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

Abstract

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)2SO4] fractionation, ii) acetone precipitation and iii)
Sephadex G-200 gel permeation chromatography. Fractions precipitated with 30 to 60% [(NH4)2SO4]
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 ~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°C. A thermal stability
at temperatures ≥ 40°C for 15 min, using casein as a substrate, with a total loss of enzymatic activity
upon heating at 70°C. Results for kinetic parameters indicated that the apparent Km and Vmax values for
AP, with casein as substrate under optimal reaction conditions (pH 10 and 55°C), were found to be 230
µg 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 microbia l alkaline proteases.