Abstract
D-psicose 3-epimerase (DPEase) catalyzes the isomerization of D-fructose to D-psicose (aka D-allulose, a low-calorie sweetener), but its industrial application has been restricted by the poor thermostability of the naturally available enzymes. Computational rational design of disulfide bridges was used to select potential sites in the protein structure of DPEase from Clostridium bolteae to engineer new disulfide bridges. Three mutants were engineered successfully with new disulfide bridges in different locations, increasing their optimum catalytic temperature from 55 to 65 °C, greatly improving their thermal stability and extending their half-lives (t1/2) at 55 °C from 0.37 h to 4−4.5 h, thereby greatly enhancing their potential for industrial application. Molecular dynamics simulation and spatial configuration analysis revealed that introduction of a disulfide bridge modified the protein hydrogen–bond network, rigidified both the local and overall structures of the mutants and decreased the entropy of unfolded protein, thereby enhancing the thermostability of DPEase.
Highlights
D-Psicose, or D-allulose, a C-3 epimer of D-fructose, is a sugar substitute with a~70% sweetness index relative to sucrose, but almost zero calories [1]
The relative B-factors are shown by color coding; B-factor values are represented by blue, green, yellow, and red in ascending order. (B) Local models of 3D structures around residues D90/A93 (A1), C175/A209 (B1) and A207/A243 (C1) residues in wild-type D-psicose 3-epimerase (DPEase) and the cysteine-substituted mutants D90C−A93C (A2), C175−A209C (B2) and Increasing the thermostability of industrial enzymes has always been of central importance to environmentally-friendly industrial production because of the advantages of thermally stable enzymes, such as the lower cost of re-usable enzymes, heat-acceleration of reactions and reduced microbial contamination [34]
Greater knowledge about the actual effect of disulfide bridges on the thermostability of DPEase is essential to accelerate the industrial production of D-allulose
Summary
D-Psicose, or D-allulose, a C-3 epimer of D-fructose, is a sugar substitute with a~70% sweetness index relative to sucrose, but almost zero calories [1]. D-Psicose, or D-allulose, a C-3 epimer of D-fructose, is a sugar substitute with a. D-psicose 3-epimerase (DPEase) catalysis has been attracting strong industrial interest [2]. Developing thermostable DPEase mutants is an urgent requirement for industrial manufacture of D-allulose. Directed evolution based on random mutagenesis and high-throughput screening approaches is a general strategy for improving enzyme thermostability [4]. This effective method has been successfully applied to, for example Pseudomonas fluorescens lipase, Agrobacterium tumefaciens uronate dehydrogenase and Bacillus clausii alkaline protease [5,6,7]
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