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, Cu 2+ and Zn 2+ induced pronounced inhibition, with loss of activity at higher metal concentrations. Combined activity, EDTA recovery, dynamic light scattering, and native–PAGE experiments reveal Zn 2+ and Cu 2+ perturb PFE's structure, promoting structural heterogeneity and the formation of aggregates, whereas for Cu 2+ computational predictions further support interactions with active site residues. Mg 2+ , Ca 2+ , Ba 2+ , Mn 2+ , Co 2+ , and Ni 2+ 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.