Isolation purification and characterization of extracellular α-amylase from Bacillus subtilis isolated from the soil with smf (submerged fermentation) method

Soil samples were taken from Makam Mountain in Ergani to be used in our study. Bacteria isolation from the soil sample taken from Makam Mountain was made in the laboratory under sterile conditions. Some biochemical analyzes were performed on the isolated microorganism and 16S rRNA was identified. As a result of the data obtained, it was determined that the isolated bacterium was Bacillus subtilis. Optimum conditions were determined by investigating the effect of various parameters on α-amylase production of Bacillus subtilis isolate produced by SmF technique. It was determined that the optimum conditions for bacterial growth were 32 hours in terms of time, 37°C in terms of temperature and pH 7.0 in terms of pH. Bacterial replication was carried out at the 48th hour, which was determined as the time during which the enzyme was produced at the maximum level. α-amylase activity study was performed by taking the supernatant from the cultured bacteria. The optimum pH at which the enzyme was active was determined as 7.0 and the optimum temperature was 37°C. The effect of nitrogen and carbon sources to be used by bacteria on enzyme production was investigated. According to the data obtained, lower amylase activity was determined from the bacteria using nitrogen sources compared to the control. Among the nitrogen sources, the highest specific activity was observed with the addition of ammonium nitrate and ammonium sulfate. When the effect of carbon sources on enzyme production was examined, it was seen that the galactose value showed a value close to the control and the enzyme activity decreased in other carbon sources. After the optimum conditions of the enzyme were determined, the purification process was started. The molecular weight of the purified amylase was determined as ~73 kDa by SDS-PAGE.To determine the effect of some detergents on the purified enzyme activity, 0.005 Tween-40, SDS, TritonX-100, Tween-8 and brige were used. It was observed that SDS, one of the detergents used, inhibited the amylase enzyme significantly. When the effect of some metals on the purified enzyme activity was examined, it was determined that CaCl2 increased the activity, no change was observed as a result of the addition of MnCl2 and MgCl2 metals, and the enzyme activity was significantly inhibited by the addition of FeCl2, CuCl2, ZnCl2 and HgCl2 metals.