Optimization of protease production conditions of Bacillus subtilis ZBP4 strain and determination of some properties of enzyme

In this study, the production of the extracellular protease enzyme by Bacillus subtilis ZBP4 strain was investigated and the effects of environmental conditions on enzyme production and microbial growth were determined. The enzymatic activity was determined in the cell-free supernatant that was obtained after the growth of the isolate. Preliminary studies showed that the isolate was able to produce protease enzyme. By taking into consideration the protease activity obtained in the basal medium, optimization studies were performed. The effects of glucose concentration (2-20 g/L), nitrogen sources (yeast extract, casein, whey powder, skimmed milk powder, soy flour, yellow lentil flour, ammonium sulfate, pepton), initial pH (5-9) of the medium and incubation temperature (30-40°C) have been determined. Enzymatic activity was determined by incubating the supernatant and casein which was dissolved in Tris HCl pH 8 buffer at 60°C for 15 minutes. In addition, some properties of the enzyme (pH, temperature) were determined. To determine the enzyme activity and microbial growth, the absorbances of the samples were measured at 280 nm and 600 nm by using a UV-VIS spectrophotometer, respectively. The enzyme activity prior to optimization was 1206 U/mL in the basal medium. The best production was achieved in the medium contained yeast extract as a nitrogen source at pH 9 and 35°C in 48 hours. Furthermore, yeast extract, soybean flour and lentil flour were the best nitrogen sources for the enzyme production. Concentration studies were performed for all three sources and it was determined that yeast extract and soy flour had the optimum 15 g/L concentration and lentil flour had the best protease activity at a concentration of 20 g/L. The optimum temperature and the pH of the protease enzyme were determined as 60°C and pH 8,0.