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Computer-Aided Molecular Modifications for Enhanced Activity and Thermal Stability of d-Allulose 3-Epimerase
Taixiao Guo1, Ming Miao1,2, Tao Zhang1,3
1State Key Laboratory of Food Science and Resources, Jiangnan University, Wuxi, Jiangsu 214122, China.
Journal of Agricultural and Food Chemistry
|March 27, 2025
Summary
Engineered D-allulose 3-epimerase (DAE) enzymes exhibit enhanced activity and thermal stability. Specific mutations significantly boost DAE performance, paving the way for improved industrial applications in D-allulose production.
Area of Science:
- Enzymology
- Protein Engineering
- Biotechnology
Background:
- D-allulose 3-epimerase (DAE) converts D-fructose to D-allulose.
- Current DAEs have limitations in activity and thermal stability for industrial use.
Purpose of the Study:
- To enhance both enzyme activity and thermal stability of DAE from *Clostridium scindens* ATCC 35704 (*Csc*-DAE).
- To identify beneficial mutations for industrial DAE applications.
Main Methods:
- Semi-rational design and site-specific mutations were employed.
- Enzyme activity and thermal stability of mutants were evaluated.
- Molecular docking was used to analyze structural changes.
Main Results:
- Six single mutants showed 1.08-1.31 fold increased activity.
- Double-point mutants exhibited 1.28-1.64 fold increased activity.
- A25V/C212N mutant demonstrated improved thermal stability (65°C optimum, 3.997h half-life at 60°C).
Conclusions:
- Semi-rational design successfully improved *Csc*-DAE activity and stability.
- Specific mutations like A25V/C212N offer significant potential for industrial D-allulose production.
- Enhanced DAEs are crucial for efficient and cost-effective manufacturing processes.
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