Montes Tamara, Grazu Valeria, López-Gallego Fernando, Hermoso Juan A, Guisan José M, Fernandez-Lafuente Roberto
Departamento de Biocatalisis, Instituto de Catalisis, CSIC, Campus Universidad Autonoma, Cantoblanco, 28049 Madrid, Spain.
Biomacromolecules. 2006 Nov;7(11):3052-8. doi: 10.1021/bm060527p.
The enzyme penicillin G acylase (PGA) is not adsorbed at pH 7 on DEAE- or PEI-coated supports, neither is it adsorbed on carboxymethyl (CM)- or dextran sulfate (DS)-coated supports. The surface of the enzyme was chemically modified under controlled conditions: chemical amination of the protein surface of carboxylic groups (using soluble carbodiimide and ethylendiamine) and chemical succinylation (using succinic anhydride) of amino groups. The full chemical modification produced some negative effects on enzyme stability and activity, although partial modification (mainly succinylation) presented negligible effects on both enzyme features. The chemical amination of the protein surface permitted the immobilization of the enzyme on CM- and DS-coated support, while the chemical succinylation permitted the enzyme immobilization on DEAE- and PEI-coated supports. Immobilization was very strong on these supports, mainly in the polymeric ones, and dependent on the degree of modification, although the enzymes still can be desorbed after inactivation by incubation under drastic conditions. Moreover, the immobilization on ionic polymeric beds allowed a significant increase in enzyme stability against the inactivation and inhibitory effects of organic solvents, very likely by the promotion of a certain partition of the organic solvent out of the enzyme environment. These results suggest that the enrichment of the surface of proteins with ionic groups may be a good strategy to take advantage of the immobilization of industrial enzymes via ionic exchange on ionic polymeric beds.
青霉素G酰化酶（PGA）在pH 7时不会吸附在涂有二乙氨基乙基（DEAE）或聚乙烯亚胺（PEI）的载体上，也不会吸附在羧甲基（CM）或硫酸葡聚糖（DS）涂层的载体上。在可控条件下对该酶的表面进行化学修饰：对羧基进行蛋白质表面化学胺化（使用可溶性碳二亚胺和乙二胺）以及对氨基进行化学琥珀酰化（使用琥珀酸酐）。尽管部分修饰（主要是琥珀酰化）对酶的稳定性和活性影响可忽略不计，但完全化学修饰对酶的稳定性和活性产生了一些负面影响。蛋白质表面的化学胺化使得该酶能够固定在CM和DS涂层的载体上，而化学琥珀酰化则使得该酶能够固定在DEAE和PEI涂层的载体上。在这些载体上的固定作用非常强，主要是在聚合物载体上，并且取决于修饰程度，不过在剧烈条件下孵育使酶失活后，酶仍可被解吸。此外，固定在离子聚合物床上可显著提高酶对有机溶剂失活和抑制作用的稳定性，这很可能是通过促进有机溶剂从酶环境中进行一定程度的分配实现的。这些结果表明，用离子基团富集蛋白质表面可能是一种利用工业酶通过离子交换固定在离子聚合物床上的良好策略。
