Alpha amylase production, purification and characterization of Bacillus mojavensis and Bacillus vallismortis

In this study, a novel thermotolerant Bacillus vallismortis and Bacillus mojavensis was isolated from thermal springs mud sample in Sirnak (Meyremderesi), Turkey. Influences of various parameters such as incubation time, temperature and pH, carbon and nitrogen sources, various metal ions, agriculture wates (banana, apple, and orange husk, barley bran, and bean pulp), NaCl concentration, different surfactants on α-amylases production were examined. The optimum incubation time, temperature and pH for production of α-amylase was found 36th, 35 °C and 7.0, respectively by B. vallismortis while the optimum temperature and pH for amylase production was found to be 35°C at pH 6.0 by B. mojavensis. When compared with control, the amylase production increased approximately 10% in presence of Mn(II) and Ca(II) and 8.2% and 12.4% with Triton-X100 and Tween-20, respectively for B. vallismortis. The highest α-amylase production was determined as 4282,6 U/mg for B. mojavensis when 0.05% Tween-20 was added to fermentation medium under the optimum conditions. Thermostable α-amylases obtained from B. vallismortis and B. mojavensis, were purified using 70% ammonium sulfate precipitation, dialysis, and diethylaminoethyl (DEAE) cellulose ion exchange chromatography, respectively. The α-amylases were purified with a 51-fold and 30% yield for B. vallismortis and 34-fold and 18% yield for B. mojavensis, respectively. The molecular masses of purified α-amylases were found as 74 kD and 73 kD for B. vallismortis and B. mojavensis, respectively. Effect of temperature and temperature stability, pH and pH stability, inhibitors and some metal ions on purified α-enzymes activity were experimented. It was revealed good hydrolyzing rate towards to raw starches of corn and wheat for both α-amylases. The end-yields of soluble starch hydrolysis were analyzed by thin-layer chromatography (TLC). The hydrolysis products were glucose (G1), maltose (G2), maltotriose (G3), maltotetraose (G4), and maltooligosaccharides. In addition to these, the usage of purified α-amylases in clarifacation of apple juice and domestic washing detergents industries were tested.