Short-loop engineering strategy for enhancing enzyme thermal stability.

Highly flexible regions were targeted for successful modification to enhance enzyme stability. However, this approach could not cover all key sites. Residues in certain rigid regions are also crucial for protein stability. This study proposed a short-loop engineering strategy that explores rigid "sensitive residues" in short-loop regions and mutated them to hydrophobic residues with large side chains to fill the cavities, thereby improving enzyme thermal stability. This strategy identified sensitive residues in the short-loop regions of three enzymes: lactate dehydrogenase from Pediococcus pentosaceus, urate oxidase from Aspergillus flavus, and D-lactate dehydrogenase from Klebsiella pneumoniae. Under the guidance of the short-loop engineering strategy, the half-life periods of these three enzymes were 9.5, 3.11, and 1.43 times higher than wild type, respectively. We also proposed a standard procedure for this strategy and developed a visualization plugin, offering new insights into enzyme stability modification.