Related Experiment Video
Updated: Oct 19, 2025
High-throughput Screening of Carbohydrate-degrading Enzymes Using Novel Insoluble Chromogenic Substrate Assay Kits
Published on: September 20, 2016
Continuous Spectrophotometric Assay for High-Throughput Screening of Predominant d-Allulose 3-Epimerases
Chao Li1, Wei Zhang1, Cancan Wei1
1Key Laboratory of Industrial Fermentation Microbiology of the Ministry of Education; Tianjin Key Laboratory of Industrial Microbiology, College of Biotechnology, Tianjin University of Science and Technology; National Engineering Laboratory for Industrial Enzymes, Tianjin 300457, P. R. China.
Researchers improved d-allulose 3-epimerases (DAEases) for better stability and activity. This advance facilitates the industrial production of d-allulose, a healthy sugar substitute.
Area of Science:
- Biotechnology
- Enzyme Engineering
- Food Science
Background:
- d-Allulose is a noncaloric sugar substitute with health benefits.
- Enzymatic biosynthesis using d-allulose 3-epimerases (DAEases) is promising but limited by enzyme instability.
- Industrial application of d-allulose requires robust and efficient DAEases.
Purpose of the Study:
- To develop a continuous spectrophotometric assay (CSA) for d-allulose analysis.
- To establish a high-throughput screening strategy for DAEase variants.
- To engineer DAEases with enhanced stability and activity for industrial applications.
Main Methods:
- Development of a continuous spectrophotometric assay (CSA).
- Coupling DAEase with NADH-dependent ribitol dehydrogenase for high-throughput screening.
- Protein engineering and characterization of DAEase variants (e.g., M15S/P40N/S209N).
- Structural modeling and molecular dynamics simulations.
Main Results:
- A robust CSA was established for efficient d-allulose quantification.
- A high-throughput screening strategy for DAEases was successfully developed.
- The engineered DAEase variant M15S/P40N/S209N showed a 22 h half-life at 60 °C and increased activity.
- Structural analysis revealed enhanced thermostability due to new hydrogen bonds.
Conclusions:
- The developed CSA and screening strategy enable efficient DAEase analysis and engineering.
- Engineered DAEases exhibit improved thermostability and activity, overcoming limitations for industrial use.
- This work provides a foundation for advancing enzymatic d-allulose biosynthesis and its market adoption.
