by Filice, Marco, Romero, Oscar, Guisan, Jose M and Palomo, Jose M
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 (textgreater95%) 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.
Reference:
trans,trans-2,4-Hexadiene incorporation on enzymes for site-specific immobilization and fluorescent labeling. (Filice, Marco, Romero, Oscar, Guisan, Jose M and Palomo, Jose M), In Organic & biomolecular chemistry, volume 9, 2011.
Bibtex Entry:
@article{filice_transtrans-24-hexadiene_2011,
	title = {trans,trans-2,4-{Hexadiene} incorporation on enzymes for site-specific immobilization and fluorescent labeling.},
	volume = {9},
	url = {http://xlink.rsc.org/?DOI=c1ob05401e},
	doi = {10.1039/c1ob05401e},
	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 ({textgreater}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.},
	language = {English},
	number = {15},
	journal = {Organic & biomolecular chemistry},
	author = {Filice, Marco and Romero, Oscar and Guisan, Jose M and Palomo, Jose M},
	month = aug,
	year = {2011},
	pmid = {21695340},
	note = {Publisher: The Royal Society of Chemistry},
	pages = {5535--5540}
}