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Engineering β-Galactosidase with Enhanced Catalytic and Transglycosylation Activity for GOS Production
Lingtong Liao1,2, Laichuang Han2, Yanfang Sun3
1Science Center for Future Foods, Jiangnan University, Wuxi 214122, China.
Journal of Agricultural and Food Chemistry
|January 2, 2026
Summary
Engineered lactase enzymes show improved activity and galactooligosaccharide (GOS) production. These enhanced biocatalysts offer greater stability and efficiency for industrial applications.
Area of Science:
- Enzymology
- Biotechnology
- Protein Engineering
Background:
- Lactases are crucial glycoside hydrolases with significant industrial utility.
- Current lactase applications are constrained by limitations in stability and transglycosylation.
- Engineering lactase is essential for enhancing its performance in food and pharmaceutical sectors.
Purpose of the Study:
- To engineer a *Kluyveromyces lactis*-derived lactase for improved hydrolytic activity and transglycosylation.
- To enhance the catalytic efficiency and galactooligosaccharide (GOS) yield of lactase.
- To investigate the structural basis for improved enzyme function using molecular dynamics simulations.
Main Methods:
- Multistrategy protein engineering, including sequence alignment, flexible region modification, and model prediction.
- Site-directed mutagenesis to create specific lactase variants (L764T and M2-3).
- Enzyme activity assays and molecular dynamics (MD) simulations to analyze performance and structural changes.
Main Results:
- Engineered lactase variants (L764T and M2-3) exhibited 2.9-fold and 4.8-fold increases in catalytic performance (kcat/KM), respectively.
- The M2-3 mutant achieved a significantly higher galactooligosaccharide (GOS) yield (47.9%) compared to the wild type (35.2%).
- MD simulations revealed enhanced substrate binding, increased domain rigidity, reinforced intersubunit interactions, and improved tetrameric compactness.
Conclusions:
- The engineered lactase mutants demonstrate superior hydrolytic activity and transglycosylation capabilities.
- Structural modifications contribute synergistically to the enhanced biocatalytic efficiency of the mutant enzymes.
- These improved lactase variants hold significant promise for efficient industrial applications.
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