Cloning and characterization of two new thermostable and alkalitolerant α-amylases from the Anoxybacillus species that produce high levels of maltose

Two genes that encode alpha-amylases from two Anoxybacillus species were cloned and expressed in Escherichia coli. The genes are 1,518 bp long and encode 506 amino acids. Both sequences are 98% similar but are distinct from other well-known alpha-amylases. Both of the recombinant enzymes, ASKA and ADTA, were purified using an alpha-CD-Sepharose column. They exhibited an optimum activity at 60A degrees C and pH 8. Both amylases were stable at pH 6-10. At 60A degrees C in the absence of Ca2+, negligible reduction in activity for up to 48 h was observed. The activity half-life at 65A degrees C was 48 and 3 h for ASKA and ADTA, respectively. In the presence of Ca2+ ions, both amylases were highly stable for at least 48 h and had less than a 10% decrease in activity at 70A degrees C. Both enzymes exhibited similar end-product profiles, and the predominant yield was maltose (69%) from starch hydrolysis. To the best of our knowledge, most alpha-amylases that produce high levels of maltose are active at an acidic to neutral pH. This is the first report of two thermostable, alkalitolerant recombinant alpha-amylases from Anoxybacillus that produce high levels of maltose and have an atypical protein sequence compared with known alpha-amylases.