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Reactivation of a thermostable lipase by solid phase unfolding/refolding: Effect of cysteine residues on refolding efficiency

Paper ID Volume ID Publish Year Pages File Format Full-Text
17287 42657 2011 7 PDF Available
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 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.

Keywords
BTL2, Lipase 2 from Geobacillus thermocathenolatus; CNBR-BTL2, BTL2 immobilized on agarose activated with cyanogen bromide groups; Gx-BTL2, BTL2 immobilized on agarose activated with glyoxylgroupsAdditives; Enzyme reactivation; Refolding; Lipase; Cysteine
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Reactivation of a thermostable lipase by solid phase unfolding/refolding: Effect of cysteine residues on refolding efficiency
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Publisher
Database: Elsevier - ScienceDirect
Journal: Enzyme and Microbial Technology - Volume 49, Issue 4, 10 September 2011, Pages 388–394
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us