Purification, characterization and application of thermoalkaliphilic proteases from Bacillus filamentosus, Lysinibacillus cresolivorans, and Bacillus subtilis

Abstract Thermophilic proteases are important industrial enzymes because they can be used at unavoidable temperature in various bioprocessing schemes. The bacterial population of Cholistan desert was explored for thermophillic proteases and their industrial applications. Three bacterial isolates K1, K5, and K7 were found promising protease producers. These isolates were preliminary identified as Bacillus based on morphological characteristics and biochemical tests (positive for catalase, oxidase, and citrate tests, and negative for indole and urease tests). The isolates K1, K5, and K7 were further identified as Bacillus filamentosus, Lysinibacillus cresolivorans, and Bacillus subtilis, respectively by phylogenetic analysis. The isolates grew best at 50oC while Bacillus filamentosus (K1), Lysinibacillus cresolivorans (K5), and Bacillus subtilis (K7) produced larger zones of hydrolysis at 37oC, 45oC and 50oC at pH 7, respectively. The optimum temperature for protease activity was 65oC for Bacillus filamentosus and Lysinibacillus cresolivorans and 55oC for Bacillus subtilis, and the optimum pH for activity was 9 for all the three strains. The protease produced by these isolates were found active at high temperature (37oC to 85oC) and high pH (5–12) which make them industrially important thermoalkaliphilic proteases. Theses proteases successfully de-haired cow’s skin and de-stained blood from cotton cloth pieces, which are rarely tested applications of these proteases.