title: Solid-phase chemical amination of a lipase from Bacillus thermocatenulatus to improve its stabilization via covalent immobilization on highly activated glyoxyl-agarose.
abstract: 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 p K 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 degrees 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.
authors.affiliation: Departamento de Microbiologia, Instituto de Fermentaciones Industriales (CSIC), C/Juan de la Cierva 3, 28006 CSIC, Madrid, Spain.
authors.forename: Gloria
authors.initials: G
authors.lastname: Fernandez-Lorente
authors.forename: Cesar A
authors.initials: CA
authors.lastname: Godoy
authors.forename: Adriano A
authors.initials: AA
authors.lastname: Mendes
authors.forename: Fernando
authors.initials: F
authors.lastname: Lopez-Gallego
authors.forename: Valeria
authors.initials: V
authors.lastname: Grazu
authors.forename: Blanca
authors.initials: B
authors.lastname: de Las Rivas
authors.forename: Jose M
authors.initials: JM
authors.lastname: Palomo
authors.forename: Juan
authors.initials: J
authors.lastname: Hermoso
authors.forename: Roberto
authors.initials: R
authors.lastname: Fernandez-Lafuente
authors.forename: Jose M
authors.initials: JM
authors.lastname: Guisan
mesh_terms: D000327:Adsorption; D000586:Amination; D001407:Bacillus / Q000145:classification / Q000201:enzymology*; D002087:Butyrates / Q000737:chemistry; D002384:Catalysis; D004795:Enzyme Stability; D004800:Enzymes, Immobilized / Q000737:chemistry* / Q000378:metabolism*; D005029:Ethylenediamines / Q000737:chemistry; D005977:Glutarates / Q000737:chemistry; D006038:Glyoxylates / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D057927:Hydrophobic and Hydrophilic Interactions; D008049:Lipase / Q000737:chemistry* / Q000378:metabolism*; D008958:Models, Molecular; D015394:Molecular Structure; D010648:Phenylacetates / Q000737:chemistry; D012685:Sepharose / Q000737:chemistry*; D013237:Stereoisomerism; D013499:Surface Properties; D013997:Time Factors
chemical_list: C534573:2-O-butyryl-2-phenylacetic acid; D002087:Butyrates; D004800:Enzymes, Immobilized; D005029:Ethylenediamines; D005977:Glutarates; D006038:Glyoxylates; D010648:Phenylacetates; C070399:glyoxyl agarose; C031234:ethylenediamine; D012685:Sepharose; D008049:Lipase