Exploiting unique NP1 interface: Oriented immobilization via electrostatic and affinity interactions in a tailored PDA/PEI microenvironment enhanced by concanavalin A.

Abstract

Enzyme immobilization techniques are crucial for enhancing enzyme stability and catalytic efficiency. Traditional methods such as physical adsorption and simple covalent binding often fail to maintain enzyme activity and stability. In this study, an innovative multi-level immobilization strategy was proposed to achieve efficient targeted immobilization of nuclease P1 (NP1) by fine-tuning the surface microenvironment. Molecular simulation results revealed that the distinctive electrostatic distribution and the specific placement of basic amino acids, such as lysine, on the NP1 surface caused dopamine to preferentially adsorb on areas away from NP1's active site. This selective adsorption facilitated the directed immobilization of NP1, while the positively charged environment generated by the co-deposited surface further enhanced NP1's adsorption capacity. This multilevel modification was found to significantly optimize the physicochemical environment of the immobilized surface through surface characterization and enzymatic testing. This strategy greatly improves enzyme activity (3590.0 U/mg), stability, and reusability (70 % after 10 cycles). In particular, NP1 on this surface exhibited an optimal Michaelis constant (K_m) of 34.0 mM and a maximum reaction rate of 5.5 mM min^-1, demonstrating the remarkable effect of the modification strategy in enhancing the enzyme catalytic performance. The present study provides an efficient and stable immobilization platform for enzyme catalytic applications by precisely modulating the surface microenvironment and the oriented immobilization strategy, which has an important potential for practical applications. This stable and reusable NP1 platform allows for efficient DNA/RNA cleavage, facilitating its application in industrial biocatalysis, biomedical enzyme-based processes, and biosensors.