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

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• 作者：Wenli Zhang ; Min Jia ; Shuhuai Yu ; Tao Zhang ; Leon Zhou ; Bo Jiang ; Wanmeng Mu
• 刊名：Journal of Agricultural and Food Chemistry
• 出版年：2016
• 出版时间：May 4, 2016
• 年：2016
• 卷：64
• 期：17
• 页码：3386-3393
• 全文大小：515K
• 年卷期：0
• ISSN：1520-5118

文摘

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 °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.