Computer-Aided Targeted Mutagenesis of Thermoclostridium caenicola d-Allulose 3-Epimerase for Improved Thermostability

d-Allulose 3-epimerase (DAEase) is a key enzyme in d-allulose bioproduction. DAEase from Thermoclostridium caenicola suffers from poor thermostability, hampering its large-scale applications in industry. In this study, mutants A70P, G107P, F155Y, and D162T with increased melting point temperature (Tm) were obtained by targeted mutagenesis based on the calculation of the free energy of folding. The optimal single-point mutant G107P showed 11.08 h, 5, and 5.70 °C increases in the values of half-life (t1/2) at 60 °C, the optimum temperature (Topt), and Tm, respectively. Beneficial mutations were combined by ordered recombination mutagenesis, and the combinational mutant Var3 (G107P/F155Y/D162T/A70P) was generated with ΔTopt of 10 °C and ΔTm of 12.25 °C. Its t1/2 value at 65 °C was more than 140 times higher than that of the wild-type enzyme. Molecular dynamics simulations and homology modeling analysis indicated that the enhanced overall rigidity, increased hydrogen bonds between subunits, and redistributed surface electrostatic charges might be responsible for the improved thermostability of the mutant Var3.