Purification and biochemical characterization of alkaline protease from an Egyptian biopesticide-producing Bacillus sphaericus strain

The objective of the current study was to purify and partially characterize an alkaline protease (AP) from a newly isolated Egyptian Bacillus sphaericus strain. The enzyme was subjected to a 3-step purification scheme involving i) ammonium sulfate [(NH4)(2)SO4] fractionation, ii) acetone precipitation and iii) Sephadex G-200 gel permeation chromatography. Fractions precipitated with 30 to 60% [(NH4)(2)SO4] saturation levels exhibited the highest enzyme activities, whereas acetone in the ranges between 50 to 75% (v/v). Gel permeation utilizing Sephadex G-200 column resulted in approx. 50 fold purification level, as compared to the original crude enzyme, with a yield recovery of 27%. The AP enzyme was successfully purified to homogeneity as a monomeric band with an estimated molecular mass of similar to 29 kDa, based on sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and zymogram activity staining analyses. While, the maximum enzymatic activity was recorded at pH 10, AP showed an optimal activity at incubation temperature range AP between 55 to 60 degrees C. A thermal stability at temperatures >= 40 degrees C for 15 min, using casein as a substrate, with a total loss of enzymatic activity upon heating at 70 degrees C. Results for kinetic parameters indicated that the apparent K-m and V-max values for AP, with casein as substrate under optimal reaction conditions (pH 10 and 55 degrees C), were found to be 230 mu g min(-1) ml(-1) and 0.05% (w/v), respectively. Thus, the potentials of AP as an industrially important enzyme were assessed in the light of our current knowledge on microbial alkaline proteases.