Improving the Thermostability and Catalytic Efficiency of the D-Psicose 3-Epimerase from Clostridium bolteae ATCC BAA-613 Using Site-Directed Mutagenesis

D-Psicose is a highly valuable rare sugar because of its excellent physiological properties and commercial potential. D-Psicose 3-epimerase (DPEase) is the key enzyme catalyzing the isomerization of D-fructose to D-psicose. However, the poor thermostability and low catalytic efficiency are serious constraints on industrial application. To address these issues, site-directed mutagenesis of Tyr68 and Gly109 of the Clostridium bolteae DPEase was performed. Compared with the wild-type enzyme, the Y68I variant displayed the highest substrate-binding affinity and catalytic efficiency, and the G109P variant showed the highest thermostability. Furthermore, the double-site Y68I/G109P variant was generated and exhibited excellent enzyme characteristics. The K-m value decreased by 17.9%; the k(cat)/K-m increased by 1.2-fold; the t(1/2) increased from 156 to 260 min; and the melting temperature (T-m) increased by 2.4 degrees C. Moreover, Co2+ enhanced the thermostability significantly, including the t(1/2) and T-m values. All of these indicated that the Y68I/G109P variant would be appropriate for the industrial production of D-psicose.