Rationally engineered self-assembling enzyme immobilization keratin platform towards multienzymatic cascade reactions

Immobilization of enzymes has the potential to improve enzyme recyclability, stability and activity. However, development of efficient enzyme immobilization, specifically for the multienzyme co-immobilization, remains a challenge. Here, we report a rational design and construction of keratin-based enzyme immobilization platform based on the principle of heterotypic keratin self-assembly. The keratin tags driven from type I keratin of K31were firstly screened through the self-assembly interaction between recombinant K86 (RK86) with different lengths of RK31, that were subsequently used to fuse with various enzymes to connect RK86 microparticles for immobilization. Furthermore, depending on the different lengths of RK31 tags, the spatial position of multienzymes can be accurately regulated, and then modulating the kinetic parameters of the cascade reactions. Our research presents a robust and efficient keratin-based platform for enzyme immobilization. By precisely adjusting the length of the self-assembly keratin tags, we ensured high enzymatic activity and seamless integration of multiple enzymes. The immobilized glucose oxidase (GOX) and horseradish peroxidase (HRP) multienzyme system showed a 33 % increase in V_max and a 22 % reduction in the Michaelis constant K_m compared to free enzymes. This study presents a novel and efficient keratin-based immobilization system that offers a precise method for regulating multienzyme spatial positioning, significantly enhancing the efficiency and reliability of enzyme cascade reactions.