Journal
PROCESS BIOCHEMISTRY
Volume 45, Issue 4, Pages 534-541Publisher
ELSEVIER SCI LTD
DOI: 10.1016/j.procbio.2009.11.014
Keywords
Chemical modification; Thiol-disulfide exchange; Tailor-made polymers; Lipase; Activity; Lipase hyperactivation
Categories
Funding
- Spanish CICYT [BIO-2005-8576]
- Comunidad Autonoma de Madrid [S0505/PPM3449]
- Spanish Ministry of Science and Innovation for Ramon y Cajal
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The activity of a lipase from Geobacillus thermocatenulatus (BTL2) can be greatly improved by site-directed chemical modification of a single external Cys64. This residue is placed in the proximity of the region where the lid is allocated when the lipase exhibits its open and active form. Thiol group of Cys64 was modified by thiol-disulfide exchange with pyridyldisulfide poly-aminated-dextrans or monocarboxylated-polyethyleneglycol. The modification was performed on the covalently immobilized lipase on CNBr-agarose or glyoxyl-agarose. The activity of modified derivatives was strongly dependent on the immobilized preparation, the polymer used and the substrate assayed. For example, the modification with PEG-COOH of BTL2 immobilized on glyoxyl-agarose increased 5-fold the enzyme activity towards the hydrolysis of 2-O-butyryl-2-phenylacetic acid. However, the modification with 3-(2-pyridyldithio)-propionyl-dextran-NH2 reduced the activity to 40%. The fact that the modified enzymes can be inhibited by an irreversible inhibitor much more rapidly than the unmodified ones suggested that the main effect of the modification is to somehow stabilize the open form of the lipase. (C) 2009 Elsevier Ltd. All rights reserved.
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