Figure 1:Strong anion exchange chromatography (Q-Sepharose Fast Flow) of B. aegyptiaca extract treated with 10% (w/v) charcoal. The Q-Sepharose column was pre-equilibrated with 50 mM Tris-HCl buffer (pH 8.1), containing 1 mM EDTA and 3 mM β-mercaptoethanol. Experiment was carried out with this buffer. Elution was performed using the same buffer containing 1 M NaCl at a flow rate of 0.5 mL/min under landing gradient of 0, 0.15, 0.4 and 1 M. (P corresponding to peak)

Figure 2: SDS-PAGE analysis of purified proteases extract from B. aegyptiaca. Samples were stained with Coomassie blue. The lane 1 corresponds to a molecular mass standards and the lane 2 to a purified protease using strong anion-exchange Q-Sepharose column (Peak 2)

Figure 3: Activity gel of purified proteases (peak 2) from B. aegyptiaca after the elution on Q-Sepharose column. The gel was Gelatin stained with Coomassie Blue

Figure 4a: SDS-PAGE electrophoregram illustrating the degradation patterns of bovine κ-casein by purified proteases extract (peak 2) from B. aegyptiaca after incubation for 1, 3 and 6h correspond to lanes 2, 3 and 4 respectively. Lane 1 represents control (κ-casein without enzyme). Commercial bovine κ-casein (2 mg) was dissolved in 1 mL of 100 mM sodium phosphate buffer (pH 6.2)

Figure 4b: SDS-PAGE electrophoregram illustrating the degradation patterns of bovine α- and β-caseins by purified proteases extract (peak 2) from B. aegyptiaca after incubation for 1, 3 and 6h correspond to lanes 2, 3 and 4 respectively. Lane 1 represents control (α, β-casein without enzyme). Commercial bovine α, β-caseins (2 mg) were dissolved in 1 mL of 100 mM sodium phosphate buffer (pH 6.2)

Figure 5: Activities of purified proteases extract from B. aegyptiaca on the synthetic substrate. N-Suc-Ala2-Pro-Phe-pNA was used as substrate and dissolved in dimethyl sulphoxide. The substrate was pre-incubated in different buffers at 25°C for 5 min: 200 mM sodium acetate (pH 5), 50 mM Tris-HCl (pH 7 and 8)

Figure 6: pH profile of purified proteases extract (peak 2) from B. aegyptiaca. Proteolytic activity was measured using azocasein as substrate. Blanks were done for each pH. The points are the means of triplicate determinations. Bars represent standard deviation

Figure 7: Temperature profile of purified proteases extract (peak 2) from B. aegyptiaca. Proteolytic activity was measured using azocasein as substrate. Blanks were done for each temperature. The points are the means of triplicate determinations. Bars represent standard deviation

Figure 8: Thermal stability of purified proteases extract (peak 2) from B. aegyptiaca, during 10 h. Residual activity was measured using azocasein as substrate. Blanks were done for each temperature. The points are the means of triplicate determinations. Bars represent standard deviation

Figure 9: MALDI-TOF mass spectra of purified proteases extract from B. aegyptiaca. 28 kDa (a), 72 kDa (b) and 85 kDa (c) spectra were recorded in positive ion reflective mode using α-cyano-4-hydroxy-trans-cinnamic acid as matrix. The test gel pieces were discolored with acetonitrile-ammonium bicarbonate (1:1). The control and test gel pieces were further in gel digested with trypsin before analysis