food preservation, the manufacture of cosmetics and pharmaceuticals, and the development of glucose monitoring devices and biofuel cells. We expressed Aspergillus niger wild type GOx and the B11 mutant, which has twice the activity of the wild type enzyme at pH 5.5, as C-terminal fusions with the Saccharomyces cerevisiae Aga2 protein, allowing the fusion proteins to be displayed on the surface of yeast EBY100 cells. After expression, we extracted the proteins from the yeast cell wall and purified them by ion-exchange chromatography and ultrafiltration. This produced a broad 100-140 kDa band by denaturing SDS-PAGE and a high-molecular-weight band by native PAGE corresponding to the activity band revealed by zymography. The wild type and B11 fusion proteins had k(cat) values of 33.3 and 61.3 s(-1) and K-m values for glucose of 33.4 and 27.9 mM, respectively. The pH optimum for both enzymes was 5.0. The kinetic properties of the fusion proteins displayed the same ratio as their native counterparts, confirming that yeast surface display is suitable for the high-throughput directed evolution of GOx using flow cytometry for selection. Aga2-GOx fusion proteins in the yeast cell wall could also be used as immobilized catalysts for the production of gluconic acid.",
publisher = "Academic Press Inc Elsevier Science, San Diego",
journal = "Protein Expression and Purification",
title = "Yeast surface display for the expression, purification and characterization of wild-type and B11 mutant glucose oxidases",
volume = "89",
number = "2",
pages = "175-180",
doi = "10.1016/j.pep.2013.03.014"
}

Blažić, M., Kovačević, G., Prodanović, O., Ostafe, R., Gavrović-Jankulović, M., Fischer, R.,& Prodanović, R.. (2013). Yeast surface display for the expression, purification and characterization of wild-type and B11 mutant glucose oxidases. in Protein Expression and Purification
Academic Press Inc Elsevier Science, San Diego., 89(2), 175-180.
https://doi.org/10.1016/j.pep.2013.03.014

Blažić M, Kovačević G, Prodanović O, Ostafe R, Gavrović-Jankulović M, Fischer R, Prodanović R. Yeast surface display for the expression, purification and characterization of wild-type and B11 mutant glucose oxidases. in Protein Expression and Purification. 2013;89(2):175-180.
doi:10.1016/j.pep.2013.03.014 .

Blažić, Marija, Kovačević, Gordana, Prodanović, Olivera, Ostafe, Raluca, Gavrović-Jankulović, Marija, Fischer, Rainer, Prodanović, Radivoje, "Yeast surface display for the expression, purification and characterization of wild-type and B11 mutant glucose oxidases" in Protein Expression and Purification, 89, no. 2 (2013):175-180,
https://doi.org/10.1016/j.pep.2013.03.014 . .