Production and characterization of extracellular protease from horse fecal–originated Bacillus amyloliquefaciens SLBD

Abstract

Proteases are valuable industrial enzymes, yet most commercial preparations in Indonesia remain imported, underscoring the need to identify local, efficient producers. This study reports, for the first time, the production and characterization of an extracellular protease from Bacillus amyloliquefaciens SLBD, isolated from horse feces. The crude extracellular enzyme was collected at different growth phases and evaluated for proteolytic index, specific activity, and catalytic properties using N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide as substrate. The crude enzyme exhibited a specific activity of 8.82 U mg-¹, Km of 0.519 mM, Vmax of 192.31 μmol min-¹, and kcat/Km of 1.23×105 M-¹ s-¹. Optimal activity was observed at pH 7–8 and 50 °C, with more than 80% stability retained between pH 6–10 and tem-peratures up to 60 °C. Activity was moderately affected by organic solvents but remained above 70% in methanol and ethanol, and was largely unaffected by nonionic detergents. Metal ions such as Zn²+ slightly enhanced activity, whereas Hg²+ and EDTA caused strong inhibition, implying partial metal dependence. Collectively, the enzyme demonstrates broad operational stability and tolerance to diverse physicochemical conditions. The findings highlight B. amyloliquefaciens SLBD as a promising feces-derived strain producing a robust, solvent- and detergent-tolerant extracellular protease suitable for bio-technological and industrial applications.