Simultaneously Enhancing the Catalytic Activity and Thermostability of <i>Pseudomonas aeruginosa</i> Aminopeptidase via Structure-based Design

Aminopeptidases are crucial hydrolases in the food and pharmaceutical industries. This study addresses the need to enhance the catalytic performance of <i>Pseudomonas aeruginosa</i> aminopeptidase (PaAps) through a multifaceted computational design strategy. We introduced single-site mutations followed by combinatorial mutations to develop a mutant library, identifying the optimal mutant S112D, which demonstrated a 5.19-fold increase in catalytic activity and nearly doubled the thermostability compared to the wild type. The kinetic parameters (<i>k</i>_cat, <i>k</i>_cat/<i>K</i>_m, and <i>V</i>_max) of S112D were found to be 4.36, 6.52, and 4.36 times greater than those of the wild type, respectively. Molecular dynamics (MD) simulations revealed that the S112D mutant induced global conformational changes, resulting in a more open active pocket that facilitated better binding with the substrate, thereby improving conformational stability. Additionally, the S112D mutant exhibited a closer nucleophilic attack distance and stronger hydrogen bonding interactions, further boosting catalytic efficiency. Remarkably, mutant S112D, as well as the wild type, showed hydrolytic activity on both corn and soybean proteins. The hydrolysis rate of corn protein by S112D was approximately 1.92 times that of PaAps, and for soybean protein, it is roughly 1.84 times. These findings offered valuable insights for developing more efficient enzyme modification strategies.