Structure-Guided Design Unlocks the Coupled Improvement of Thermostability and Activity in Lipoxygenase

Lipoxygenase (LOX) is widely used in the food and pharmaceutical industries, yet its industrial application has been limited by low catalytic activity and poor thermostability. Here, we employed a B-factor-guided rational design strategy to engineer Nostoc sphaeroides LOX (NsLOX). Through site-saturation and combinatorial mutagenesis, several thermostable mutants were obtained. Among them, the double mutant T2C/S36P exhibited a half-life at 50 °C of 264.36 ± 21.66 min, which is 30.78 times longer than that of the wild-type enzyme. Its melting temperature also increased by 5.53 °C, from 59.81 ± 0.17 °C to 65.34 ± 1.74 °C. Notably, the specific activity of T2C/S36P reached 197832.16 ± 3860.89 U/mg, representing a 1.53-fold enhancement over the wild-type. Molecular dynamics and structural analyses revealed that the enhanced thermostability originated from increased global conformational rigidity and optimized surface properties. Building on this stabilized scaffold, redesign of the substrate-binding pocket toward an electrostatic–hydrophobic balance─by preserving key electrostatic interactions while remodeling hydrophobic topology─synergistically boosted catalytic activity. To our knowledge, this is among the first reports on simultaneously improving both thermostability and activity of LOX via B-factor-guided design, providing an efficient strategy for engineering industrially relevant enzymes.