Purification and chemical characterization of thermostable amylases produced by Bacillus stearothermophilus

Abstract

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 α-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 α- 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. Fe 3+, Cd 2+, Pb 2+, Hg 2+, Ni 2+ and Ag 1+ were potent inhibitors whereas Zn 2+, Mg 2+, Mn 2+ and Al 3+ were mild inhibitors. Ca 2+, Ba 2+, Sr 2+ and K + stimulated amylase activity in the order of Ca 2+ > Ba 2+ > Sr 2+ > 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 α- as well as β-amylase activities. EDTA inhibition was reversed by the addition of Ca 2+ and PCMB inhibition by the addition of glutathione (reduced). The K m for α- and β-amylases were found to be 1.05 and 1.25 mg starch per ml, respectively.