Abstract: To identify the raw starch binding region of the thermoacidiphilic raw starch degrading α-amylase Gt-amy, C-terminal domain (CTD) truncated mutant Gt-amy-T was constructed and its enzymatic properties were characterized and compared with those of Gt-amy. The abilities of Gt-amy and Gt-amy-T to bind to and hydrolyze raw corn starch to evaluate the role of CTD as a raw starch binding domain. Both Gt-amy and CTD displayed comparable affinities for raw corn starch whereas Gt-amy-T was unable to bind to raw corn starch. Gt-amy hydrolyzed raw corn starch efficiently, while Gt-amy-T did not. The kcat of Gt-amy-T was 77.9% of that of Gt-amy with soluble starch as the substrate. Phylogenetic analysis of CTD revealed that CTD was not a typical starch binding domain, but Gt-amy’s CTD was experimentally verified to be raw starch binding domain, which played an important role in raw starch binding and hydrolysis by Gt-amy. CTD contributed to the hydrolysis of soluble starch by Gt-amy. In addition, mutational analysis of Tyr residues in CTD was performed to identify the raw starch binding sites in CTD. The binding capacities of CTD and the mutants to raw corn starch were assayed. The raw corn starch binding and hydrolysis capacities of Gt-amy and Gt-amy W501A/W514A were investigated. The results revealed that the residues W501 and W514 were probably raw starch binding sites in CTD.

Key words: thermoacidiphilic raw starch degrading α-amylase, raw starch binding domain, raw starch binding site, mutational analysis