Departamento de Biocatálisis, Instituto de Catálisis (CSIC), c/marie curie 2, Cantoblanco, Campus UAM, 28049 Madrid, Spain.
Org Biomol Chem. 2011 Aug 7;9(15):5535-40. doi: 10.1039/c1ob05401e. Epub 2011 Jun 22.
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.
脂肪酶 B 来自南极假丝酵母（CAL-B），通过在脂肪酶分子上引入反式，反式-己二烯部分，从而实现定点修饰，通过 MALDI-TOF 鉴定。这种修饰使得 CAL-B 可以在 pH 7 和 25°C 时，在 Q-琼脂糖载体上以优异的收率（>95%）固定化，而天然脂肪酶则不能固定化。在整个修饰过程之后，蛋白质在固体载体上的催化活性出人意料地提高了 2 倍。一种特制的马来酰亚胺-荧光染料衍生物通过在水介质中的选择性 Diels-Alder 反应以高产率特异性地共价连接到蛋白质上。此外，NBD 标记的-CAL-B 也可以通过离子载体固定化，保留约 80%的比活性。通过固相上的 Diels-Alder 反应也可以以优异的收率制备这种标记的-CAL-B。
