PLS‐Guided Mutant Recombination to Improve the Stability of Bovine Enterokinases Obtained by Directed Evolution

Abstract Activity and thermostability are critical yet challenging to improve simultaneously in enzymes. Using directed evolution, we previously identified bovine enterokinase (EK L ) variants with enhanced soluble expression and thermal stability. Partial least‐squares (PLS) analysis of 321 EK L variants revealed the impact of individual mutations and identified neutral or detrimental mutations in top‐performing variants. Leveraging PLS rankings, we created new variants with fewer mutations and enhanced stability. Most original and PLS‐guided variants exhibited an activity‐stability trade‐off. However, two new triple‐ and quadruple‐mutants improved both activity and stability, surpassing the trade‐off limit. Recombining PLS‐guided mutations likely eliminated neutral or harmful mutations, enhancing stability. MD simulations linked residue‐specific dynamics with stability, pinpointing critical structural regions near aggregation‐prone areas. Our findings validate PLS as a potent strategy to enhance enzyme properties, complementing directed evolution.