Optimization and partial characterization of culture conditions for the production of alkaline protease from Bacillus licheniformis P003

Abstract

Proteolytic enzymes have occupied a pivotal position for their practical applications. The present study was carried out under shake flask conditions for the production of alkaline protease from Bacillus licheniformis P003 in basal medium containing glucose, peptone, K2HPO4, MgSO4 and Na2CO3 at pH 10. The effect of culture conditions and medium components for maximum production of alkaline protease was investigated using one factor constant at a time method along with its characterization. Maximum level of enzyme production was obtained after 48h of incubation with 2% inoculum size at 42°C, under continuous agitation at 150 rpm, in growth medium of pH 9. Highest enzyme production was obtained using 1% rice flour as carbon source and 0.8% beef extract as organic nitrogen source. Results indicated that single organic nitrogen source alone was more suitable than using in combinations and there was no significant positive effect of adding inorganic nitrogen sources in basal medium. After optimization of the parameters, enzyme production was increased about 20 fold than that of in basal medium. The crude enzyme was highly active at pH 10 and stable from pH 7-11. The enzyme showed highest activity (100%) at 50°C, and retained 78% relative activity at 70°C. Stability studies showed that the enzyme retained 75% of its initial activity after heating at 60°C for 1h. The enzyme retained about 66% and 46% of its initial activity after 28 days of storage at 4°C and room temperature (25°C) respectively. Mn(2+) and Mg(2+) increased the residual activity of the enzyme, whereas Fe(2+) moderately inhibited its residual activity. When pre-incubated with Tween-20, Tween-80, SDS and H2O2, each at 0.5% concentration, the enzyme showed increased residual activity. These characteristics may make the enzyme suitable for several industrial applications, especially in leather industries.

Keywords: Alkaline protease; Bacillus licheniformis; Production optimization; Shake flask culture.

Figure 1

Effect of carbon source (rice flour) concentration on protease production by Bacillus licheniformis P-003. The effect of rice flour concentration on enzyme production was investigated by using 1% inoculum (w/v) in 100 ml basal medium. The fermentation was carried out at 37°C at 150 rpm for 48 h. Absorbance was measured at 660 nm with spectrophotometer and enzyme activity was presented on the y axis and carbon source concentration was on x axis. Bars represent means ± standard errors for three replicates.

Figure 2

Effect of inorganic nitrogen source on protease production by Bacillus licheniformis P-003. To determine the effect of inorganic nitrogen sources on enzyme production, different inorganic nitrogen sources were used (1.0% w/v) in 100 ml of basal medium. The fermentation was carried out at 37°C at 150 rpm for 48 h. Bars represent means ± standard deviations for three replicates.

Figure 3

Effect of pH on protease activity. For determination of optimum assay pH of the enzyme reaction, 0.05M Na₂HPO₄-NaH₂PO₄ (pH 6.5 to 7.0), Tris- HCl (pH 7.5 to 8.5) and Glycine-NaOH (pH 9.0 to 12) buffers were used. The reaction was carried out for 20 min at 50°C in shaking water bath. Enzyme activity was measured and the results were presented on graph. Bars represent means ± standard deviations for three replicates.

Figure 4

Effect of pH stability on protease activity. The effect of pH on the stability on protease was studied by pre-incubating the enzyme with 0.05 M buffers covering the pH range of 6.5-12.0. 0.5 ml enzyme samples were added to 1 ml of different buffer and pre-incubated at 4°C for 24 h. Then residual activity in each sample was determined by standard protease assay and compared with the control sample kept at 4°C without any buffer. Bars represent means ± standard deviations for three replicates.

Figure 5

Effect of temperature on protease activity. To study the effect of temperature on enzyme reaction activity, enzyme reaction was carried out at different temperatures for 20 min in a shaking water bath and results are presented on graph. Bars represent means ± standard deviations for three replicates.

Figure 6

Effect of temperature on protease stability. For the determination of thermostability of protease, 0.5 ml of enzyme solutions were pre heated at different temperatures for different time intervals in a shaking water bath. Then enzyme activity of the heat treated enzymes were then measured and the results are presented on graph. Bars represent means ± standard deviations for three replicates.

Figure 7

Effect of reaction time on enzyme activity.

Figure 8

Effect of reaction time on enzyme activity. To investigate the optimum reaction time of the enzyme solution, reaction was carried out at 50°C in a water bath at different time intervals and the enzyme activity was then measured. The results are presented on graph. Bars represent means ± standard deviations for three replicates.

Figure 9

Effect of storage temperature on stability. To determine the storage stability of protease enzyme, crude enzyme solution was stored at 4°C and room temperature. Enzyme activity was measured at 7 days interval over 28 days by standard assay method described previously. The results are presented on graph. Bars represent means ± standard deviations for three replicates.