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://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",
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. 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  - DATA
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://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
UR  - https://hdl.handle.net/21.15107/rcub_cherry_2900
ER  - 

@misc{
author = "Blažić, Marija and Balaž, Ana Marija and Tadić, Vojin and Draganić, Bojana and Ostafe, Raluca and Fischer, Rainer and Prodanović, Radivoje",
year = "2019",
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",
url = "https://hdl.handle.net/21.15107/rcub_cherry_2900"
}

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. in Biochemical Engineering Journal
Elsevier..
https://hdl.handle.net/21.15107/rcub_cherry_2900

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. in Biochemical Engineering Journal. 2019;.
https://hdl.handle.net/21.15107/rcub_cherry_2900 .

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" in Biochemical Engineering Journal (2019),
https://hdl.handle.net/21.15107/rcub_cherry_2900 .

TY  - JOUR
AU  - Tadić, Vojin
AU  - Tadić, Jovan
AU  - Milošević, Snežana
AU  - Cingel, Aleksandar
AU  - Prodanović, Olivera
AU  - Ćosić, Tatjana
AU  - Vujčić, Zoran
PY  - 2018
UR  - https://cherry.chem.bg.ac.rs/handle/123456789/2090
AB  - In this study we investigated physiological parameters of stress (enzymatic and non-enzymatic) in lettuce (Lactuca sativa L.) and its hairy roots induced by water solution of phenol. Two varieties of lettuce were examined, Ljubljanska ledenka and Nansen. Plants were grown in water with phenol concentration of 200 mgL(-1), during 10 days. We monitored activity of peroxidases, catalases, polyphenol oxidase and superoxide dismutase, as well as proline and chlorophyll content. We observed a decrease in peroxidases, and increase in activity of catalase, polyphenol oxidase and superoxide dismutase compared to control plants. The concentration of praline was constantly increasing in both lettuce varieties over the course of the experiment. We detected an increase in activity of all monitored enzymes, except polyphenol oxidases, in hairy roots. The hydroponic system provides a useful framework for studying the effect of different harmful substances and its elimination. In such a system, as used in this work for the study of physiological processes in antioxidant protection activated when plant was exposed to phenol, lettuce and its hairy roots can be viewed as tools for water remediation.
PB  - Elsevier Science Bv, Amsterdam
T2  - Scientia Horticulturae
T1  - Phenol induced physiological stress in hydroponically grown lettuce (Lactuca sativa L.)- Part 2
VL  - 232
SP  - 71
EP  - 83
DO  - 10.1016/j.scienta.2017.12.024
ER  - 

@article{
author = "Tadić, Vojin and Tadić, Jovan and Milošević, Snežana and Cingel, Aleksandar and Prodanović, Olivera and Ćosić, Tatjana and Vujčić, Zoran",
year = "2018",
abstract = "In this study we investigated physiological parameters of stress (enzymatic and non-enzymatic) in lettuce (Lactuca sativa L.) and its hairy roots induced by water solution of phenol. Two varieties of lettuce were examined, Ljubljanska ledenka and Nansen. Plants were grown in water with phenol concentration of 200 mgL(-1), during 10 days. We monitored activity of peroxidases, catalases, polyphenol oxidase and superoxide dismutase, as well as proline and chlorophyll content. We observed a decrease in peroxidases, and increase in activity of catalase, polyphenol oxidase and superoxide dismutase compared to control plants. The concentration of praline was constantly increasing in both lettuce varieties over the course of the experiment. We detected an increase in activity of all monitored enzymes, except polyphenol oxidases, in hairy roots. The hydroponic system provides a useful framework for studying the effect of different harmful substances and its elimination. In such a system, as used in this work for the study of physiological processes in antioxidant protection activated when plant was exposed to phenol, lettuce and its hairy roots can be viewed as tools for water remediation.",
publisher = "Elsevier Science Bv, Amsterdam",
journal = "Scientia Horticulturae",
title = "Phenol induced physiological stress in hydroponically grown lettuce (Lactuca sativa L.)- Part 2",
volume = "232",
pages = "71-83",
doi = "10.1016/j.scienta.2017.12.024"
}

Tadić, V., Tadić, J., Milošević, S., Cingel, A., Prodanović, O., Ćosić, T.,& Vujčić, Z.. (2018). Phenol induced physiological stress in hydroponically grown lettuce (Lactuca sativa L.)- Part 2. in Scientia Horticulturae
Elsevier Science Bv, Amsterdam., 232, 71-83.
https://doi.org/10.1016/j.scienta.2017.12.024

Tadić V, Tadić J, Milošević S, Cingel A, Prodanović O, Ćosić T, Vujčić Z. Phenol induced physiological stress in hydroponically grown lettuce (Lactuca sativa L.)- Part 2. in Scientia Horticulturae. 2018;232:71-83.
doi:10.1016/j.scienta.2017.12.024 .

Tadić, Vojin, Tadić, Jovan, Milošević, Snežana, Cingel, Aleksandar, Prodanović, Olivera, Ćosić, Tatjana, Vujčić, Zoran, "Phenol induced physiological stress in hydroponically grown lettuce (Lactuca sativa L.)- Part 2" in Scientia Horticulturae, 232 (2018):71-83,
https://doi.org/10.1016/j.scienta.2017.12.024 . .

TY  - JOUR
AU  - Tadić, Vojin
AU  - Petric, Marija
AU  - Uzelac, Branka
AU  - Milošević, Snežana
AU  - Vujčić, Zoran
AU  - Stevanovic, Jasmina
AU  - Tadić, Jovan
PY  - 2018
UR  - https://cherry.chem.bg.ac.rs/handle/123456789/2106
AB  - We investigated the removal of phenol from water solutions (200 mg L-1) using two varieties of lettuce (Lactuca sativa L) and their hairy roots. Experiments were done in a hydroponic system where adult plants were grown in phenol solutions for 10 days. The solution was refreshed every two days in order to maintain the constant concentration of phenol. Hairy roots were also cultivated in a solution containing phenol at concentrations varying from 25 to 125 mg L-1 in order to determine the maximum concentration of phenol that can be removed by hairy roots. Both varieties of lettuce reduced the concentration of phenol below the detection limit after six days at the initial phenol concentration of 200 mg L-1. Transformed roots completely removed phenol at the initial concentrations of 100 mg L-1, but were not able to remove phenol at constant concentration above 25 mg L-1. Lettuce plants and hairy roots are excellent candidates for the process of phenol removal from wastewaters. This plant is good choice for bioremediation of water and represents a potentially efficient and inexpensive system for water purification. The performance of lettuce plants and hairy roots to remove phenol from water solutions under real conditions, depleted nutrients or presence of other compounds should be examined further.
PB  - Elsevier Science Bv, Amsterdam
T2  - Scientia Horticulturae
T1  - Phenol removal from solution using different varieties of lettuce (Lactuca sativa L.) - Part 1
VL  - 231
SP  - 210
EP  - 218
DO  - 10.1016/j.scienta.2017.12.025
ER  - 

@article{
author = "Tadić, Vojin and Petric, Marija and Uzelac, Branka and Milošević, Snežana and Vujčić, Zoran and Stevanovic, Jasmina and Tadić, Jovan",
year = "2018",
abstract = "We investigated the removal of phenol from water solutions (200 mg L-1) using two varieties of lettuce (Lactuca sativa L) and their hairy roots. Experiments were done in a hydroponic system where adult plants were grown in phenol solutions for 10 days. The solution was refreshed every two days in order to maintain the constant concentration of phenol. Hairy roots were also cultivated in a solution containing phenol at concentrations varying from 25 to 125 mg L-1 in order to determine the maximum concentration of phenol that can be removed by hairy roots. Both varieties of lettuce reduced the concentration of phenol below the detection limit after six days at the initial phenol concentration of 200 mg L-1. Transformed roots completely removed phenol at the initial concentrations of 100 mg L-1, but were not able to remove phenol at constant concentration above 25 mg L-1. Lettuce plants and hairy roots are excellent candidates for the process of phenol removal from wastewaters. This plant is good choice for bioremediation of water and represents a potentially efficient and inexpensive system for water purification. The performance of lettuce plants and hairy roots to remove phenol from water solutions under real conditions, depleted nutrients or presence of other compounds should be examined further.",
publisher = "Elsevier Science Bv, Amsterdam",
journal = "Scientia Horticulturae",
title = "Phenol removal from solution using different varieties of lettuce (Lactuca sativa L.) - Part 1",
volume = "231",
pages = "210-218",
doi = "10.1016/j.scienta.2017.12.025"
}

Tadić, V., Petric, M., Uzelac, B., Milošević, S., Vujčić, Z., Stevanovic, J.,& Tadić, J.. (2018). Phenol removal from solution using different varieties of lettuce (Lactuca sativa L.) - Part 1. in Scientia Horticulturae
Elsevier Science Bv, Amsterdam., 231, 210-218.
https://doi.org/10.1016/j.scienta.2017.12.025

Tadić V, Petric M, Uzelac B, Milošević S, Vujčić Z, Stevanovic J, Tadić J. Phenol removal from solution using different varieties of lettuce (Lactuca sativa L.) - Part 1. in Scientia Horticulturae. 2018;231:210-218.
doi:10.1016/j.scienta.2017.12.025 .

Tadić, Vojin, Petric, Marija, Uzelac, Branka, Milošević, Snežana, Vujčić, Zoran, Stevanovic, Jasmina, Tadić, Jovan, "Phenol removal from solution using different varieties of lettuce (Lactuca sativa L.) - Part 1" in Scientia Horticulturae, 231 (2018):210-218,
https://doi.org/10.1016/j.scienta.2017.12.025 . .

TY  - JOUR
AU  - Petric, Marija
AU  - Subotic, Angelina
AU  - Jevremovic, Sladana
AU  - Trifunović-Momcilov, Milana
AU  - Tadić, Vojin
AU  - Grujić, Marica
AU  - Vujčić, Zoran
PY  - 2017
UR  - https://cherry.chem.bg.ac.rs/handle/123456789/2543
AB  - The aim of this study was to determine the enzymatic profile of esterases and peroxidases during early stages of somatic embryogenesis of Fritillaria meleagris L. Somatic embryogenesis was induced using the leaf base as explant on a medium supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D). Zymography showed the presence of different moieties, six isoforms of esterases and peroxidases, during morphogenesis as compared to control explants. One isoform of esterases was detected only during the process of somatic embryogenesis, and one isoform was detected in control explants. Analysis of esterases with 1-naphthyl butyrate proved that esterases, which participate in somatic embryogenesis of F. meleagris, belong to the family of aryl esterases. For the first time it was proved that five isoforms of esterases, which are involved in morphogenesis of F. meleagris, belong to the family of aryl esterases, while two isoforms are carboxyl esterases. One isoform of carboxyl esterases was visible in control explants. This is also the first description of peroxidases during the morphogenetic process, and of the difference between aryl and carboxyl esterases. More isoforms of esterases during morphogenesis as compared to control explants are probably responsible for some early physiological process during somatic embryogenesis of F. meleagris.
PB  - Inst Bioloska Istrazivanja Sinisa Stankovic, Beograd
T2  - Archives of biological sciences
T1  - Esterase and peroxidase isoforms during initial stages of somatic embryogenesis in Fritillaria meleagris L. leaf base
VL  - 69
IS  - 4
SP  - 619
EP  - 625
DO  - 10.2298/ABS161130007P
ER  - 

@article{
author = "Petric, Marija and Subotic, Angelina and Jevremovic, Sladana and Trifunović-Momcilov, Milana and Tadić, Vojin and Grujić, Marica and Vujčić, Zoran",
year = "2017",
abstract = "The aim of this study was to determine the enzymatic profile of esterases and peroxidases during early stages of somatic embryogenesis of Fritillaria meleagris L. Somatic embryogenesis was induced using the leaf base as explant on a medium supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D). Zymography showed the presence of different moieties, six isoforms of esterases and peroxidases, during morphogenesis as compared to control explants. One isoform of esterases was detected only during the process of somatic embryogenesis, and one isoform was detected in control explants. Analysis of esterases with 1-naphthyl butyrate proved that esterases, which participate in somatic embryogenesis of F. meleagris, belong to the family of aryl esterases. For the first time it was proved that five isoforms of esterases, which are involved in morphogenesis of F. meleagris, belong to the family of aryl esterases, while two isoforms are carboxyl esterases. One isoform of carboxyl esterases was visible in control explants. This is also the first description of peroxidases during the morphogenetic process, and of the difference between aryl and carboxyl esterases. More isoforms of esterases during morphogenesis as compared to control explants are probably responsible for some early physiological process during somatic embryogenesis of F. meleagris.",
publisher = "Inst Bioloska Istrazivanja Sinisa Stankovic, Beograd",
journal = "Archives of biological sciences",
title = "Esterase and peroxidase isoforms during initial stages of somatic embryogenesis in Fritillaria meleagris L. leaf base",
volume = "69",
number = "4",
pages = "619-625",
doi = "10.2298/ABS161130007P"
}

Petric, M., Subotic, A., Jevremovic, S., Trifunović-Momcilov, M., Tadić, V., Grujić, M.,& Vujčić, Z.. (2017). Esterase and peroxidase isoforms during initial stages of somatic embryogenesis in Fritillaria meleagris L. leaf base. in Archives of biological sciences
Inst Bioloska Istrazivanja Sinisa Stankovic, Beograd., 69(4), 619-625.
https://doi.org/10.2298/ABS161130007P

Petric M, Subotic A, Jevremovic S, Trifunović-Momcilov M, Tadić V, Grujić M, Vujčić Z. Esterase and peroxidase isoforms during initial stages of somatic embryogenesis in Fritillaria meleagris L. leaf base. in Archives of biological sciences. 2017;69(4):619-625.
doi:10.2298/ABS161130007P .

Petric, Marija, Subotic, Angelina, Jevremovic, Sladana, Trifunović-Momcilov, Milana, Tadić, Vojin, Grujić, Marica, Vujčić, Zoran, "Esterase and peroxidase isoforms during initial stages of somatic embryogenesis in Fritillaria meleagris L. leaf base" in Archives of biological sciences, 69, no. 4 (2017):619-625,
https://doi.org/10.2298/ABS161130007P . .

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  - https://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",
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). Cloning of the gene for a carbohydrate oxidase from Lactuca sativa in the yeasts Saccharomyces cerevisiae and Pichia pastoris. in 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. Cloning of the gene for a carbohydrate oxidase from Lactuca sativa in the yeasts Saccharomyces cerevisiae and Pichia pastoris. in Hemijska industrija. 2015;69(6):689-701.
doi:10.2298/HEMIND140823003T .

Tadić, Vojin, Balaž, Ana Marija, Petric, Marija P., Milošević, Snežana, Zelenović, Nevena D., Raspor, Martin Z., Tadić, Jovan, Prodanović, Radivoje, "Cloning of the gene for a carbohydrate oxidase from Lactuca sativa in the yeasts Saccharomyces cerevisiae and Pichia pastoris" in Hemijska industrija, 69, no. 6 (2015):689-701,
https://doi.org/10.2298/HEMIND140823003T . .

TY  - JOUR
AU  - Petric, Marija
AU  - Subotic, Angelina
AU  - Jevremovic, Sladana
AU  - Trifunović-Momcilov, Milana
AU  - Tadić, Vojin
AU  - Grujić, Marica
AU  - Vujčić, Zoran
PY  - 2015
UR  - https://cherry.chem.bg.ac.rs/handle/123456789/2032
AB  - Morphogenesis in vitro is a complex and still poorly defined process. We investigated esterase and peroxidase isoforms detected in bulb scale, during Fritillaria meleagris morphogenesis. Bulbs were grown either at 4 degrees C or on a medium with an increased concentration of sucrose (4.5%) for 30 days. After these pre-treatments, the bulb scales were further grown on nutrient media that contained different concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D) and kinetin (KIN) or thidiazuron (TDZ). Regeneration of somatic embryos and bulblets occurred at the same explant. The highest numbers of somatic embryos and bulblets were regenerated on the medium containing 2,4-D and KIN (1 mg/L each), while morphogenesis was most successful at a TDZ concentration between 0.5 and 1 mg/L. Monitoring of esterases and peroxidases was performed by growing bulb scales on a medium enriched with 2,4-D and KIN or TDZ (1 mg/L), and the number and activity of isoforms were followed every 7 days for 4 weeks. In control explants, six isoforms of esterase were observed. Three isoforms of peroxidase were not detected in the control bulb scale, which has not begun its morphogenesis process. 2015 Academie des sciences. (C) Published by Elsevier Masson SAS. All rights reserved.
PB  - Elsevier France-Editions Scientifiques Medicales Elsevier, Issy-Les-Moulineaux
T2  - Comptes Rendus Biologies
T1  - Esterase and peroxidase isoforms in different stages of morphogenesis in Fritillaria meleagris L. in bulb-scale culture
VL  - 338
IS  - 12
SP  - 793
EP  - 802
DO  - 10.1016/j.crvi.2015.08.002
ER  - 

@article{
author = "Petric, Marija and Subotic, Angelina and Jevremovic, Sladana and Trifunović-Momcilov, Milana and Tadić, Vojin and Grujić, Marica and Vujčić, Zoran",
year = "2015",
abstract = "Morphogenesis in vitro is a complex and still poorly defined process. We investigated esterase and peroxidase isoforms detected in bulb scale, during Fritillaria meleagris morphogenesis. Bulbs were grown either at 4 degrees C or on a medium with an increased concentration of sucrose (4.5%) for 30 days. After these pre-treatments, the bulb scales were further grown on nutrient media that contained different concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D) and kinetin (KIN) or thidiazuron (TDZ). Regeneration of somatic embryos and bulblets occurred at the same explant. The highest numbers of somatic embryos and bulblets were regenerated on the medium containing 2,4-D and KIN (1 mg/L each), while morphogenesis was most successful at a TDZ concentration between 0.5 and 1 mg/L. Monitoring of esterases and peroxidases was performed by growing bulb scales on a medium enriched with 2,4-D and KIN or TDZ (1 mg/L), and the number and activity of isoforms were followed every 7 days for 4 weeks. In control explants, six isoforms of esterase were observed. Three isoforms of peroxidase were not detected in the control bulb scale, which has not begun its morphogenesis process. 2015 Academie des sciences. (C) Published by Elsevier Masson SAS. All rights reserved.",
publisher = "Elsevier France-Editions Scientifiques Medicales Elsevier, Issy-Les-Moulineaux",
journal = "Comptes Rendus Biologies",
title = "Esterase and peroxidase isoforms in different stages of morphogenesis in Fritillaria meleagris L. in bulb-scale culture",
volume = "338",
number = "12",
pages = "793-802",
doi = "10.1016/j.crvi.2015.08.002"
}

Petric, M., Subotic, A., Jevremovic, S., Trifunović-Momcilov, M., Tadić, V., Grujić, M.,& Vujčić, Z.. (2015). Esterase and peroxidase isoforms in different stages of morphogenesis in Fritillaria meleagris L. in bulb-scale culture. in Comptes Rendus Biologies
Elsevier France-Editions Scientifiques Medicales Elsevier, Issy-Les-Moulineaux., 338(12), 793-802.
https://doi.org/10.1016/j.crvi.2015.08.002

Petric M, Subotic A, Jevremovic S, Trifunović-Momcilov M, Tadić V, Grujić M, Vujčić Z. Esterase and peroxidase isoforms in different stages of morphogenesis in Fritillaria meleagris L. in bulb-scale culture. in Comptes Rendus Biologies. 2015;338(12):793-802.
doi:10.1016/j.crvi.2015.08.002 .

Petric, Marija, Subotic, Angelina, Jevremovic, Sladana, Trifunović-Momcilov, Milana, Tadić, Vojin, Grujić, Marica, Vujčić, Zoran, "Esterase and peroxidase isoforms in different stages of morphogenesis in Fritillaria meleagris L. in bulb-scale culture" in Comptes Rendus Biologies, 338, no. 12 (2015):793-802,
https://doi.org/10.1016/j.crvi.2015.08.002 . .