Studies on Alkaline Protease Production from Bacillus sp

The aim of this study was optimization of the conditions for the production of alkaline protease from the Bacillus strains coded as L18, L21 and I18, which were isolated from our natural habitats. Additionally, the goal was also to optimize the production of alkaline protease from Bacillus sp. L21 in a new low-cost media formulation by employing design of experiments and response surface methodology. Lastly, the focus was given to the determination of the characteristic properties of the crude alkaline protease of Bacillus sp. L21. The strains L21 and L18 were supplied by İYTE Department of Biology, whereas I18 was supplied by Ege University Department of Biology. These potential alkaline protease producer strains identified as Bacillus sp. were isolated from the by products of a leather factory (strains L21 and L18) and from the soil of the Ege University Campus (strain I18). Specific growth rates calculated from the slope of the logarithmic phase were as 0.49h, 0.60 h, 0.70 h for I18, L18 and L21 respectively. When the effects of environmental conditions on alkaline protease production from strains L21, L18 and I18 were studied, the optimum temperatures were determined as 30°C for strain I18 and 37°C for the strains L18 and L21. Similarly the optimum agitation speeds were 100 rpm for I18 and 180 rpm for L18 and L21. The optimum inoculation ratio was determined as 5% (v/v) for all three strains. The optimum incubation time was determined as 96 hours for the strains I18 and L21 and 125 hours for L18. The optimum initial media pH was estimated as pH 10 for strain L18 and L21. The constant medium constituents in the development of a low-cost medium study were peptone, KH2PO4, Mg2SO4.7H2O, and Na2CO3. At the end of screening experiments, the factors considered include, soybean meal and corn steep liquor as nitrogen source, maltose as carbon source, CaCl2 and Tween 80 as elements. The relative importance of these factors on alkaline protease production was investigated by using a resolution IV fractional factorial design with single replicate of treatment combinations of four factors (soybean meal, corn steep liquor, CaCl2, Tween 80). Soybean meal, maltose, Tween 80 concentrations and initial pH were found to significantly influence the alkaline protease production. For obtaining the mutual interaction between the variables and optimizing these variables, Box-Behnken design and central composite design (CCD) using response surface methodology was employed. Among six interaction terms only those of soybean meal and maltose (X1*X2) and Tween80 and pH (X3*X4) were found to be significant. The neutral pH values of the medium and Tween 80 around its maximum level has a positive effect on proteolytic activity. Additionally, 4 sets of validation experiments were employed. The validation experiment, which state soybean meal, maltose, Tween 80 and pH as 2.5 g/L, 15 g/L, 0.35 g/L and 8.5, respectively, yielded an actual protease activity of 294.3 U/mL, where the model estimated a value of 338 U/mL. The BoxBehnken design and validation experiments were not sufficient to determine the true optimum values for the significant factors because of the saddle nature of the response surfaces. Therefore pH and Tween 80 were kept constant and a central composite design with two factors (soybean meal and maltose concentrations) was conducted. The adjusted R2 of the model was 93.3% with an insignificant lack of fit (p value=0.141). The CCD was able to determine the optimum value of soybean meal as 3.0 g/L, however it could be determined for maltose concentration after a set of additional experiments. Consequently, the optimum values of the four factors were determined as 8.0 for pH, 0.35 g/L for Tween 80, 3.0 g/L for soybean meal and 30-40 g/L for maltose concentration. The maximum activity under these conditions was 269.2 U/mL. In the characterization part of this study, the crude alkaline protease of Bacillus sp. L21 displayed a pH optimum of 11.0 and retained about 73-78% of its original activity between pH 4.0 and 11.0 in the stability studies. The optimum temperature for protease activity was found to be 60oC and retained about 90% of the original activity even at 80°C. By analyzing the thermal stability, the alkaline protease was found to be stable in a temperature range of 30°C to 50°C but lost about 30% of its activity at 60°C, after 30 min and 1 hour incubations both in the presence and absence of Ca2+. The protease from strain L21 showed high stability against both 5% and 15% (v/v) concentrations of H2O2, which is a bleaching agent, and retained approximately 82% and 94% of its activity, respectively, therefore, the present alkaline protease is thought to be a bleach-stable enzyme, so that it can be used in detergent formulations. There was no inhibitory effect observed from EDTA that further show that the enzyme is not a metalloprotease. However, PMSF (phenylmethylsulphonyl fluoride) strongly inhibited the protease activity, suggesting that the enzyme is a serine protease. In addition, CaCl2 showed inhibitory effect on the enzyme, decreasing the activity to 90% of the control and this can be explained that the enzyme do not require the presence of Ca ions to be active and stable.