Metal-Dependent Effects and Crowding Robustness of Pseudomonas fluorescens Esterase I

Industrial biocatalysts are strongly influenced by their physicochemical environment, yet systematic studies on how metals and macromolecular crowding influence metal-independent esterases remain scarce. Here, Pseudomonas fluorescens esterase I (PFE), an α/β-hydrolase, is employed to evaluate how divalent cations and molecular crowding modulate catalysis and structural integrity. PFE retained >85% activity for most metals at 2 mM, demonstrating notable robustness. By contrast, Cu²⁺ and Zn²⁺ induced pronounced inhibition, with loss of activity at higher metal concentrations. Combined activity, EDTA recovery, dynamic light scattering, and native-PAGE experiments reveal Zn²⁺ and Cu²⁺ perturb PFE's structure, promoting structural heterogeneity and the formation of aggregates, whereas for Cu²⁺ computational predictions further support interactions with active site residues. Mg²⁺, Ca²⁺, Ba²⁺, Mn²⁺, Co²⁺, and Ni²⁺ exert weaker and variable effects. Unlike chemical perturbation, macro- and micromolecular crowding was idle to PFE's activity, despite increases in viscocity, underscoring resilience to physical crowding. Overall, PFE responses to metals are governed by metal-specific coordination chemistry and distinct deactivation mechanisms, providing a framework for understanding esterase performance in metal-rich and crowded environments and highlighting physicochemical tuning as a complementary strategy to protein engineering in biocatalysis.