TY  - JOUR
AU  - Bezbradica, Dejan
AU  - Mateo, Cesar
AU  - Guisan, Jose M.
PY  - 2014
UR  - http://TechnoRep.tmf.bg.ac.rs/handle/123456789/2813
AB  - Immobilization of enzymes on glutaraldehyde-activated supports has been largely used on supports previously activated with amine groups. Therefore, the supports are positively charged hence usually the immobilization is promoted through a two step mechanism: in a first step the enzyme is adsorbed on the support via an anionic exchange mechanism and then, the covalent immobilization occurs. In this paper a new glutaraldehyde activated support without a net charge is presented and characterized in immobilizations of trypsin, penicillin acylase G, lipase and E. coli BL21 cell extract. Immobilization mechanism was studied and this was produced without an adsorption step. This support promoted initially a reversible immobilization, converting into irreversible after incubation of the enzyme-support for several days or after a reduction step. In addition the stability of glutaraldehyde groups was studied retaining around 50 and 25% of its immobilization capacity for 24 h at pH 7 and 10 respectively. This fact allows the incubation of the enzyme with the support even at alkaline pH promoting an extra stabilization factor for trypsin on this support.
PB  - Elsevier, Amsterdam
T2  - Journal of Molecular Catalysis B-Enzymatic
T1  - Novel support for enzyme immobilization prepared by chemical activation with cysteine and glutaraldehyde
EP  - 224
SP  - 218
VL  - 102
DO  - 10.1016/j.molcatb.2014.02.021
ER  - 

@article{
author = "Bezbradica, Dejan and Mateo, Cesar and Guisan, Jose M.",
year = "2014",
abstract = "Immobilization of enzymes on glutaraldehyde-activated supports has been largely used on supports previously activated with amine groups. Therefore, the supports are positively charged hence usually the immobilization is promoted through a two step mechanism: in a first step the enzyme is adsorbed on the support via an anionic exchange mechanism and then, the covalent immobilization occurs. In this paper a new glutaraldehyde activated support without a net charge is presented and characterized in immobilizations of trypsin, penicillin acylase G, lipase and E. coli BL21 cell extract. Immobilization mechanism was studied and this was produced without an adsorption step. This support promoted initially a reversible immobilization, converting into irreversible after incubation of the enzyme-support for several days or after a reduction step. In addition the stability of glutaraldehyde groups was studied retaining around 50 and 25% of its immobilization capacity for 24 h at pH 7 and 10 respectively. This fact allows the incubation of the enzyme with the support even at alkaline pH promoting an extra stabilization factor for trypsin on this support.",
publisher = "Elsevier, Amsterdam",
journal = "Journal of Molecular Catalysis B-Enzymatic",
title = "Novel support for enzyme immobilization prepared by chemical activation with cysteine and glutaraldehyde",
pages = "224-218",
volume = "102",
doi = "10.1016/j.molcatb.2014.02.021"
}

Bezbradica, D., Mateo, C.,& Guisan, J. M.. (2014). Novel support for enzyme immobilization prepared by chemical activation with cysteine and glutaraldehyde. in Journal of Molecular Catalysis B-Enzymatic
Elsevier, Amsterdam., 102, 218-224.
https://doi.org/10.1016/j.molcatb.2014.02.021

Bezbradica D, Mateo C, Guisan JM. Novel support for enzyme immobilization prepared by chemical activation with cysteine and glutaraldehyde. in Journal of Molecular Catalysis B-Enzymatic. 2014;102:218-224.
doi:10.1016/j.molcatb.2014.02.021 .

Bezbradica, Dejan, Mateo, Cesar, Guisan, Jose M., "Novel support for enzyme immobilization prepared by chemical activation with cysteine and glutaraldehyde" in Journal of Molecular Catalysis B-Enzymatic, 102 (2014):218-224,
https://doi.org/10.1016/j.molcatb.2014.02.021 . .

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 . .