Enhancing the thermostability of d-allulose 3-epimerase from Clostridium cellulolyticum H10 via directed evolution

d-allulose, the epimer at C-3 position of d-fructose, is a low-calorie functional rare sugar, which is regarded as one of the most potential sweeteners. At present, the main production method of d-allulose is epimerization of d-fructose by d-allulose 3-epimerase (DAE). However, industrial applications of DAE are still limited by its poor thermostability. Herein, directed evolution was applied to improve the thermostability of DAE from Clostridium cellulolyticum H10 (CcDAE). Two optimal mutants D281G and C289R, exhibiting 13.80-fold and 13.88-fold t1/2 values as that of wild type at 65 ℃, respectively, were obtained. To further enhance the thermostability, the triple mutant A107P/D281G/C289R was constructed after combination of mutants D281G, C289R, and previously identified thermostability-enhanced mutant A107P. The Tm and optimal temperature of triple mutant were increased by 14.39 ℃ and 5 ℃, respectively, compared to the wild type, meanwhile, the half-life of triple mutant was 58.85-fold as that of wild type at 65 ℃. Furthermore, the conversion rate of triple mutant was increased from 24.76% of wild type to 27.53% using 300 g/L d-fructose as substrate at 70 ℃. The effectiveness of directed evolution was verified and the triple mutant with enhanced thermostability had great application value in the large-scale production of d-allulose.