Improved thermostability of D-allulose 3-epimerase from Clostridium bolteae ATCC BAA-613 by proline residue substitution

D-allulose, a rare sugar that is scarce in nature, exerts several beneficial effects and has commercial potential. D-allulose 3-epimerase (DAEase) plays a vital role in catalyzing the isomerization from D-fructose to D-allulose. However, the industrial application of DAEase for D-allulose production is hindered by its poor long-term thermostability. In the present research, we introduced a proline residue (i) to restrict its spatial conformation and (ii) to reduce the entropy of the unfolded state of DAEase. The t(1/2) value of the double-site Clostridium bolteae DAEase mutant Cb-51P/89P was prolonged to 58 min at 55 degrees C, a 2.32-fold increase compared with wild-type DAEase. The manipulation did not cause obvious changes in the enzymatic properties, including optimum pH, optimal temperature, optimum metal ion, and enzymatic activity. As the accumulation of multiple small effects through proline substitution could dramatically improve the thermostability of the mutant protein, our method to improve the thermostability while roughly retaining the original enzymatic properties is promising.