Co-Engineering Vapor-Deposited Polycationic Microenvironment and Immobilization Strategy to Enhance β-Galactosidase Kinetics at Acidic pH

The industrial application of enzymes, such as β-galactosidase (LacZ) for processing acidic whey, is often constrained by their poor activity and stability at suboptimal acidic pH. Here we present novel material-based strategies to overcome this limitation by immobilizing LacZ on vapor-deposited polycationic polymer supports that create an electrostatically tuned microenvironment. We systematically investigated the effects of enzyme immobilization strategy (random vs. site-directed via SpyCatcher/SpyTag) and polycationic polymer supports (initiated chemical vapor deposition (iCVD) vs. self-assembled monolayers (SAM)). Using LacZ as enzyme model, we showed that site-directed immobilization consistently outperformed random methods, mainly by enhancing the enzyme's intrinsic turnover rate ( ). But this alone was insufficient to prevent enzyme deactivation under low pHs (e.g., pH ≤ 4). Polycationic support via iCVD is the key to effectively preserving enzyme activity at low pH by providing local electrostatic shielding, reducing hydronium ion concentration near the enzyme active sites. As a result, LacZ activity increased by 40%-842% on different polycationic supports. Additionally, we showed that the film's physical supporting structure proved as important as its chemical feature. Even with identical surface chemistry, thick iCVD films (200nm) demonstrated significantly enhanced enzyme activity compared to thin SAM films (7nm) at acidic pH, due to higher areal density of protonatable groups. Furthermore, crosslinking the polycationic support improved catalytic efficiency by decreasing the substrate affinity constant ( ). These findings highlight the interplay between polymer chemistry, immobilization method, and support structure, demonstrating the synergistic integration of enzyme orientation control and microenvironment engineering as a powerful strategy for enhancing enzyme performance under challenging reaction conditions.