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Rational Engineering of Cellobiose 2-Epimerase Through Flexible Loop Modulation and Structure-Guided Sequence
Xinyan Mao1, Hongbin Zhang1, Chao Hu1
1School of Food and Biological Engineering, Hefei University of Technology, Hefei 230009, China.
Biomolecules
|February 27, 2026
Summary
Researchers enhanced lactulose production using enzyme engineering of cellobiose 2-epimerase (CsCE). Optimized mutants and conditions achieved high yields and purity for this functional disaccharide.
Area of Science:
- Biotechnology
- Enzyme Engineering
- Biocatalysis
Background:
- Lactulose is a functional disaccharide with significant pharmaceutical and food applications.
- Enzymatic isomerization of lactose is an efficient method for lactulose synthesis.
Purpose of the Study:
- To enhance lactulose production through protein engineering of cellobiose 2-epimerase (CsCE) and process optimization.
- To develop superior CsCE variants with improved activity, stability, and substrate affinity.
Main Methods:
- Employed a dual-track protein engineering strategy involving flexible loop modulation and structure-guided sequence alignment.
- Created and characterized two superior CsCE mutants: R17Q/L184S and R17Q/S142T.
- Optimized reaction conditions including temperature, pH, substrate concentration, and enzyme loading.
Main Results:
- The R17Q/L184S mutant showed a 37% increase in activity, enhanced thermostability, and improved substrate affinity.
- The R17Q/S142T mutant exhibited the highest specific activity (24.08 U/mg), a 21% increase.
- Optimized conditions yielded 75.6% lactulose with >95% purity.
Conclusions:
- Synergistic enzyme engineering and process intensification successfully boosted lactulose biosynthesis efficiency.
- Developed viable enzyme variants and system solutions for industrial-scale lactulose production.
- Structural insights revealed mechanisms for improved enzyme performance, including enhanced rigidity and allosteric regulation.
