Purification and Kinetic Parameters Characterization of an Alkaline Protease Produced from Bacillus subtilis through Submerged Fermentation Technique

This paper reported the purification and kinetic parameters characterization of an alkaline protease produced from Bacillus subtilis through submerged fermentation process using rice husk as growth supporting substrate (by-product of rice industry) collected from Pearl Rice Mill Hafiz Abad, Faisalabad. Bacillus subtilis was cultured in fermentation medium under some pre-optimized growth conditions. Maximum alkaline protease activity of 216±4.32U/mL was obtained when fermentation medium of rice husk was inoculated with 4% (4mL/100mL) inoculum size, 7% substrate concentration at pH 11 for 48 h fermentation time period with 2% molasses as additional supplement material for fermentation medium. An alkaline protease was purified 1.49-fold with specific activity of 74.66 U/mg in comparison to crude enzyme extract using ammonium sulfate precipitation, dialysis and Sephadex-G-100 column chromatography. The enzyme was shown to have a relative low molecular weight of 27kDa by sodium dodecyl sulphate poly-acrylamide gel electrophoresis (SDS-PAGE). The enzyme displayed 10 and 45°C as an optimum pH and temperature respectively. Using casein as substrate, the enzyme showed maximum activity (V ) of 148U/mL with its corresponding K value of 58μM. Among max M activators/inhibitors EDTA and Ca gave enhancing effect on purified alkaline protease where as SDS, Tween2+ 81, Na and Hg caused enzyme inhibition and inactivation to variable extents. The specific activity and + 2+ substrate affinity of this alkaline protease from Bacillus subtilis is greater than those of other reported Bacillus sp; therefore, it was concluded that it may be potentially useful for industrial purposes.