Torres Rodrigo, Mateo Cesar, Fernández-Lorente Gloria, Ortiz Claudia, Fuentes Manuel, Palomo Jose M, Guisan Jose M, Fernández-Lafuente Roberto
Departamento de Biocatálisis, Instituto de Catalisis, CSIC, Campus Universidad Autonoma, Cantoblanco, 28049 Madrid, Spain.
Biotechnol Prog. 2003 May-Jun;19(3):1056-60. doi: 10.1021/bp025771g.
The properties of a new and commercially available amino-epoxy support (amino-epoxy-Sepabeads) have been compared to conventional epoxy supports to immobilize enzymes, using the beta-galactosidase from Aspergillus oryzae as a model enzyme. The new support has a layer of epoxy groups over a layer of ethylenediamine that is covalently bound to the support. This support has both a great anionic exchanger strength and a high density of epoxy groups. Epoxy supports require the physical adsorption of the proteins onto the support before the covalent binding of the enzyme to the epoxy groups. Using conventional supports the immobilization rate is slow, because the adsorption is of hydrophobic nature, and immobilization must be performed using high ionic strength (over 0.5 M sodium phosphate) and a support with a fairly hydrophobic nature. Using the new support, immobilization may be performed at moderately low ionic strength, it occurs very rapidly, and it is not necessary to use a hydrophobic support. Therefore, this support should be specially recommended for immobilization of enzymes that cannot be submitted to high ionic strength. Also, both supports may be expected to yield different orientations of the proteins on the support, and that may result in some advantages in specific cases. For example, the model enzyme became almost fully inactivated when using the conventional support, while it exhibited an almost intact activity after immobilization on the new support. Furthermore, enzyme stability was significantly improved by the immobilization on this support (by more than a 12-fold factor), suggesting the promotion of some multipoint covalent attachment between the enzyme and the support (in fact the enzyme adsorbed on an equivalent cationic support without epoxy groups was even slightly less stable than the soluble enzyme).
将一种新型且市售的氨基环氧载体（氨基环氧 - 西帕玻珠）的特性与传统环氧载体进行了比较，以固定化酶，使用米曲霉的β - 半乳糖苷酶作为模型酶。这种新型载体在与载体共价结合的乙二胺层上有一层环氧基团。该载体兼具强大的阴离子交换剂强度和高密度的环氧基团。环氧载体在酶与环氧基团共价结合之前，需要蛋白质物理吸附到载体上。使用传统载体时，固定化速率较慢，因为吸附具有疏水性，且固定化必须在高离子强度（超过0.5M磷酸钠）和具有相当疏水性的载体上进行。使用新型载体时，固定化可在适度低的离子强度下进行，速度非常快，且无需使用疏水性载体。因此，对于不能承受高离子强度的酶的固定化，应特别推荐这种载体。此外，预计两种载体上蛋白质的取向会有所不同，这在特定情况下可能会带来一些优势。例如，使用传统载体时模型酶几乎完全失活，而固定在新型载体上后它表现出几乎完整的活性。此外，通过固定在这种载体上，酶的稳定性显著提高（提高了12倍以上），这表明酶与载体之间促进了一些多点共价连接（事实上，吸附在不含环氧基团的等效阳离子载体上的酶甚至比可溶性酶稍微不稳定一些）。
