Improving the Catalytic Efficiency of Aspergillus fumigatus Glucoamylase toward Raw Starch by Engineering Its N‑Glycosylation Sites and Saturation Mutation

Raw starch glucoamylase (RSGA) can degrade the raw starch below the starch gelatinization temperature. In this study, to improve the catalytic activity of raw corn starch, N-glycosylation was introduced into the RSGA from Aspergillus fumigatus through site-directed mutation and the recombinant expression in Komagataella phaffii. Among them, the mutants G101S (N99-L100-S101) and Q113T (N111-S112-T113) increased the specific activity of raw corn starch by 1.19- and 1.21-fold, respectively. The optimal temperature of Q113T decreased from 70 to 60 °C. Notably, the combined mutant G101S/Q113T increased the specific activity toward raw starch by 1.22-fold and reduced the optimal temperature from 70 to 60 °C. Moreover, the mutant Q113M with a 1.5-fold increase in the catalytic activity was obtained via saturation mutation at site 113. Thus, the N-glycosylation site engineering is an efficient method to improve the activity of RSGA toward raw starch.