Computer-Aided Multidimensional Strategy To Improve the Thermostability of d-Allulose 3-Epimerase from Christensenellaceae Bacterium for the Production of d-Allulose

d-Allulose, a rare sugar with broad applications, is produced from d-fructose by d-allulose 3-epimerase (DAE). However, a high temperature is needed for producing d-allulose, and it is always a challenge to improve both the thermostability and catalytic efficiency of DAEs. In this study, a weakly acidophilic d-allulose 3-epimerase (CbDAE) from Christensenellaceae bacterium with good specific activity 223.5 U/mg was successfully characterized. Subsequently, a multidimensional computer-aided engineering/iterative saturation mutagenesis (MCAE/ISM) strategy was employed to improve the thermostability of CbDAE based on analysis of flexibility and secondary structure of the protein, as well as the calculation of free energy changes of folding (ΔΔG_fold). Finally, the best variant M4 (A13S/V235I/D100N/I242V) exhibited a 2310.49 min half-life at 70 °C, a 19 °C increase in T_m, and a 2.84-fold higher activity. The conversion ratio could reach up to 38% with 200 g/L d-fructose, and even at high concentrations of 700 g/L, it could still reach 32%. When M4 was used in combination with glucose isomerase, the yield of 19.7% of d-allulose was achieved from d-glucose. Additionally, molecular dynamics simulations and structural analysis indicated that the improved thermostability and catalytic activity resulted from optimized protein conformations, redistributed surface charge networks, and enhanced inter-residue interactions. This study shows that variant M4 is a promising biocatalyst for the production of d-allulose.