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.

Adaptive Steered Molecular Dynamics Combined With Protein Structure Networks Revealing the Mechanism of Y68I/G109P Mutations That Enhance the Catalytic Activity of D-psicose 3-Epimerase From Clostridium Bolteae

“…And Y6 Cc DPEase /Y6 Cb DPEase , E150 Cc DPEase /E152 Cb DPEase , R215 Cc DPEase /R217 Cb DPEase , and E244 Cc DPEase /E246 Cb DPEase can make hydrogen bonds with D-fructose (Chan et al, 2012 ). Moreover, the Y68I/G109P mutant enzyme is more thermostable and it demonstrates outstanding catalytic ability, which is 1.7-fold for t 1/2 value and 1.2-fold for k cat /K m of the wild-type Cb DPEase correspondently (Zhang et al, 2016 ).…”

“…D-psicose, as a C-3 epimer of D-fructose, has plentiful significant physiological functions, covering anti-hyperglycemia and anti-obesity effects (Granström et al, 2004 ; Fukada et al, 2010 ; Iida et al, 2010 ; Zhang et al, 2016 ; Shintani et al, 2017 ). It has a sucrose sweetness of 70%, and contributes less calories for its poor absorption in the digestive tract (Granström et al, 2004 ; Fukada et al, 2010 ).…”

“…D-psicose is rare in nature, and its chemical synthesis is difficult to get (Zhang et al, 2016 ). Therefore, the technology of D-psicose production by the enzymatic reaction of ketose 3-epimerase has been widely concerned in commercial production (Kim et al, 2006 ; Yoshida et al, 2007 ; Mu et al, 2011 ; Chan et al, 2012 ).…”

“…Recently, Jia et al reported the characterization study of the wild-type Clostridium bolteae DPEase ( Cb DPEase) (Jia et al, 2014 ). Also, the catalytic activity of the wild-type Cb DPEase was promoted by mutations in the Y68 and G109 sites (Zhang et al, 2016 ). Compared with the wild-type, the Y68I/G109P mutant would be more appropriate for the industrial D-psicose production.…”

Production of d‐allulose from d‐glucose by Escherichia coli transformant cells co‐expressing d‐glucose isomerase and d‐psicose 3‐epimerase genes

“…Through random and site‐directed mutagenesis, the double‐site I33L/S213C variant of DPEase from A. tumefaciens was constructed, which showed obvious enhancement in thermostability, including a 7.5 °C increase in optimal temperature, a 29.9‐fold increase in half‐life ( t ½ , 50 °C) and 7.5 °C increase in melting temperature ( T m ) . Recently, by site‐directed mutagenesis, the Y68I/G109P variant of C. bolteae DPEase was obtained, which improved the catalytic efficiency and thermostability with a 17.9% decrease in K m value, a 108 min increase in t ½ (55 °C) and a 2.4 °C increase in T m. Based on the systematic analysis of subunit–subunit interface interactions and the residues around active site development of iterative saturation mutagenesis and high‐efficient screening results in beneficial A. tumefaciens DPEase variants with greatly improved thermostability and substrate specificity, respectively . In addition, the food‐grade expression of DPEase from Clostridium scindens 35704 in Bacillus subtilis based on an antibiotic resistance gene‐independent replicative plasmid was constructed, producing 196 g L −1 d ‐allulose from 700 g L −1 of d ‐fructose …”