Purification and characterization of the extracellular α-amylase from Streptococcus bovis JB1

Abstract

The extracellular α-amylase (1,4-α-D-glucanglucanohydrolase; EC 3.2.1.1) from maltose-grown Streptococcus bovis JB1 was purified to apparent homogeneity by ion-exchange chromatography (Mono Q). The enzyme had an isoelectric point of 4.50 and an apparent molecular mass of 77,000 Da, as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme was rich in acidic and hydrophobic amino acids. The 15-amino-acid NH2-terminal sequence was 40% homologous with the Bacillus subtilis saccharifying α-amylase and 27% homologous with the Clostridium acetobutylicum α-amylase. α-Amylase activity on soluble starch was optimal at pH 5.0 to 6.0. The enzyme was relatively stable between pH 5.5 and 8.5 and at temperatures below 50°C. When soluble potato starch was used as the substrate, the enzyme had a K(m) of 0.88 mg · ml-1 and a k(cat) of 2,510 μmol of reducing sugar · min-1 · mg of protein-1. The enzyme exhibited neither pullulanase nor dextranase activity and was 40 to 70% as active on amylopectin as on amylose. The major end products of amylose hydrolysis were maltose, maltotriose, and maltotetraose.