Propeptide engineering of Aspergillus niger-derived aspartic protease PepA for enhanced catalytic activity

The propeptide of aspartic proteases plays a critical role in correct folding and functional maturation, yet its precise regulatory mechanism remains to be fully elucidated. This study systematically investigated the structure-function relationship of the propeptide in Aspergillus niger-derived aspartic protease PepA via propeptide engineering. We found that an intact propeptide structure is indispensable for PepA to attain catalytic activity. Rational design of the autocleavage site (P1) revealed that substituting alanine with tryptophan enhanced enzyme activity by 39.97 %, likely through strengthened hydrophobic interactions. Furthermore, sequence alignment and site-directed mutagenesis identified four key regulatory residues (L26, S33, L34, F37) on the propeptide α-helix. Notably, the F37Y mutation dramatically increased activity by 92.53 %, potentially through the formation of a novel hydrogen-bonding network. Ultimately, combinatorial optimization of these beneficial sites yielded the optimal mutant (L26G/L34Y/F37Y), which exhibited a 2.83-fold increase in catalytic activity compared to the wild-type. Homology modeling and structural analysis suggested that these mutations synergistically modulate the propeptide conformation and its interface with the mature enzyme, thereby facilitating a more efficient folding and activation pathway. Our work not only significantly enhances the catalytic performance of PepA but also provides novel insights into the molecular mechanism and engineering of aspartic protease propeptides.