Isolation, Production, Partial Purification, and Biochemical Characterization of a Thermophilic Alkaline Protease from Tuwa Hot Spring with Industrial Applications

Thermophilic bacteria were isolated from the Tuwa hot spring region of Gujarat, India, to evaluate extracellular thermostable alkaline protease production. Total twenty-five isolates were screened on skim milk agar, and five potent strains were selected for submerged fermentation. The 16S rRNA gene sequence of Bacillus licheniformis MT_1 has been deposited in the NCBI GenBank database under accession number PZ221934.1. Among them, Bacillus licheniformis MT_1 showed the highest enzyme yield and was selected for further study. Partial purification using ammonium sulfate precipitation (20–70% saturation) resulted in 1.08-fold purification, 90.82% yield, and specific activity of 65.00 U/mg. SDS-PAGE analysis revealed a predominant protein band corresponding to the expected molecular weight of the partially purified protease. The enzyme exhibited maximum activity at pH 9.0 and 80°C, confirming its alkaline and thermophilic nature. It retained over 92% activity after prolonged exposure to elevated temperatures and remained stable across alkaline pH conditions. Substrate specificity studies showed highest activity with bovine serum albumin, followed by gelatin and casein. Kinetic analysis using the Lineweaver–Burk plot gave a Km of 0.25 g% and V _max of 5000 U/mL. Enzyme activity was enhanced by Mg²⁺ and Mn²⁺ ions and retained partial activity in the presence of organic solvents, surfactants, NaCl, and urea, with tolerance varying by agent and concentration. This protease shows strong potential for detergent and industrial applications.