Production, Optimization, Purification, Kinetic analysis and applications of alkaline proteases produced from Bacillus subtilis through solid state fermentation of agricultural byproducts

Abstract

Proteases have gained more commercial value to date due to multiple applications in different industrial sectors. Current research was aimed to use the cheaper agricultural waste for optimal protease production. Maximum level of protease production was achieved at 37 °C, incubation period of 24 h, pH 9.0, inoculum size 3%, 1.5 g sucrose as a carbon source and 30% moisture content by using solid-state fermentation. Among the various inorganic and organic nitrogen sources, ammonium nitrate and yeast extract tremendously increased the production of protease. Among metal ions and surfactants tested, Ca2+ and Tween 40 showed the optimal protease production. The purification of protease was carried out by ammonium salt precipitation followed by sephadex G-100 gel filtration chromatography. The purification resulted in 1.3 fold of purified protease with a specific activity of 51.5 and a total yield of 37.5 %. Molecular weight of purified protease was predicted upon SDS-PAGE with a single band of ~36 kDa. The protease was quite stable over a temperature range of 35-45 °C and pH 7-9 with maximal activity at 40 °C and pH 9. The kinetic parameters Vmax (maximum rate) and Km (Michaelis-Menten constant) were calculated as 0.307 U/g and 11.2 mg/mL, respectively. The alkaline protease significantly de-hair the goat skin and successfully removed the animal blood stain from cotton cloth.