Partitioning purification, biochemical characterization, and milk coagulation efficiency of protease from a newly Streptomyces sp. isolate.

An actinobacteria strain was isolated from an olive waste mill and tested for protease production on skimmed milk media. The strain identification was achieved through both 16 S rDNA sequencing and phenotypic characterization. The enzyme was purified using the ammonium sulfate/t-butanol three-phase partitioning (TPP) method, followed by characterization to investigate the effect of pH, temperature, and various chemical agents. Subsequently, the enzyme was assessed for its milk coagulation activity. The strain belonging to the Streptomyces genera, exhibits significant phylogenetic and phenotypic differences from the aligned species, suggesting its novelty as a new strain. The enzyme was best separated in the TPP aqueous phase with a 5.35 fold and 56.25% yield. Optimal activity was observed at pH 9.0 and 60 °C, with more than half of the activity retained within the pH range of 7-10 over one hour. The protease demonstrated complete stability between 30 and 60 °C. While metallic ions enhanced enzyme activity, EDTA acted as an inhibitor. The enzyme displayed resistance to H2O2, SDS, Tween 80, and Triton X-100. Notably, it was activated in organic solvents (ethyl acetate, petroleum ether, and xylene), maintaining > 75% of its original activity in butanol, ethanol, and methanol. Additionally, the enzyme yielded high milk coagulant activity of 11,478 SU/mL. The new Streptomyces sp. protease revealed high activity and stability under a wide range of biochemical conditions. Its use in the dairy industry appears particularly promising. Further industrial process investigations will be valuable in determining potential uses for this enzyme.