TY  - CONF
AU  - García-Sanz, Carla
AU  - Andreu, Alicia
AU  - de las Rivas, Blanca
AU  - Muñoz, Rosario
AU  - Vukoičić, Ana
AU  - Milivojević, Ana
AU  - Bezbradica, Dejan
AU  - Palomo, Jose Miguel
PY  - 2023
UR  - http://TechnoRep.tmf.bg.ac.rs/handle/123456789/6949
AB  - Laccase (EC 1.10.3.2, p-diphenol: dioxygen oxidoreductase) is a blue copper oxidase
of great industrial interest due to its ability to catalyseoxidation processes of phenols and
persistent organic pollutants. However, it is susceptible to denaturation by high temperatures,
and sensitive to pH and the presence of high concentrations of solvents, which is a problem
for industrial use. To solve this problem, this project develops the synthesis in aqueous
medium of a new Mn metalloenzyme with laccase oxidase mimetic catalytic activity. To do
this, Geobacillus thermocatenulatus lipase (GTL) is used as a "scaffold" enzyme, which is
mixed with a manganese salt at 50 ºC in an aqueous medium. In this way, the in situ
formation of manganese (IV) oxide nanowires is generated, interacting with the enzyme and
obtaining the GTL-Mn bioconjugate. On the other hand, its oxidative activity was evaluated
using the ABTS assay, obtaining a specificity 300 times greater than the laccase from
Trametes versicolor and 2 times more than the laccase from Myceliophthora thermophila
expressed in Aspergillus oryzae (Novozym 51003®). In addition, the new metalloenzyme
turned out to be 2 times more stable at 40 ºC, 3 times more stable in thepresence of 10% can,
and 10 times more stable at 20% and 30% AcN than laccase (Novozym 51003®) by
evaluating it at 2 hour incubation. Moreover, it was shown that the use of immobilization
strategies improves the stabilization of this artificial metalloenzyme two times more. Finally,
the novel Mn metalloenzymes were effective in the reaction of phlorizin oligomerization.
Future research will aim to extend this study for the use of flavonoids as prebiotic molecules
in the cosmetics industry.
PB  - Belgrade : University, Faculty of Technology and Metallurgy
C3  - Book of Abstracts / International Conference Biochemical Engineering and Biotechnology for Young Scientists, 7-8 December, 2023, Belgrade
T1  - STUDY AND PREPARATION OF ARTIFICIAL MANGANESE METALLOENZYMES WITH LACCASE LIKE ACTIVITY
SP  - 35
UR  - https://hdl.handle.net/21.15107/rcub_technorep_6949
ER  - 

@conference{
author = "García-Sanz, Carla and Andreu, Alicia and de las Rivas, Blanca and Muñoz, Rosario and Vukoičić, Ana and Milivojević, Ana and Bezbradica, Dejan and Palomo, Jose Miguel",
year = "2023",
abstract = "Laccase (EC 1.10.3.2, p-diphenol: dioxygen oxidoreductase) is a blue copper oxidase
of great industrial interest due to its ability to catalyseoxidation processes of phenols and
persistent organic pollutants. However, it is susceptible to denaturation by high temperatures,
and sensitive to pH and the presence of high concentrations of solvents, which is a problem
for industrial use. To solve this problem, this project develops the synthesis in aqueous
medium of a new Mn metalloenzyme with laccase oxidase mimetic catalytic activity. To do
this, Geobacillus thermocatenulatus lipase (GTL) is used as a "scaffold" enzyme, which is
mixed with a manganese salt at 50 ºC in an aqueous medium. In this way, the in situ
formation of manganese (IV) oxide nanowires is generated, interacting with the enzyme and
obtaining the GTL-Mn bioconjugate. On the other hand, its oxidative activity was evaluated
using the ABTS assay, obtaining a specificity 300 times greater than the laccase from
Trametes versicolor and 2 times more than the laccase from Myceliophthora thermophila
expressed in Aspergillus oryzae (Novozym 51003®). In addition, the new metalloenzyme
turned out to be 2 times more stable at 40 ºC, 3 times more stable in thepresence of 10% can,
and 10 times more stable at 20% and 30% AcN than laccase (Novozym 51003®) by
evaluating it at 2 hour incubation. Moreover, it was shown that the use of immobilization
strategies improves the stabilization of this artificial metalloenzyme two times more. Finally,
the novel Mn metalloenzymes were effective in the reaction of phlorizin oligomerization.
Future research will aim to extend this study for the use of flavonoids as prebiotic molecules
in the cosmetics industry.",
publisher = "Belgrade : University, Faculty of Technology and Metallurgy",
journal = "Book of Abstracts / International Conference Biochemical Engineering and Biotechnology for Young Scientists, 7-8 December, 2023, Belgrade",
title = "STUDY AND PREPARATION OF ARTIFICIAL MANGANESE METALLOENZYMES WITH LACCASE LIKE ACTIVITY",
pages = "35",
url = "https://hdl.handle.net/21.15107/rcub_technorep_6949"
}

García-Sanz, C., Andreu, A., de las Rivas, B., Muñoz, R., Vukoičić, A., Milivojević, A., Bezbradica, D.,& Palomo, J. M.. (2023). STUDY AND PREPARATION OF ARTIFICIAL MANGANESE METALLOENZYMES WITH LACCASE LIKE ACTIVITY. in Book of Abstracts / International Conference Biochemical Engineering and Biotechnology for Young Scientists, 7-8 December, 2023, Belgrade
Belgrade : University, Faculty of Technology and Metallurgy., 35.
https://hdl.handle.net/21.15107/rcub_technorep_6949

García-Sanz C, Andreu A, de las Rivas B, Muñoz R, Vukoičić A, Milivojević A, Bezbradica D, Palomo JM. STUDY AND PREPARATION OF ARTIFICIAL MANGANESE METALLOENZYMES WITH LACCASE LIKE ACTIVITY. in Book of Abstracts / International Conference Biochemical Engineering and Biotechnology for Young Scientists, 7-8 December, 2023, Belgrade. 2023;:35.
https://hdl.handle.net/21.15107/rcub_technorep_6949 .

García-Sanz, Carla, Andreu, Alicia, de las Rivas, Blanca, Muñoz, Rosario, Vukoičić, Ana, Milivojević, Ana, Bezbradica, Dejan, Palomo, Jose Miguel, "STUDY AND PREPARATION OF ARTIFICIAL MANGANESE METALLOENZYMES WITH LACCASE LIKE ACTIVITY" in Book of Abstracts / International Conference Biochemical Engineering and Biotechnology for Young Scientists, 7-8 December, 2023, Belgrade (2023):35,
https://hdl.handle.net/21.15107/rcub_technorep_6949 .

TY  - JOUR
AU  - Godoy, Cesar A.
AU  - de las Rivas, Blanca
AU  - Bezbradica, Dejan
AU  - Bolivar, Juan M.
AU  - Lopez-Gallego, Fernando
AU  - Fernandez-Lorente, Gloria
AU  - Guisan, Jose M.
PY  - 2011
UR  - http://TechnoRep.tmf.bg.ac.rs/handle/123456789/1875
AB  - Lipase from Geobacillus thermocatenulatus (BTL2) was immobilized in two different matrixes. In one derivative, the enzyme was immobilized on agarose activated with cyanogen bromide (CNBr-BTL2) via its most reactive superficial amino group, whereas the other derivative was covalently immobilized on glyoxyl agarose supports (Gx-BTL2). The latter immobilization protocol leads to intense multipoint covalent attachment between the lysine richest region of enzyme and the glyoxyl groups on the support surface. The resulted solid derivatives were unfolded by incubation under high concentrations of guanidine and then resuspended in aqueous media under different experimental conditions. In both CNBr-BTL2 and Gx-BTL2 derivatives, the oxidation of Cys residues during the unfolding/refolding processes led to inefficient folding for the enzyme because only 25-30% of its initial activity was recovered after 3 h in refolding conditions. Dithiothreitol (DTT), a very mild reducing agent, prevented Cys oxidation during the unfolding/refolding process, greatly improving activity recovery in the refolded forms. In parallel, other variables such as pH, buffer composition and the presence of polymers and other additives, had different effects on refolding efficiencies and refolding rates for both derivatives. In the case of solid derivatives of BTL2 immobilized on CNBr-agarose, the surface's chemistry was crucial to guarantee an optimal protein refolding. In this way, uncharged protein vicinities resulted in better refolding efficiencies than those charged ones.
PB  - Elsevier Science Inc, New York
T2  - Enzyme and Microbial Technology
T1  - Reactivation of a thermostable lipase by solid phase unfolding/refolding Effect of cysteine residues on refolding efficiency
EP  - 394
IS  - 4
SP  - 388
VL  - 49
DO  - 10.1016/j.enzmictec.2011.06.018
ER  - 

@article{
author = "Godoy, Cesar A. and de las Rivas, Blanca and Bezbradica, Dejan and Bolivar, Juan M. and Lopez-Gallego, Fernando and Fernandez-Lorente, Gloria and Guisan, Jose M.",
year = "2011",
abstract = "Lipase from Geobacillus thermocatenulatus (BTL2) was immobilized in two different matrixes. In one derivative, the enzyme was immobilized on agarose activated with cyanogen bromide (CNBr-BTL2) via its most reactive superficial amino group, whereas the other derivative was covalently immobilized on glyoxyl agarose supports (Gx-BTL2). The latter immobilization protocol leads to intense multipoint covalent attachment between the lysine richest region of enzyme and the glyoxyl groups on the support surface. The resulted solid derivatives were unfolded by incubation under high concentrations of guanidine and then resuspended in aqueous media under different experimental conditions. In both CNBr-BTL2 and Gx-BTL2 derivatives, the oxidation of Cys residues during the unfolding/refolding processes led to inefficient folding for the enzyme because only 25-30% of its initial activity was recovered after 3 h in refolding conditions. Dithiothreitol (DTT), a very mild reducing agent, prevented Cys oxidation during the unfolding/refolding process, greatly improving activity recovery in the refolded forms. In parallel, other variables such as pH, buffer composition and the presence of polymers and other additives, had different effects on refolding efficiencies and refolding rates for both derivatives. In the case of solid derivatives of BTL2 immobilized on CNBr-agarose, the surface's chemistry was crucial to guarantee an optimal protein refolding. In this way, uncharged protein vicinities resulted in better refolding efficiencies than those charged ones.",
publisher = "Elsevier Science Inc, New York",
journal = "Enzyme and Microbial Technology",
title = "Reactivation of a thermostable lipase by solid phase unfolding/refolding Effect of cysteine residues on refolding efficiency",
pages = "394-388",
number = "4",
volume = "49",
doi = "10.1016/j.enzmictec.2011.06.018"
}

Godoy, C. A., de las Rivas, B., Bezbradica, D., Bolivar, J. M., Lopez-Gallego, F., Fernandez-Lorente, G.,& Guisan, J. M.. (2011). Reactivation of a thermostable lipase by solid phase unfolding/refolding Effect of cysteine residues on refolding efficiency. in Enzyme and Microbial Technology
Elsevier Science Inc, New York., 49(4), 388-394.
https://doi.org/10.1016/j.enzmictec.2011.06.018

Godoy CA, de las Rivas B, Bezbradica D, Bolivar JM, Lopez-Gallego F, Fernandez-Lorente G, Guisan JM. Reactivation of a thermostable lipase by solid phase unfolding/refolding Effect of cysteine residues on refolding efficiency. in Enzyme and Microbial Technology. 2011;49(4):388-394.
doi:10.1016/j.enzmictec.2011.06.018 .

Godoy, Cesar A., de las Rivas, Blanca, Bezbradica, Dejan, Bolivar, Juan M., Lopez-Gallego, Fernando, Fernandez-Lorente, Gloria, Guisan, Jose M., "Reactivation of a thermostable lipase by solid phase unfolding/refolding Effect of cysteine residues on refolding efficiency" in Enzyme and Microbial Technology, 49, no. 4 (2011):388-394,
https://doi.org/10.1016/j.enzmictec.2011.06.018 . .