Overcoming the Stability-Activity Trade-Off in Lipoxygenase by Integrated Computational-Assisted Structure-Guided Design.

The industrial potential of lipoxygenase (LOX) in food processing and pharmaceutical research is often hampered by the persistent trade-off between the enzymatic activity and thermostability. Herein, we applied the integrated computational-assisted structure-guided design of a mutant (CP-G194 V/L235T) in Nostoc sphaeroides LOX (NsLOX) that effectively decouples this constraint. The engineered variant demonstrated a remarkable 45.29-fold extension in half-life at 50 °C (389.08 ± 36.62 min) with a Tm of 67.85 ± 0.40 °C and a 2.89-fold increase in specific activity toward linoleic acid (215881.25 ± 5024.47 U/mg) compared to the wild type. Structural and molecular dynamic analyses revealed that the mutations reinforce structural rigidity through strengthened hydrophobic interactions and optimized surface electrostatics while simultaneously inducing subtle rearrangements in α-helices that enlarge the substrate-binding cavity and improve substrate channel accessibility. This study demonstrates the effective application of established semirational design tools to overcome a key limitation in LOX engineering and, to our knowledge, represents the first reported success in simultaneously enhancing both thermostability and specific activity in a LOX enzyme.