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

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

Organic & biomolecular chemistry (2011-06-23)
Marco Filice, Oscar Romero, Jose M Guisan, Jose M Palomo
摘要

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.

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Sigma-Aldrich
4-氯-7-硝基苯并-2-氧杂-1,3-二唑, 98%