Production of Alkaline protease from Bacillus amyloliquefaciens TAS-2 and Optimizing fermentation conditions

Abstract

The aim of the present investigation was to standardize the conditions of batch fermentation for the production of a commercially significant alkaline protease from Bacillus amyloliquefaciens TAS-2. The B. amyloliquefaciens TAS-2 exhibited a distinct zone of clearance on the skim milk agar medium and demonstrated an enzyme activity of 38.6 U/mL after 24 hours of incubation at a pH of 7 and a temperature of 37°C. B. amyloliquefaciens TAS-2 was identified through genotypic analysis using 16S rRNA sequencing. The highest protease activity was obtained after 24 hours of incubation time, at pH 9, and temperature of 40°C. Similarly, maximum activity was observed with an agitation speed of 150 rpm, an inoculum age of 24 hours, an inoculum volume of 3% v/v, a substrate concentration of 3%, and a flask capacity of 250 mL. The activity was positively enhanced with addition of various nitrogen and carbon sources. Similarly, the presence of amino acids and metal ions induced protease production. However, the addition of Fe2 + and Zn2 + ions at specific concentrations in the medium was found to be inhibitory. Conversely, the addition of Mg2 + and Ca2 + ions had a stimulating effect on protease production. All the optimized parameters were incorporated into the basal medium, and fermentation was conducted under optimal conditions. The precipitation of the maximum amount of protein was achieved at 70–80% saturation of ammonium sulfate. The protease activity was 1.56 time higher for the partially purified protease compared to the crude supernatant. The partially purified protease exhibited optimum activity at a temperature of 55°C and a pH of 9. At 5 mM, PMSF significantly suppressed enzyme activity, whereas Triton X-100 and CTAB increased enzyme activity. Among the different metal ions tested, Ca2+ (5 mM), Mg2+ (5 mM), and Mn2+ (5 mM) stimulated enzyme activity, while Zn2 + and Fe2 + decreased protease activity. The enzyme demonstrated remarkable stability, retaining its activity even after being heated to 60°C for 60 minutes and remaining stable within a pH range of 8 to 11. The study suggests that the alkaline protease of B. amyloliquefaciens TAS-2 that is thermotolerant and surfactant stable can have potential applications across different industries due to its ability to improve yield and properties.