Trans,trans-2,4-Hexadiene incorporation on enzymes for site-specific immobilization and fluorescent labeling

Marco Filice, OSCAR ENRIQUE ROMERO ORMAZABAL, Jose M. Guisan, Jose M. Palomo

Research output: Contribution to journalArticlepeer-review

19 Scopus citations

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.

Original languageEnglish
Pages (from-to)5535-5540
Number of pages6
JournalOrganic and Biomolecular Chemistry
Volume9
Issue number15
DOIs
StatePublished - 7 Aug 2011

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