Comparative characterization of complete and truncated forms of Lactobacillus amylovorus α-amylase and role of the C-terminal direct repeats in raw-starch binding

Two constructs derived from the ol-amylase gene (amyA) of Lactobacillus amylovorus were expressed in Lactobacillus plantarum, and their expression products were purified, characterized, and compared, These products correspond to the complete (AmyA) and truncated (AmyA Delta) forms of ru-amylase; AmyA Delta lacks the 66-kDa carboxyl-terminal direct-repeating-unit region. AmyA and AmyA Delta exhibit similar amylase activities towards a range of soluble substrates (amylose, amylopectin and alpha-cyclodextrin, and soluble starch). The specific activities of the enzymes towards soluble starch are similar, but the K-M and V-max values of AmyA Delta were slightly higher than those of AmyA whereas the thermal stability of AmyA Delta was lower than that of AmyA. In contrast to AmyA, AmyA Delta is unable to bind to beta-cyclodextrin and is only weakly active towards glycogen. More striking is the fact that AmyA Delta cannot bind or hydrolyze raw starch, demonstrating that the carboxyl-terminal repeating-unit domain of AmyA is required for raw-starch binding activity.