Engineering stable and reusable biocatalysts through electrostatic immobilization of (S)-norcoclaurine synthase on...

Immobilization of enzymes onto solid supports is a key strategy for enhancing their stability, reusability, and performance under harsh conditions, thereby enabling broader applications in sustainable catalysis. Here, we investigate the immobilization of (S)-Norcoclaurine Synthase ((S)-NCS), a highly promising yet scarcely studied biocatalyst capable of catalyzing the Pictet-Spengler reaction with high enantioselectivity for the synthesis of tetrahydroisoquinolines. We explored its immobilization onto low-cost mesoporous materials, Kaolin and Celite 545, characterized by distinct surface properties. Nitrogen sorption isotherms and BET analysis revealed Kaolin to possess a significantly higher surface area and mesopore volume than Celite 545. Notably, zeta potential measurements confirmed the more negative surface charge of Kaolin, which correlated with higher enzyme adsorption and changes in surface electrostatics after immobilization. Using zeta potential measurements and coarse-grained Monte Carlo simulations, we demonstrate that adsorption is predominantly driven by electrostatic interactions between the cationic disordered N-terminal of (S)-NCS and the negatively charged Kaolin surface. Immobilization on Kaolin yielded a robust biocatalyst with enhanced thermostability and DMSO tolerance, while preserving enantioselectivity and enabling multiple reuse cycles. Our findings highlight the pivotal roles of surface charge and porosity in protein-matrix interactions, providing mechanistic insights to guide the rational design of enzyme-matrix systems. This work establishes Kaolin as a versatile and accessible mesoporous support for biocatalyst development, contributing to a more predictive framework for enzyme immobilization.