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

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.