Reactivation of a thermostable lipase by solid phase unfolding/refolding Effect of cysteine residues on refolding efficiency
Само за регистроване кориснике
2011
Аутори
Godoy, Cesar A.de las Rivas, Blanca
Bezbradica, Dejan
Bolivar, Juan M.
Lopez-Gallego, Fernando
Fernandez-Lorente, Gloria
Guisan, Jose M.
Чланак у часопису (Објављена верзија)
Метаподаци
Приказ свих података о документуАпстракт
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 o...xidation 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.
Кључне речи:
Additives / Enzyme reactivation / Refolding / Lipase / Cysteine oxidation / ImmobilizationИзвор:
Enzyme and Microbial Technology, 2011, 49, 4, 388-394Издавач:
- Elsevier Science Inc, New York
Финансирање / пројекти:
- Comunidad Autonoma de Madrid (CAM)Comunidad de Madrid [S0505/PPQ/0344]
- CICYTConsejo Interinstitucional de Ciencia y Tecnologia (CICYT) [BIO-2005-8576, AGL-2009-07625]
- MCI
- Развој биотехнолошких поступака за производњу адитива и нових формулација за прехрамбену индустрију (RS-MESTD-MPN2006-2010-20064)
DOI: 10.1016/j.enzmictec.2011.06.018
ISSN: 0141-0229
PubMed: 22112565
WoS: 000295107300009
Scopus: 2-s2.0-80051794269
Институција/група
Tehnološko-metalurški fakultetTY - 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 . .