Balaž, Ana Marija

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orcid::0000-0003-1762-2183
  • Balaž, Ana Marija (11)
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Author's Bibliography

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

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

(Elsevier, 2020)

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

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

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

(2020)

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

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

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

(Belgrade : Serbian Chemical Society, 2020)

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

Proteinski inženjering celobioza - dehidrogenaze iz Phanerochaete chrysosporium u cilju povećanja oksidativne stabilnosti za primenu u biokatalizi

Balaž, Ana Marija

(Универзитет у Београду, Хемијски факултет, 2019)

TY  - BOOK
AU  - Balaž, Ana Marija
PY  - 2019
UR  - http://eteze.bg.ac.rs/application/showtheses?thesesId=7704
UR  - https://fedorabg.bg.ac.rs/fedora/get/o:22916/bdef:Content/download
UR  - http://vbs.rs/scripts/cobiss?command=DISPLAY&base=70036&RID=23928841
UR  - https://nardus.mpn.gov.rs/handle/123456789/17616
UR  - http://cherry.chem.bg.ac.rs/handle/123456789/4383
AB  - Celobioza – dehidrogenaza poreklom iz Phanerochaete chrysosporium, gljive bele truleži, pripada ekstracelularnim oksidoredukcionim enzimima i katalizuje oksidaciju β – 1,4 – glikozidno vezanih oligosaharida poput celobioze i laktoze. Oksidacijom laktoze dolazi do formiranja laktobionske kiseline koja pronalazi veliku primenu u farmaceutskoj i kozmetičkoj industriji gde se koristi prilikom distribucije lekova i za hidrataciju kože kao sastavni deo različitih krema, gde zajedno sa hijaluronskom kiselinu ima ulogu u smanjenju bora.Celobioza – dehidrogenaza prilikom oksidacije laktoze ili celobioze, kao prirodnih supstrata, katalizuje redukciju jednog dvoelektronskog ili dva jednoelektronska akceptora elektrona. Jedan od najkorišćenijih dvoelektronskih akceptora elektrona je upravo dihlor fenol indofenol (DCIP), dok jednoelektronski akceptori elektrona mogu biti citohrom c, ABTS, ali i Fe3+ i Mn3+ joni. Redukcijom Fe3+ jona u prisustvu molekulskog kiseonika dolazi do formiranja vodonik peroksida i posredstvom Fentonove reakcije do generisanja hidroksil radikala.Polazeći od ove činjenice, iskoristili smo upravo Fentonovu reakciju za razvoj fluorescentnog eseja za visoko efikasnu pretragu biblioteka gena celobioza – dehidrogenaze, baziranog na detekciji proizvedenih hidroksil radikala fluorogenom probom aminofenil – fluoresceinom (APF).Primena celobioza – dehidrogenaze u konstruisanju biosenzora i biogorivnih ćelija leži upravo u njenoj sposobnosti da katalizuje oksidaciju laktoze, celobioze i β – 1,4 – vezanih oligosaharida do odgovarajućih laktona koji potom spontano hidrolizuju do aldonskih kiselina. Enzimi koji nalaze primenu u konstruisanju biosenzora i biogorivnih ćelija, moraju da zadovoljavaju nekoliko kriterijuma,odnosno moraju da imaju veliku osetljivost i supstratnu specifičnost, ali i da pokazuju povećanu stabilnost...
AB  - Cellobiose dehydrogenase from Phanerochaete chrysosporium, a white rot fungus, belongs to the extracellular oxidoreductive group of enzymes and catalyzes the oxidation of the β – 1,4 – glycoside bond of oligosaccharides such as cellobiose and lactose. During oxidation of lactose, formation of lactobionic acid occurs which has many applications in pharmaceutical and cosmetic industry. Such applications include the distribution of medicine, a component responsible for skin hydration and when combined with hyaluronic acid as an agent against wrinkles.During oxidation of lactose or cellobiose by cellobiose dehydrogenase, reduction catalysis occurs of one two electron or two one electron acceptors. One of the most utilized two electron acceptors is DCIP, while one electron acceptors are usually cytochrome c, ABTS, Fe3+ and Mn3+ ions. During reduction of Fe3+ ions in the presence of molecular oxygen, H2O2 is formed and due to the Fenton reaction formation of hydroxyl radicals occurs. Due to this occurrence we wanted to use the Fenton reaction in order to develop a fluorescent assay based on the production of hydroxyl radicals and the fluorescence of aminophenyl fluorescein (APF). This would allow us to efficiently analyze cellobiose dehydrogenase gene libraries.With this fact in mind, the Fenton reaction was used to develop a fluorescent assay for the high throughput screening of cellobiose dehydrogenase genes, based on the detection of hydroxyl radicals with the fluorescent probe APF.The possible application of cellobiose dehydrogenase in the construction of various biosensors and biofuel cells is due the its ability to catalyze the oxidation of lactose, cellobiose and similar β – 1,4 – oligosaccharides do their corresponding lactones which then spontaneously hydrolyze to aldonic acids. Enzymes used in suchapplications need to satisfy certain criteria, such as exceptional sensitivity, substrate selectivity, stability and activity...
PB  - Универзитет у Београду, Хемијски факултет
T2  - Универзитет у Београду
T1  - Proteinski inženjering celobioza - dehidrogenaze iz Phanerochaete chrysosporium u cilju povećanja oksidativne stabilnosti za primenu u biokatalizi
ER  - 
@phdthesis{
author = "Balaž, Ana Marija",
year = "2019",
url = "http://eteze.bg.ac.rs/application/showtheses?thesesId=7704, https://fedorabg.bg.ac.rs/fedora/get/o:22916/bdef:Content/download, http://vbs.rs/scripts/cobiss?command=DISPLAY&base=70036&RID=23928841, https://nardus.mpn.gov.rs/handle/123456789/17616, http://cherry.chem.bg.ac.rs/handle/123456789/4383",
abstract = "Celobioza – dehidrogenaza poreklom iz Phanerochaete chrysosporium, gljive bele truleži, pripada ekstracelularnim oksidoredukcionim enzimima i katalizuje oksidaciju β – 1,4 – glikozidno vezanih oligosaharida poput celobioze i laktoze. Oksidacijom laktoze dolazi do formiranja laktobionske kiseline koja pronalazi veliku primenu u farmaceutskoj i kozmetičkoj industriji gde se koristi prilikom distribucije lekova i za hidrataciju kože kao sastavni deo različitih krema, gde zajedno sa hijaluronskom kiselinu ima ulogu u smanjenju bora.Celobioza – dehidrogenaza prilikom oksidacije laktoze ili celobioze, kao prirodnih supstrata, katalizuje redukciju jednog dvoelektronskog ili dva jednoelektronska akceptora elektrona. Jedan od najkorišćenijih dvoelektronskih akceptora elektrona je upravo dihlor fenol indofenol (DCIP), dok jednoelektronski akceptori elektrona mogu biti citohrom c, ABTS, ali i Fe3+ i Mn3+ joni. Redukcijom Fe3+ jona u prisustvu molekulskog kiseonika dolazi do formiranja vodonik peroksida i posredstvom Fentonove reakcije do generisanja hidroksil radikala.Polazeći od ove činjenice, iskoristili smo upravo Fentonovu reakciju za razvoj fluorescentnog eseja za visoko efikasnu pretragu biblioteka gena celobioza – dehidrogenaze, baziranog na detekciji proizvedenih hidroksil radikala fluorogenom probom aminofenil – fluoresceinom (APF).Primena celobioza – dehidrogenaze u konstruisanju biosenzora i biogorivnih ćelija leži upravo u njenoj sposobnosti da katalizuje oksidaciju laktoze, celobioze i β – 1,4 – vezanih oligosaharida do odgovarajućih laktona koji potom spontano hidrolizuju do aldonskih kiselina. Enzimi koji nalaze primenu u konstruisanju biosenzora i biogorivnih ćelija, moraju da zadovoljavaju nekoliko kriterijuma,odnosno moraju da imaju veliku osetljivost i supstratnu specifičnost, ali i da pokazuju povećanu stabilnost..., Cellobiose dehydrogenase from Phanerochaete chrysosporium, a white rot fungus, belongs to the extracellular oxidoreductive group of enzymes and catalyzes the oxidation of the β – 1,4 – glycoside bond of oligosaccharides such as cellobiose and lactose. During oxidation of lactose, formation of lactobionic acid occurs which has many applications in pharmaceutical and cosmetic industry. Such applications include the distribution of medicine, a component responsible for skin hydration and when combined with hyaluronic acid as an agent against wrinkles.During oxidation of lactose or cellobiose by cellobiose dehydrogenase, reduction catalysis occurs of one two electron or two one electron acceptors. One of the most utilized two electron acceptors is DCIP, while one electron acceptors are usually cytochrome c, ABTS, Fe3+ and Mn3+ ions. During reduction of Fe3+ ions in the presence of molecular oxygen, H2O2 is formed and due to the Fenton reaction formation of hydroxyl radicals occurs. Due to this occurrence we wanted to use the Fenton reaction in order to develop a fluorescent assay based on the production of hydroxyl radicals and the fluorescence of aminophenyl fluorescein (APF). This would allow us to efficiently analyze cellobiose dehydrogenase gene libraries.With this fact in mind, the Fenton reaction was used to develop a fluorescent assay for the high throughput screening of cellobiose dehydrogenase genes, based on the detection of hydroxyl radicals with the fluorescent probe APF.The possible application of cellobiose dehydrogenase in the construction of various biosensors and biofuel cells is due the its ability to catalyze the oxidation of lactose, cellobiose and similar β – 1,4 – oligosaccharides do their corresponding lactones which then spontaneously hydrolyze to aldonic acids. Enzymes used in suchapplications need to satisfy certain criteria, such as exceptional sensitivity, substrate selectivity, stability and activity...",
publisher = "Универзитет у Београду, Хемијски факултет",
journal = "Универзитет у Београду",
title = "Proteinski inženjering celobioza - dehidrogenaze iz Phanerochaete chrysosporium u cilju povećanja oksidativne stabilnosti za primenu u biokatalizi"
}
Balaž, A. M. (2019). Proteinski inženjering celobioza - dehidrogenaze iz Phanerochaete chrysosporium u cilju povećanja oksidativne stabilnosti za primenu u biokatalizi.
Универзитет у Београду
Универзитет у Београду, Хемијски факултет..
Balaž AM. Proteinski inženjering celobioza - dehidrogenaze iz Phanerochaete chrysosporium u cilju povećanja oksidativne stabilnosti za primenu u biokatalizi. Универзитет у Београду. 2019;
Balaž Ana Marija, "Proteinski inženjering celobioza - dehidrogenaze iz Phanerochaete chrysosporium u cilju povećanja oksidativne stabilnosti za primenu u biokatalizi" Универзитет у Београду (2019)

Protein engineering of cellobiose dehydrogenase from Phanerochaete chrysosporium in yeast Saccharomyces cerevisiae InvSc1 for increased activity and stability

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

(Elsevier, 2019)

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  - http://cherry.chem.bg.ac.rs/handle/123456789/2898
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 hyper-glycosylation and the best S137 N mutant showed 2.2 times increased k cat 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 S137 N mutant retained 30% of it's original activity after 15 min at 70 °C compared to 10% of activity that the wild type enzyme retained. Mutants M65S and S137 N showed also 1.6 and 1.5 times increased productivity of hydrogen peroxide in the presence of 30 mM 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",
url = "http://cherry.chem.bg.ac.rs/handle/123456789/2898",
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 hyper-glycosylation and the best S137 N mutant showed 2.2 times increased k cat 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 S137 N mutant retained 30% of it's original activity after 15 min at 70 °C compared to 10% of activity that the wild type enzyme retained. Mutants M65S and S137 N showed also 1.6 and 1.5 times increased productivity of hydrogen peroxide in the presence of 30 mM 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.
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. Biochemical Engineering Journal. 2019;146:179-185
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" Biochemical Engineering Journal, 146 (2019):179-185,
https://doi.org/10.1016/j.bej.2019.03.025 .
6
1
6

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

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

(Elsevier, 2019)

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

Directed evolution of cellobiose dehydrogenase on the surface of yeast cells using resazurin-based fluorescent assay

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

(Applied sciences, 2019)

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  - http://cherry.chem.bg.ac.rs/handle/123456789/2909
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  - Applied sciences
T2  - Applied Sciences (Switzerland)
T1  - Directed evolution of cellobiose dehydrogenase on the surface of yeast cells using resazurin-based fluorescent assay
VL  - 9
IS  - 7
SP  - 1
EP  - 15
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",
url = "http://cherry.chem.bg.ac.rs/handle/123456789/2909",
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 = "Applied sciences",
journal = "Applied Sciences (Switzerland)",
title = "Directed evolution of cellobiose dehydrogenase on the surface of yeast cells using resazurin-based fluorescent assay",
volume = "9",
number = "7",
pages = "1-15",
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.
Applied Sciences (Switzerland)
Applied sciences., 9(7), 1-15.
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. Applied Sciences (Switzerland). 2019;9(7):1-15
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" Applied Sciences (Switzerland), 9, no. 7 (2019):1-15,
https://doi.org/10.3390/app9071413 .
6
4
6

Supplementary data for the article: Blažić, M.; Balaž, A. M.; Prodanović, O.; Popović, N.; Ostafe, R.; Fischer, R.; Prodanović, R. Directed Evolution of Cellobiose Dehydrogenase on the Surface of Yeast Cells Using Resazurin-Based Fluorescent Assay. Applied Sciences (Switzerland) 2019, 9 (7). https://doi.org/10.3390/app9071413

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

(Applied sciences, 2019)

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

Development of GFP-based high-throughput screening system for directed evolution of glucose oxidase

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

(Elsevier, 2018)

TY  - JOUR
AU  - Kovačević, Gordana
AU  - Ostafe, Raluca
AU  - Balaž, Ana Marija
AU  - Fischer, Rainer
AU  - Prodanović, Radivoje
PY  - 2018
UR  - http://cherry.chem.bg.ac.rs/handle/123456789/336
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. © 2018 The Society for Biotechnology, Japan
PB  - Elsevier
T2  - Journal of Bioscience and Bioengineering
T1  - Development of GFP-based high-throughput screening system for directed evolution of glucose oxidase
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 = "2018",
url = "http://cherry.chem.bg.ac.rs/handle/123456789/336",
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. © 2018 The Society for Biotechnology, Japan",
publisher = "Elsevier",
journal = "Journal of Bioscience and Bioengineering",
title = "Development of GFP-based high-throughput screening system for directed evolution of glucose oxidase",
doi = "10.1016/j.jbiosc.2018.07.002"
}
Kovačević, G., Ostafe, R., Balaž, A. M., Fischer, R.,& Prodanović, R. (2018). Development of GFP-based high-throughput screening system for directed evolution of glucose oxidase.
Journal of Bioscience and Bioengineering
Elsevier..
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. Journal of Bioscience and Bioengineering. 2018;
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" Journal of Bioscience and Bioengineering (2018),
https://doi.org/10.1016/j.jbiosc.2018.07.002 .
1
12
8
10

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

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

(Elsevier, 2018)

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

Cloning of the gene for a carbohydrate oxidase from Lactuca sativa in the yeasts Saccharomyces cerevisiae and Pichia pastoris

Tadić, Vojin; Balaž, Ana Marija; Petric, Marija P.; Milošević, Snežana; Zelenović, Nevena D.; Raspor, Martin Z.; Tadić, Jovan; Prodanović, Radivoje

(Assoc Chemical Engineers Serbia, Belgrade, 2015)

TY  - JOUR
AU  - Tadić, Vojin
AU  - Balaž, Ana Marija
AU  - Petric, Marija P.
AU  - Milošević, Snežana
AU  - Zelenović, Nevena D.
AU  - Raspor, Martin Z.
AU  - Tadić, Jovan
AU  - Prodanović, Radivoje
PY  - 2015
UR  - http://cherry.chem.bg.ac.rs/handle/123456789/2040
AB  - We have cloned the gene for carbohydrate oxidase (CHO) from Lactuca sativa in two species of yeasts (Saccharomyces cerevisiae and Pichia pastoris). The synthetic gene for the carbohydrate oxidase (1821 bp) from L. sativa cloned into the vector pUC57 and inserted into plasmids pYES2 and pGAP using Escherichia coli DH5 alpha strain. The P. pastoris strain X-33 and the S. cerevisiae strain InvSC1 were used for extracellular expression of CHO. After transformation of P. pastoris X-33 with CHO-pGAP construct none of the colonies showed CHO activity. Two samples displayed a band which did not exist in the sample with the empty vector similar to the molecular weight of CHO. The S. cerevisiae strain InvSC1 has been also transformed with CHO-pYES constructs. Three colonies grew on the plate with cells transformed with the construct. One of the samples showed a band corresponding to about 110 kDa, but no CHO activity was recorded in this case either. Cloning of the foreign genes and heterologous expression in yeasts is widely used in biotechnology, but sometimes can be very dependent on the gene sequence and strain used. In order to obtain active CHO enzyme the further studies on purification and refolding of expressed protein are necessary.
AB  - Ugljeni hidrat-oksidaza (CHO) iz zelene salate (Lactuca sativa) je enzim koji je do danas nedovoljno ispitan. Vrlo se malo zna o njegovoj strukturi i funkciji. CHO pripada velikoj familiji ugljenihidrat-oksidaza, koje oksiduju šećere. Svaki od članova ove velike familije dobio je ime po supstratu koji oksiduje. Oksidaze iz ove familije enzima imaju kako sličnu ulogu tako i sličnu strukutru. Sve ili većina ovih enzima su monomeri, čiji se polipeptidni lanac uvija u dva domena. Jedan od domena vezuje flavinski kofaktor, a drugi domen je supstrat vezujući. Većina njih oksidaciju supstrata vrši po takozvanom ping-pong mehanizmu. Sve oksidaze iz karbohidrat-oksidaza familije, pa među njima i enzim koji je predmet ove studije (CHO), danas su našle veliku primenu u industriji. CHO se može primenjivati kako u medicinskoj djagnostici, konkretno u biosenzorima za određivanje glukoze u krvi, u prehrambenoj industriji, poljoprivredi, proizvodnji hleba, deterdženata i u raznim drugim industrijskim oblastima. Problem sa ovim enzimom, kao i sa ostalim članovima ove familije, jeste niska koncentracija u prirodnim izvorima. Zato su danas razvijene različite metode rekombinantne tehnologije, kojima se dobijaju ovi enzimi. U ovom radu opisano je kloniranje gena za CHO iz zelene salate u dve vrste kvasaca (Saccharomyces cerevisiae i Pichia pastoris). Sintetički gen za CHO (1821 bp) iz zelene salate kloniran je u vektor pUC57. Escherichia coli soj DH5α korišćen je za kloniranje gena i održavanje plazmida. P. pastoris soj X-33 i S. cerevisiae soj InvSC1 korišćeni su za ekstracelularnu ekspresiju CHO. Aktivnost CHO određena je ABST esejom, a promena absorbance merena je na 405 nm. Potvrda prisustva enzima rađena je na DNK agaroznoj elektroforezi i SDS-PAGE. Posle transformacije P. pastoris X-33, nijedan od klonova nije pokazivao aktivnost CHO. Posle prve fermentacije, kolonije su testirane na SDS-PAGE. Kako su dva uzorka pokazala trake, koje ne postoje na praznom vektoru, ove trake bi mogle odgovarati željenom enzimu, CHO. Traka se nalazi na molekulskoj masi koja je veća od teoretske (više od 120 kDa). Enzim bi mogao biti glikolizovan i zbog toga pokazivati ovako velike vrednosti za molekulsku masu. S. cerevisiae soj InvSC1 transformisan je konstruktom CHO-pZES. Posle 24 sata, tri kolonije su porasle na ploči na kojoj su bile ćelije transformisane pomenutim konstruktom. Uzorci su testirani na SDS-PAGE. Jedan uzorak je pokazao traku na oko 110 kDa, ali aktivnost CHO nije potvrđena takođe. Cilj ove studije je bio kloniranje CHO u kvascima S. cerevisiae i P. pastoris, kao i njena ekspresija u ovim, danas široko primenjivanim ekspresionim sistemima.
PB  - Assoc Chemical Engineers Serbia, Belgrade
T2  - Hemijska industrija
T1  - Cloning of the gene for a carbohydrate oxidase from Lactuca sativa in the yeasts Saccharomyces cerevisiae and Pichia pastoris
T1  - Kloniranje gena za ugljeni hidrat oksidazu iz biljke Lactuca sativa u kvasce saccharomyces cerevisiae i Pichia pastoris
VL  - 69
IS  - 6
SP  - 689
EP  - 701
DO  - 10.2298/HEMIND140823003T
ER  - 
@article{
author = "Tadić, Vojin and Balaž, Ana Marija and Petric, Marija P. and Milošević, Snežana and Zelenović, Nevena D. and Raspor, Martin Z. and Tadić, Jovan and Prodanović, Radivoje",
year = "2015",
url = "http://cherry.chem.bg.ac.rs/handle/123456789/2040",
abstract = "We have cloned the gene for carbohydrate oxidase (CHO) from Lactuca sativa in two species of yeasts (Saccharomyces cerevisiae and Pichia pastoris). The synthetic gene for the carbohydrate oxidase (1821 bp) from L. sativa cloned into the vector pUC57 and inserted into plasmids pYES2 and pGAP using Escherichia coli DH5 alpha strain. The P. pastoris strain X-33 and the S. cerevisiae strain InvSC1 were used for extracellular expression of CHO. After transformation of P. pastoris X-33 with CHO-pGAP construct none of the colonies showed CHO activity. Two samples displayed a band which did not exist in the sample with the empty vector similar to the molecular weight of CHO. The S. cerevisiae strain InvSC1 has been also transformed with CHO-pYES constructs. Three colonies grew on the plate with cells transformed with the construct. One of the samples showed a band corresponding to about 110 kDa, but no CHO activity was recorded in this case either. Cloning of the foreign genes and heterologous expression in yeasts is widely used in biotechnology, but sometimes can be very dependent on the gene sequence and strain used. In order to obtain active CHO enzyme the further studies on purification and refolding of expressed protein are necessary., Ugljeni hidrat-oksidaza (CHO) iz zelene salate (Lactuca sativa) je enzim koji je do danas nedovoljno ispitan. Vrlo se malo zna o njegovoj strukturi i funkciji. CHO pripada velikoj familiji ugljenihidrat-oksidaza, koje oksiduju šećere. Svaki od članova ove velike familije dobio je ime po supstratu koji oksiduje. Oksidaze iz ove familije enzima imaju kako sličnu ulogu tako i sličnu strukutru. Sve ili većina ovih enzima su monomeri, čiji se polipeptidni lanac uvija u dva domena. Jedan od domena vezuje flavinski kofaktor, a drugi domen je supstrat vezujući. Većina njih oksidaciju supstrata vrši po takozvanom ping-pong mehanizmu. Sve oksidaze iz karbohidrat-oksidaza familije, pa među njima i enzim koji je predmet ove studije (CHO), danas su našle veliku primenu u industriji. CHO se može primenjivati kako u medicinskoj djagnostici, konkretno u biosenzorima za određivanje glukoze u krvi, u prehrambenoj industriji, poljoprivredi, proizvodnji hleba, deterdženata i u raznim drugim industrijskim oblastima. Problem sa ovim enzimom, kao i sa ostalim članovima ove familije, jeste niska koncentracija u prirodnim izvorima. Zato su danas razvijene različite metode rekombinantne tehnologije, kojima se dobijaju ovi enzimi. U ovom radu opisano je kloniranje gena za CHO iz zelene salate u dve vrste kvasaca (Saccharomyces cerevisiae i Pichia pastoris). Sintetički gen za CHO (1821 bp) iz zelene salate kloniran je u vektor pUC57. Escherichia coli soj DH5α korišćen je za kloniranje gena i održavanje plazmida. P. pastoris soj X-33 i S. cerevisiae soj InvSC1 korišćeni su za ekstracelularnu ekspresiju CHO. Aktivnost CHO određena je ABST esejom, a promena absorbance merena je na 405 nm. Potvrda prisustva enzima rađena je na DNK agaroznoj elektroforezi i SDS-PAGE. Posle transformacije P. pastoris X-33, nijedan od klonova nije pokazivao aktivnost CHO. Posle prve fermentacije, kolonije su testirane na SDS-PAGE. Kako su dva uzorka pokazala trake, koje ne postoje na praznom vektoru, ove trake bi mogle odgovarati željenom enzimu, CHO. Traka se nalazi na molekulskoj masi koja je veća od teoretske (više od 120 kDa). Enzim bi mogao biti glikolizovan i zbog toga pokazivati ovako velike vrednosti za molekulsku masu. S. cerevisiae soj InvSC1 transformisan je konstruktom CHO-pZES. Posle 24 sata, tri kolonije su porasle na ploči na kojoj su bile ćelije transformisane pomenutim konstruktom. Uzorci su testirani na SDS-PAGE. Jedan uzorak je pokazao traku na oko 110 kDa, ali aktivnost CHO nije potvrđena takođe. Cilj ove studije je bio kloniranje CHO u kvascima S. cerevisiae i P. pastoris, kao i njena ekspresija u ovim, danas široko primenjivanim ekspresionim sistemima.",
publisher = "Assoc Chemical Engineers Serbia, Belgrade",
journal = "Hemijska industrija",
title = "Cloning of the gene for a carbohydrate oxidase from Lactuca sativa in the yeasts Saccharomyces cerevisiae and Pichia pastoris, Kloniranje gena za ugljeni hidrat oksidazu iz biljke Lactuca sativa u kvasce saccharomyces cerevisiae i Pichia pastoris",
volume = "69",
number = "6",
pages = "689-701",
doi = "10.2298/HEMIND140823003T"
}
Tadić, V., Balaž, A. M., Petric, M. P., Milošević, S., Zelenović, N. D., Raspor, M. Z., Tadić, J.,& Prodanović, R. (2015). Kloniranje gena za ugljeni hidrat oksidazu iz biljke Lactuca sativa u kvasce saccharomyces cerevisiae i Pichia pastoris.
Hemijska industrija
Assoc Chemical Engineers Serbia, Belgrade., 69(6), 689-701.
https://doi.org/10.2298/HEMIND140823003T
Tadić V, Balaž AM, Petric MP, Milošević S, Zelenović ND, Raspor MZ, Tadić J, Prodanović R. Kloniranje gena za ugljeni hidrat oksidazu iz biljke Lactuca sativa u kvasce saccharomyces cerevisiae i Pichia pastoris. Hemijska industrija. 2015;69(6):689-701
Tadić Vojin, Balaž Ana Marija, Petric Marija P., Milošević Snežana, Zelenović Nevena D., Raspor Martin Z., Tadić Jovan, Prodanović Radivoje, "Kloniranje gena za ugljeni hidrat oksidazu iz biljke Lactuca sativa u kvasce saccharomyces cerevisiae i Pichia pastoris" Hemijska industrija, 69, no. 6 (2015):689-701,
https://doi.org/10.2298/HEMIND140823003T .