Purification and chemical characterization of thermostable amylases produced by Bacillus stearothermophilus

The amylases produced by a Bacillus stearothermophilus were purified through a series of four steps. Two separable enzyme fractions having starch hydrolysing activity were eluted from a DEAE-cellulose column by NaCl gradient elution. The homogeneity of the purified enzymes was checked on polyacrylamide gel electrophoresis. The product formation studies indicated that fraction I was an -amylase whereas fraction II was a β-amylase. The molecular weights were determined to be 48 000 and 57 000 and the carbohydrate moiety was found to be 13.2 and 0.8% for - and β-amylase, respectively. The protein digest of these enzymes indicated a total number of 15 amino acids with aspartic and glutamic acid showing the highest value. The purified amylase showed maximal activity at 80°C and pH 6.9. Fe³⁺, Cd²⁺, Pb²⁺, Hg²⁺, Ni²⁺ and Ag¹⁺ were potent inhibitors whereas Zn²⁺, Mg²⁺, Mn²⁺ and Al³⁺ were mild inhibitors. Ca²⁺, Ba²⁺, Sr²⁺ and K⁺ stimulated amylase activity in the order of Ca²⁺ > Ba²⁺ > Sr²⁺ > K⁺. PCMB, EDTA and sodium iodoacetate were inhibitory whereas glutathione (GSH) and cysteine afforded protection of enzyme activity. EDTA showed dose-dependent noncompetitive inhibition of both - as well as β-amylase activities. EDTA inhibition was reversed by the addition of Ca²⁺ and PCMB inhibition by the addition of glutathione (reduced). The K_m for - and β-amylases were found to be 1.05 and 1.25 mg starch per ml, respectively.