4.7 Article

Enhanced Thermostability of D-Psicose 3-Epimerase from Clostridium bolteae through Rational Design and Engineering of New Disulfide Bridges

Journal

Publisher

MDPI
DOI: 10.3390/ijms221810007

Keywords

disulfide bonds; cysteine; molecular dynamics simulation; cold-shock

Funding

  1. National Natural Science Foundation of China [31760469]
  2. Natural Science Foundation of Guangxi Province [2018GXNSFAA050126, 2020GXNSFAA297104]
  3. Young and Middle-aged Backbone Teachers Training Project in Colleges and Universities of Guangxi province
  4. China Agriculture Research System of MOF and MARA
  5. Science and technology project of Chongzuo [FA2020001]

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By computationally designing disulfide bridges, mutants of D-psicose 3-epimerase were successfully engineered to increase their optimum catalytic temperature, greatly enhance their thermal stability, and extend their half-lives, thereby significantly improving their potential for industrial application.
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 degrees C, greatly improving their thermal stability and extending their half-lives (t(1/2)) at 55 degrees 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.

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