Improved production of extremophilic protease using low-cost substrate by Bacillus subtilis ZB isolated from extreme environment for tannery effluent treatment.

A halotolerant alkaline protease-producing Bacillus subtilis ZB was isolated from the extreme environment. It was characterized by morphological, biochemical, and 16S rDNA gene sequencing. Halotolerant protease production by the strain ZB was performed using low-cost substrate to reduce the production cost of the enzyme. Enzyme production was optimized using cow dung substrate in solid-state fermentation. In this study, the important process parameters were determined by the traditional one-variable-at-a-time approach, followed by response surface methodology. Three key factors including pH, moisture, and calcium chloride significantly influenced halotolerant protease activity (p < 0.05) in a two-level full factorial experimental design. Then, the central composite design was subjected to analyze the optimum concentration. The regression coefficient value, R² value, was 0.9227 and the corresponding F-value was 26.21. The pinnacle of protease production was reached under the following optimized conditions: pH 8.85, 118.4% moisture, and 0.913% CaCl₂. As compared with the unoptimized medium, the optimized medium showed an overall 2.5-fold increase in the yield of halotolerant protease. The protease enzyme was purified and the molecular weight was 49 kDa. The pH, thermo, and salt tolerance of protease enzyme were stable between pH 6–9, 30–40 °C, and 1–5% NaCl, respectively. Of the tested divalent ions, Mg²⁺, Na⁺, Mn²⁺, and Ca²⁺ showed protease activity (%) of 122.5, 115.4, 107.6, and 103.5, respectively. The ability of the organism to reduce the chemical oxygen demand (COD) of tannery wastewater was also studied. The crude enzyme reduced > 70% of COD content after 24 h of enzyme treatment at 4% salinity. The present study suggested the application of salt-tolerant bacterium for the potential degradation of tannery effluent. [ABSTRACT FROM AUTHOR]