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dc.creatorGodoy, Cesar A.
dc.creatorde las Rivas, Blanca
dc.creatorBezbradica, Dejan
dc.creatorBolivar, Juan M.
dc.creatorLopez-Gallego, Fernando
dc.creatorFernandez-Lorente, Gloria
dc.creatorGuisan, Jose M.
dc.date.accessioned2021-03-10T11:34:19Z
dc.date.available2021-03-10T11:34:19Z
dc.date.issued2011
dc.identifier.issn0141-0229
dc.identifier.urihttp://TechnoRep.tmf.bg.ac.rs/handle/123456789/1875
dc.description.abstractLipase 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.en
dc.publisherElsevier Science Inc, New York
dc.relationComunidad Autonoma de Madrid (CAM)Comunidad de Madrid [S0505/PPQ/0344]
dc.relationCICYTConsejo Interinstitucional de Ciencia y Tecnologia (CICYT) [BIO-2005-8576, AGL-2009-07625]
dc.relationMCI
dc.relationinfo:eu-repo/grantAgreement/MESTD/MPN2006-2010/20064/RS//
dc.rightsrestrictedAccess
dc.sourceEnzyme and Microbial Technology
dc.subjectAdditivesen
dc.subjectEnzyme reactivationen
dc.subjectRefoldingen
dc.subjectLipaseen
dc.subjectCysteine oxidationen
dc.subjectImmobilizationen
dc.titleReactivation of a thermostable lipase by solid phase unfolding/refolding Effect of cysteine residues on refolding efficiencyen
dc.typearticle
dc.rights.licenseARR
dc.citation.epage394
dc.citation.issue4
dc.citation.other49(4): 388-394
dc.citation.rankM22
dc.citation.spage388
dc.citation.volume49
dc.identifier.doi10.1016/j.enzmictec.2011.06.018
dc.identifier.pmid22112565
dc.identifier.rcubconv_3662
dc.identifier.scopus2-s2.0-80051794269
dc.identifier.wos000295107300009
dc.type.versionpublishedVersion


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