title: Improving the Thermostability and Catalytic Efficiency of the d-Psicose 3-Epimerase from Clostridium bolteae ATCC BAA-613 Using Site-Directed Mutagenesis.
abstract: 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 Km value decreased by 17.9%; the kcat/Km increased by 1.2-fold; the t1/2 increased from 156 to 260 min; and the melting temperature (Tm) increased by 2.4 °C. Moreover, Co(2+) enhanced the thermostability significantly, including the t1/2 and Tm values. All of these indicated that the Y68I/G109P variant would be appropriate for the industrial production of d-psicose.
authors.forename: Wenli
authors.initials: W
authors.lastname: Zhang
authors.forename: Min
authors.initials: M
authors.lastname: Jia
authors.forename: Shuhuai
authors.initials: S
authors.lastname: Yu
authors.forename: Tao
authors.initials: T
authors.lastname: Zhang
authors.affiliation: Roquette America , 1003 Commercial Street, Keokuk, Iowa 52632, United States.
authors.forename: Leon
authors.initials: L
authors.lastname: Zhou
authors.forename: Bo
authors.initials: B
authors.lastname: Jiang
authors.forename: Wanmeng
authors.initials: W
authors.lastname: Mu
mesh_terms: D000595:Amino Acid Sequence; D002238:Carbohydrate Epimerases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002384:Catalysis; D004795:Enzyme Stability; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D017386:Sequence Homology, Amino Acid
chemical_list: D002238:Carbohydrate Epimerases; C000595678:D-psicose 3-epimerase, Clostridium cellulolyticum
keywords: catalytic efficiency; d-psicose; d-psicose 3-epimerase (DPEase); site-directed mutagenesis; thermostability