Cross-linked enzyme aggregate (CLEA) methodology has been applied to immobilize cytochrome P450 BM3 variants (F87A and 21B3) with peroxygenase activity. Several Ru(II)-diimine complexes were found to be suitable cross-linking agents, surpassing the traditional glutaraldehyde and dextran aldehyde. They offer modular numbers of aldehyde functionalities and a more rigid framework than their organic counterparts. The F87A CLEAs display significant activity loss compared to the protein in solution. Meanwhile, for the 21B3 CLEAs, high activity recovery (up to 95%) is obtained. In order to minimize enzyme leaching from the CLEA, sodium cyanoborohydride was used to reduce the CLEAs imine bonds. The reduced CLEAs were active for several rounds of reactions leading to an overall increase in protein activity of 170% compared to the free protein in solution.

交联酶聚集体（CLEA）方法已用于固定具有过加氧酶活性的细胞色素P450 BM3变体（F87A和21B3）。发现几种Ru（II）-二亚胺配合物是合适的交联剂，超过了传统的戊二醛和葡聚糖醛。与有机同类产品相比，它们提供了醛官能团的模块化数量和更严格的框架。与溶液中的蛋白质相比，F87A CLEAs表现出明显的活性损失。同时，对于21B3 CLEA，获得了高活性回收率（高达95％）。为了使酶从CLEA的浸出最小化，使用氰基硼氢化钠还原CLEA的亚胺键。与溶液中的游离蛋白质相比，还原的CLEA在数轮反应中均具有活性，从而使蛋白质活性总体提高了170％。