title: Reactivation of a thermostable lipase by solid phase unfolding/refolding effect of cysteine residues on refolding efficiency.
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 3h 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.
authors: Departamento de Biocatálisis, Instituto de Catálisis, CSIC, Campus UAM, Cantoblanco, 28049 Madrid, Spain.
authors: César A
authors: CA
authors: Godoy
authors: Blanca
authors: B
authors: de las Rivas
authors: Dejan
authors: D
authors: Bezbradica
authors: Juan M
authors: JM
authors: Bolivar
authors: Fernando
authors: F
authors: López-Gallego
authors: Gloria
authors: G
authors: Fernandez-Lorente
authors: Jose M
authors: JM
authors: Guisan
mesh_terms: D001426:Bacterial Proteins; D001709:Biotechnology / Q000379:methods; D003545:Cysteine / Q000737:chemistry / Q000494:pharmacology; D004229:Dithiothreitol / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects*; D004795:Enzyme Stability; D004800:Enzymes, Immobilized; D056506:Geobacillus / Q000201:enzymology; D006038:Glyoxylates; D008049:Lipase / Q000737:chemistry / Q000378:metabolism*; D008958:Models, Molecular; D010084:Oxidation-Reduction; D058849:Protein Refolding / Q000187:drug effects; D012685:Sepharose
chemical_list: D001426:Bacterial Proteins; D004800:Enzymes, Immobilized; D006038:Glyoxylates; C070399:glyoxyl agarose; D012685:Sepharose; D008049:Lipase; C428386:thermostable lipase; D003545:Cysteine; D004229:Dithiothreitol