The study involves the isolation and characterization of a serine peptidase, named SP, from Pseudomonas aeruginosa. In addition to basic characterization, the protein was engineered, by site-directed mutagenesis of selected non-catalytic residues, to increase its thermal stability and catalytic activity. Among the eight-point mutations, predicted by FireProt, two mutants, A29G and V336I, yielded a positive impact. The Tm of A29G and V336I showed an increase by 5 °C and also a substantial increase in residual activity of the enzyme at elevated temperature. Moreover, the catalytic activity of A29G and V336I also showed an increase of 1.4-fold activity, compared to the wild-type (WT). Moreover, molecular docking simulations also predicted better substrate affinity of themutants. Wehave also performed molecular dynamics (MD) simulations at 315 and 345 K, and theMDdata at 345 K demonstrates improved thermostability for the mutants, compared to theWT. Our findings not only contribute to a better understanding of the structure-stability-activity relationship of SP but also highlights, that modification of non-catalytic residues could also promote favourable catalytic behaviour.