As a natural sweetener with low calories and various physiological activities, d-allulose has drawn worldwide attention. Currently, d-allulose 3-epimerase (DAEase) is mainly used to catalyze the epimerization of d-fructose to d-allulose. Therefore, it is quite necessary to enhance the food-grade expression of DAEase to meet the surging market demand for d-allulose. In this study, initially, the promising variant H207L/D281G/C289R of Clostridium cellulolyticum H10 DAEase (CcDAEase) was generated by protein engineering, the specific activity and the T 1/2 of which were 2.24-fold and 13.45-fold those of the CcDAEase wild type at 60 °C, respectively. After that, PamyE was determined as the optimal promoter for the recombinant expression of CcDAEase in Bacillus subtilis, and catabolite-responsive element (CRE) box engineering was further performed to eliminate the carbon catabolite repression (CCR) effect. Lastly, high-density fermentation was carried out and the final activity peaked at 4971.5 U mL–1, which is the highest expression level and could effectively promote the industrial production of DAEase. This research provides a theoretical basis and technical support for the molecular modification of DAEase and its efficient fermentation preparation.

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