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Solid-Phase Chemical Amination of a Lipase from Bacillus thermocatenulatus To Improve Its Stabilization via Covalent Immobilization on Highly Activated Glyoxyl-Agarose

2008; American Chemical Society; Volume: 9; Issue: 9 Linguagem: Inglês

10.1021/bm800609g

1526-4602

Gloria Fernández‐Lorente, César A. Godoy, Adriano A. Méndes, Fernándo López-Gallego, Valeria Grazú, Blanca de las Rivas, José M. Palomo, J.A. Hermoso, Roberto Fernandéz‐Lafuente, José M. Guisán,

Pancreatic function and diabetes

In this paper, the stabilization of a lipase from Bacillus thermocatenulatus (BTL2) by a new strategy is described. First, the lipase is selectively adsorbed on hydrophobic supports. Second, the carboxylic residues of the enzyme are modified with ethylenediamine, generating a new enzyme having 4-fold more amino groups than the native enzyme. The chemical amination did not present a significant effect on the enzyme activity and only reduced the enzyme half-life by a 3−4-fold factor in inactivations promoted by heat or organic solvents. Next, the aminated and purified enzyme is desorbed from the support using 0.2% Triton X-100. Then, the aminated enzyme was immobilized on glyoxyl-agarose by multipoint covalent attachment. The immobilized enzyme retained 65% of the starting activity. Because of the lower pK of the new amino groups in the enzyme surface, the immobilization could be performed at pH 9 (while the native enzyme was only immobilized at pH over 10). In fact, the immobilization rate was higher at this pH value for the aminated enzyme than that of the native enzyme at pH 10. The optimal stabilization protocol was the immobilization of aminated BTL2 at pH 9 and the further incubation for 24 h at 25 °C and pH 10. This preparation was 5-fold more stable than the optimal BTL2 immobilized on glyoxyl agarose and around 1200-fold more stable than the enzyme immobilized on CNBr and further aminated. The catalytic properties of BTL2 could be greatly modulated by the immobilization protocol. For example, from (R/S)-2-O-butyryl-2-phenylacetic acid, one preparation of BTL2 could be used to produce the S-isomer, while other preparation produced the R-isomer.