title: trans,trans-2,4-Hexadiene incorporation on enzymes for site-specific immobilization and fluorescent labeling.
abstract: Lipase B from Candida antarctica (CAL-B) has been site-directedly modified by the introduction of a trans,trans-hexadiene moiety onto lipase molecules, identified by MALDI-TOF. This modification on CAL-B permitted its immobilization on Q-Sepharose supports in excellent yields (>95%) when native lipase was not immobilized at pH 7 and 25 °C. After the entire modification procedure, the catalytic activity of the protein on the solid support was surprisingly increased 2-fold. A tailor-made maleimide-fluorophore derivative was specifically covalently linked to the protein in high yield via a selective Diels-Alder reaction in aqueous media. Furthermore, the NBD-labeled-CAL-B was also immobilized on the ionic support, retaining around 80% of the specific activity. The preparation of this labeled-CAL-B was also possible by a Diels-Alder reaction on solid phase in excellent yields.
authors.affiliation: Departamento de Biocatálisis, Instituto de Catálisis (CSIC), c/marie curie 2, Cantoblanco, Campus UAM, 28049 Madrid, Spain.
authors.forename: Marco
authors.initials: M
authors.lastname: Filice
authors.forename: Oscar
authors.initials: O
authors.lastname: Romero
authors.forename: Jose M
authors.initials: JM
authors.lastname: Guisan
authors.forename: Jose M
authors.initials: JM
authors.lastname: Palomo
mesh_terms: D000595:Amino Acid Sequence; D002175:Candida / Q000201:enzymology*; D004800:Enzymes, Immobilized / Q000737:chemistry*; D005456:Fluorescent Dyes / Q000737:chemistry*; D008049:Lipase / Q000737:chemistry*; D008969:Molecular Sequence Data; D015394:Molecular Structure; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D004800:Enzymes, Immobilized; D005456:Fluorescent Dyes; D008049:Lipase