Proteases are an important group of industrial enzymes and require to break long-chain polypeptides. Conventionally, proteases are classified into six different groups. Among them, serine proteases are most widely studied. Right now, these enzymes are used as the catalyst in many different industries, including leather, poultry, waste management, and, chemical. Despite a lot of merits, the majority of naturally occurring enzymes cannot be used at industrial scale due to their incompatibility with industrial conditions. Moreover, the high enzyme production cost is another bottleneck to large-scale application of enzyme in industry. Protein engineering is an effective way to address these problems. There are two protein engineering approaches available, directed evolution and rational design. Later is preferred due to accelerated process and small mutants’ library size. Furthermore, this is an efficient approach to add desirable characteristics through in-depth analysis of structure. There are many studies in which this approach has been successfully applied to improve catalytic efficiency and thermostability of enzymes. Thus, by applying this approach, we can produce cheaper and effective enzymes for large-scale industrial use. In this work, serine peptidase from locally isolated Pseudomonas aeruginosa strain was identified. Later on, to make enzyme thermostable site-directed mutagenesis of non-catalytic was carried out to increase temperature resistance and activity. The protease producing bacteria were isolated from tanneries waste and screened for their proteolytic activity using casein as the substrate. The bacterial strain, showing high caseinolytic activity was selected. The type of the protease, optimum pH and temperature was identified using the crude extract of bacterial culture. The PMSF assay revealed that the enzyme was serine protease. The bacterial strain was identified using 16S rRNA sequencing. It came to know that our bacterial strain belonged to Pseudomonas aeruginosa and named Pseudomonas aeruginosa BMB1. The 16S rRNA sequence was deposited to NCBI nucleotide database under accession number KY285994. Primers were designed using homologous sequences to amplify and sequence the serine protease (SP) gene from the bacterial strain. The gene sequence was submitted to NCBI nucleotide database under accession no MH045598. The computational characterization of the gene sequence deduced 50 kDa molecular weight serine protease with 7.01 PI along with 25 amino acid residues long N-terminal signal peptide sequence. Moreover, it was also deduced that there are three domains in the serine protease, including N-terminal trypsin domain, and two C-terminal PDZ domains (PDZ1 and PDZ2). The SP gene was cloned and expressed in three different expression vectors, including pET32(a) with 6X His tag, pET32 with TRX tag and pGEX 6p-1 with GST tag with and without N-terminal signal peptide sequence. As expected, we could not get the soluble expression of the SP with the signal peptide. The highest expression of the SP was observed in pET32(a) with 6X His tag. The Ni+2 affinity column was used to purify the SP after expression in BL21(DE3). The purified serine protease was characterized. The residual activity analysis of serine protease showed the small half-life; therefore, the rational protein engineering was used to elevate thermostability and half-life of SP. We used I-TASSER and FireProt to predict the 3D structure and stabilizing substitutions in the protein structure respectively. FireProt predicted eight stabilizing substitutions using consensus and energy-based approaches. Only two substitutions A29G and V336I were proved to be stable among eight. There was the substantial increase in the half-life of A29G and V336I as compared to their template. Moreover, both mutants exhibited 5 oC increase in Tm value compared to wild-type in thermal denaturation CD analysis. Furthermore, MD simulation also showed the same type of trends in thermal stability of both mutants. Interestingly, along with thermal stability, there was also an increase in catalytic efficiency of both mutants. The Km values of both mutants were smaller than wild-type, indicating the strong binding of the substrate with enzymes. The molecular docking analysis also showed a similar type of results. The increase in thermostability and catalytic efficiency of the mutants was possibly due to stabilization of the oligomeric state and strong intra-molecular interactions.