Purification of alkaline serine protease from local Bacillus subtilis M33 by two steps: a novel organic solvent and detergent tolerant enzyme

Abstract

Alkaline proteases are important from an industrial perspective due to their wide scale applications and obtained from different sources. In this study, an alkaline protease from a newly isolated Bacillus subtilis M33 was purified by ammonium sulfate precipitation and DEAE cellulose anion exchange chromatography with %38.66 yield and 15.50 fold. The molecular mass of purified enzyme was determined approximately 39 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) and gel filtration chromatography. The enzyme exhibited pH and temperature optima of 10.0 and 55°C respectively, and was stable between a wide pH range of 8.0 and 11.0 for 7 days. Some spesific protease inhibitors such as phenylmethyl sulfonyl fluoride (PMSF) completely inhibited the enzyme activity. However, the protease activity was increased in the presence of 2-mercaptoethanol and dithiothreitol, suggesting it to be a thiol-dependent serine protease.  The enzyme was also stable towards laboratory bleaches (H²O²), surfactants (Tween 80, Triton X-100, SDS) and organic solvents such as benzene, toluene, acetone. Different commercially avaliable detergents were used to study the compatibility of the purified alkaline protease. The kinetic parameters Km and Vmax of the protease were determined by measuring the protease activity casein as a substrate 0.706 mg/ml, 3000 μM.min^-1 respectively.

Keywords

• Bacillus subtilis,Alkaline protease,Bacillus subtilis,Enzyme purification,DEAE

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