Production of an alkaline protease using Bacillus pumilus D3 without inactivation by SDS, its characterization and purification

Figures & data

Figure 1. Effect of pH on protease production (experimental conditions: incubation time 18 h, incubation temperature 35 °C, inoculum size 5%, glucose as carbon source, yeast extract as nitrogen source).

Figure 2. Effect of inoculum size on protease production (experimental conditions: incubation time 18 h, incubation temperature 35 °C, glucose as carbon source, yeast extract as nitrogen source, pH 11).

Figure 3. Effect of carbon sources on protease production (experimental conditions: incubation time 18 h, incubation temperature 35 °C, inoculum size 1%, yeast extract as nitrogen source, pH 11.0).

Figure 4. Effect of nitrogen sources on protease production (incubation time 18 h, incubation temperature 35 °C, inoculum size 1%, lactose as carbon source, pH 11.0).

Figure 5. Effect of temperature on protease production (experimental conditions: incubation time 18 h, inoculum size 1%, lactose as carbon source, yeast extract as nitrogen source, pH 11.0).

Figure 6. Effect of incubation time on protease production (incubation temperature 35 °C, inoculum size 1%, lactose as carbon source, yeast extract as nitrogen source, pH 11.0).

Table 1. Summary of the purification of an alkaline protease.

Purification step	Volume (ml)	Protein (mg/ml)	Total protein (mg/ml)	Activity (U/ml/min)	Total activity (U)	Specific activity (U/mg)	Yield(%)	Purification fold
Culture filtrate	750	0.22	165	78.30	58 725	355.9	100	1
(NH4)2SO4 precipitation (75%) and dialysis	17	1.2735	21.64	731.61	12 432	574.49	21	1.61
Anion-exchange column	8	076	6.08	1366.6	10 932	1798.15	18	5.05

Figure 7. Effects of temperature on protease activity (experimental conditions: reaction pH 10.5, incubation time 20 min, lactose as carbon source, yeast extract as nitrogen source, pH 10.5).

Figure 8. Effects of pH on protease activity (experimental conditions: reaction temperature 30 °C, incubation time 20 min, lactose carbon source, yeast extract as nitrogen source).

Table 2. Effects of heavy metals on protease activity (experimental conditions: reaction temperature 30 °C, reaction pH 10.5, incubation time 20 min).

Metal salts	Relative activity (%)
None	100
CuCl2	65.3
NiCl2	65.24
CuSO4	71.87
FeCl3	43.82
CdCl2	45.47
HgCl2	50.04

The activity in the absence of metal salt (none), referred to 100% relative activity.

Figure 9. Effect of SDS concentration on relative activity of protease (the activity in the absence of SDS, referred to 100% relative activity).

Figure 10. (A) Electrophoretic profiles of dialysate (lane 1) and purified enzyme (lane 2) by 12% SDS–PAGE under reducing conditions. Molecular weight markers are shown on the left. (B) Caseinolytic profiles of dialysate (lane 1) were performed by 12% SDS–PAGE–zymogram assay containing casein as a substrate. Zymograms were developed overnight at 37 °C in the presence of protease inhibitors as PMSF (lane 2) and EDTA (lane 3) at pH 10.5. Caseinolytic activity was observed as colorless bands.