Cross-linked cytochrome P450 BM3 aggregates promoted by Ru(II)-diimine complexes bearing aldehyde groups

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.