In this paper, a novel procedure for the immobilization and stabilization of enzymes is proposed: the multipoint covalent attachment of bi-molecular enzyme aggregates. This immobilization protocol allows the “capture” and fixation of the enzyme aggregate on the support surface. In addition to stabilization by multipoint attachment, enzyme aggregation promotes very interesting stabilizing effects. In the presence of low concentrations of polyethylene glycol (30%) the dimeric amine oxidase from Pisum sativum forms soluble bi-molecular aggregates. Enzyme aggregates were analyzed by Dynamic Light Scattering and by full chemical loading of a mesoporous support (10% agarose gels activated with glyoxyl groups). The soluble aggregate was immobilized by multipoint attachment on glyoxyl- agarose at pH 8.5 though the four amino termini of the two dimeric molecules (Lys residues are not reactive at this pH). The immobilized aggregated structure cannot undergo any movement (translational or rotational) after multipoint attachment and the aggregate is “fixed” on the support surface even after the removal of PEG. The immobilized aggregate was further incubated at pH 10 in order to allow the Lys residues to react with the glyoxyl groups on the support. Enzyme aggregation has an important effect on enzyme stabilization: the aggregated derivative was 40 fold more stable than a similar derivative of the isolated enzyme and 200 fold more than native enzymes in experiments of thermal inactivation.

在本文中，提出了一种固定和稳定酶的新方法：双分子酶聚集体的多点共价连接。该固定方案允许将酶聚集体“捕获”并固定在支持表面上。除了通过多点连接实现稳定之外，酶聚集还促进了非常有趣的稳定作用。在低浓度聚乙二醇 (30%) 存在下，豌豆中的二聚胺氧化酶形成可溶性双分子聚集体。酶聚集体通过动态光散射和介孔载体（用乙醛基活化的 10% 琼脂糖凝胶）的完全化学加载进行分析。尽管两个二聚体分子的四个氨基末端（赖氨酸残基在该 pH 值下没有反应性），但可溶性聚集体通过多点连接固定在 pH 8.5 的乙醛琼脂糖上。固定的聚集结构在多点附着后不能进行任何运动（平移或旋转），即使在去除 PEG 后，聚集体也“固定”在支撑表面上。固定的聚集体在 pH 10 下进一步温育，以允许 Lys 残基与支持物上的乙醛基反应。酶聚集对酶的稳定性有重要影响：