Purification and characterization of a novel thermostable β-amylase from Clostridium thermosulphurogenes

Abstract

An extracellular β-amylase from Clostridium thermosulphurogenes was purified 811-fold to homogeneity, and its general molecular, physico-chemical and catalytic properties were determined. The native enzyme was a tetramer of 210 kDa composed of a single type subunit; its 20 amino acid N-terminus displayed 45% homology with Bacillus polymyxa β-amylase. The β-amylase was enriched in both acidic and hydrophobic amino acids. The pure enzyme displayed an isoelectric point of 5.1 and a pH activity optimum of 5.5. The optimum temperature for β-amylase activity was 75°C, and enzyme thermostability at 80°C was enhanced by substrate and Ca2+ addition. The β-amylase hydrolysed amylose to maltose and amylopectin and glycogen to maltose and limit dextrins, and it was inhibited by α- and β-cyclodextrins. The enzyme displayed κ(cat). and K(m) values for boiled soluble starch of 400,000 min-1 per mol and 1.68 mg/ml, respectively. The enzyme was antigenically distinct from plant β-amylases.