Biochemical and structural characterization of a cold-active Psychrobacter nivimaris esterase and its catalytically...

Cold-active enzymes from Antarctic microorganisms are highly valuable for low-temperature industrial processes, yet the enzymatic capabilities of the bacterium Psychrobacter nivimaris remain largely unexplored. This study identifies a novel cold-active esterase, EstPN8, from P. nivimaris and engineers it for improved performance. EstPN8, with a molecular weight of 25.8 kDa in its recombinant form, exhibits a low activation energy (E_a) of 4.70 kcal/mol and retains at least 40% of its maximum activity at 10 °C. Its optimal activity occurs at 50 °C and pH 9.0, with a strong preference for p-nitrophenyl caproate (C6) and halotolerance up to 1.0 M NaCl. To enhance efficiency, a quadruple variant, F1 (D35A/P174D/M142L/Q205F), was developed, resulting in a 3.9- and 4.8-fold increase in catalytic efficiency at 10 °C and 40 °C, respectively. Notably, F1 retained cold-active characteristics of EstPN8 while exhibiting enhanced thermostability at 50 °C. Molecular dynamics simulations revealed the intrinsic catalytic plasticity of the EstPN8 scaffold, while substrate analogue-induced dimerization suggested a potential mechanism by which transient intermolecular association provides structural confinement for substrate retention. These findings provide structural insights into the molecular basis of cold-activity in EstPN8 and support its potential as a high-performance biocatalyst for sustainable low-temperature bioprocesses.