Engineering dextranase for the directed preparation of low-molecular-weight dextran

Area of Science:

• Biochemistry
• Enzyme Engineering
• Industrial Biotechnology

Background:

• Dextranase is crucial for producing isomaltooligosaccharides (IMOs) and low-molecular-weight dextran (LMWD).
• The natural dextranase from *Chaetomium gracile* (CgDEX) has limited catalytic activity, restricting its industrial use.

Purpose of the Study:

• To enhance the catalytic activity of CgDEX through protein engineering.
• To investigate the impact of mutations on enzyme performance and substrate interactions.

Main Methods:

• Multiple sequence alignment (MSA) to identify potential mutation sites.
• Site-directed mutagenesis and heterologous expression in *Pichia pastoris*.
• Enzyme activity assays, molecular docking, and kinetic analysis.

Main Results:

• Engineered CgDEX exhibited a 17.5% increase in specific activity (5185 ± 70 U/mg).
• Mutations strengthened hydrogen-bond interactions and increased substrate affinity.
• The mutant enzyme primarily produced IMOs (DP 2-4) and synthesized LMWD synergistically.

Conclusions:

• Protein engineering can significantly enhance dextranase catalytic performance.
• Engineered CgDEX enables green manufacturing of functional oligosaccharides and LMWD.
• This study validates a strategy for improving biocatalyst efficiency for industrial applications.