Production of a thermostable α-amylase from Bacillus sp. PS-7 by solid state fermentation and its synergistic use in the hydrolysis of malt starch for alcohol production

Bacillus sp. PS-7, isolated from the hot springs of Manikaran (H.P.), India, produced very high levels of thermostable α-amylase by solid state fermentation (SSF) in Erlenmeyer flasks and enamel coated metallic trays. Productivity was affected by the nature of the solid substrate, nature of the moistening agent, level of moisture content, incubation temperature, presence or absence of surfactant, carbon, nitrogen, mineral, amino acid and vitamin supplements. Maximum enzyme production of 4,64,000 U/g dry bacterial bran was obtained on wheat bran supplemented with glycerol (1.0%, w/w), soyabean meal (1.0%, w/w), l-proline (0.1%, w/w), vitamin B-complex (0.01%) and moistened with tap water containing 1% Tween-40 and 1 mM MgSO4·7H2O at ratio of 1:1.5 after 48 h incubation at 37 °C. The enzyme was partially purified to 12.7-fold by ammonium sulphate precipitation, gel filtration on Sephadex G-75 column followed by Phenyl agarose hydrophobic interaction chromatography. The kinetic characterisation of the partially purified enzyme exhibited maximum activity at 60 °C and pH 6.5. The thermal stability profile revealed the half-life of more than 6 h at 60° and 5(1/2) h at 70 °C respectively in presence of Ca2+. The enzyme did not exhibit marked increase in activity in presence of any metal ion. It could effectively act in synergism with the commercial amyloglucosidase during direct malt starch hydrolysis for ethanol fermentation.