Functional study of C-terminal domain of the thermoacidophilic raw starch-hydrolyzing α-amylase Gt-amy

Since the thermoacidophilic raw-starch hydrolyzing α-amylase Gt-amy can effectively hydrolyze corn starch under starch liquefaction conditions, it has potential for many industrial applications. To identify the raw starch-binding domain of Gt-amy, a C-terminal domain (CTD)-truncated mutant (Gt-amy-T) was constructed, and its enzymatic properties were compared with Gt-amy. In comparison to CTD of Gt-amy, which could effectively bind corn starch, the Gt-amy-T could not bind to and hydrolyze corn starch under similar conditions. In addition, Gt-amy-T showed significantly lower thermal activity and thermal stability. Using soluble starch as the substrate, the k _cat of Gt-amy-T at 80 °C was approximately 77.9% of that of Gt-amy. The half-life of Gt-amy at 80 °C was 3 h, while that of Gt-amy-T was 2 h. These results reveal that the CTD plays a vital role in raw starch binding and degradation by Gt-amy and helps Gt-amy maintain thermal activity and stability.