Improving the Thermostability of Raw-Starch-Digesting Amylase from a Cytophaga sp. by Site-Directed Mutagenesis

A heat-stable raw-starch-digesting amylase (RSDA) was generated through PCR-based site-directed mutagenesis. At 65°C, the half-life of this mutant RSDA, which, compared with the wild-type RSDA, lacks amino acids R178 and G179, was increased 20-fold. While the wild type was inactivated completely at pH 3.0, the mutant RSDA still retained 41% of its enzymatic activity. The enhancement of RSDA thermostability was demonstrated to be via a Ca²⁺-independent mechanism.