Subtilisin is a non-specific protease enzyme (Protein digesting enzyme) initially isolated and characterized in an around Kalasalingam University campus from volatile substances. Total 14 isolate was characterized KLU119, KLU-121, KLU- 122, KLU-137, KLU-138, KLU-153, KLU-161, KLU-166, KLU-178, KLU-191, KLU- 211, KLU-221, KLU-243, and KLU-300 based on the zone formation in the solid media for extracellular subtilisin enzyme express. The PCR analysis of all the 14 isolates carried out of them KLU119, KLU161, KLU166 and KLU187 was amplified in specific primer extracellular subtilisin enzyme producing gene. In the present study, KLU119 was further developed a recombinant enzyme, an alkaline serine protease from halo alkaliphilic bacteria, which has been cloned and expressed in DH5α (E. coli) as host. The enzymes BamHI/EcoRI were used for restriction and digestion of the gene 1170 bp. The gene was further subjected to 39 kDa protein expression and analysis. pET24a was used as a potential cloning expression vector. The properties of the enzyme were then compared with the native one. Subtilisin being a protease was studied for its enzyme activity against a tyrosine standard. The maximum concentration of 1.059 Units of enzyme/ml and 18.250 μg protein/ ml of broth was found in KLU119 compare to other isolates. The feasibility of exploiting this recombinant enzyme to various novel applications other than those that already exist was also deduced.