Structure-Guided Remodeling of β-Glucosidase Catalytic Performance toward Reverse Hydrolysis for Enhanced Kinsenoside...

Area of Science:

• Biocatalysis and Enzyme Engineering
• Synthetic Biology
• Green Chemistry

Background:

• Kinsenoside, a bioactive C10 glucoside, faces production challenges due to limited plant sources and inefficient chemical synthesis.
• Enzymatic reverse hydrolysis using β-glucosidase offers a greener alternative but suffers from low yields and product degradation due to hydrolytic bias.

Purpose of the Study:

• To engineer a β-glucosidase from *Dictyoglomus thermophilum* (DtBGL) for enhanced kinsenoside production via structure-guided semirational design.
• To investigate the structural and mechanistic basis for improved catalytic performance in the engineered enzyme.
• To enhance the stability and yield of kinsenoside production through enzyme immobilization.

Main Methods:

• Structure-guided semirational design was employed to engineer DtBGL.
• The optimal mutant (M6) was characterized for its catalytic activity and specificity.
• Molecular dynamics simulations and substrate tunnel analysis were used to understand the mechanism of improvement.
• Immobilization of the engineered enzyme onto UiO-66-NH₂ was performed to assess stability and production yield.

Main Results:

• The engineered DtBGL mutant M6 significantly increased kinsenoside yield from 17.90% (wild type) to 42.07%.
• Mutant M6 exhibited reduced hydrolytic specific activity (182 U/mg vs. 395 U/mg for wild type), indicating a shift towards glycosylation.
• Structural modifications, including a remodeled hydrophobic pocket and optimized catalytic tunnel, were identified as key factors for improved performance.
• Immobilization of M6 on UiO-66-NH₂ enhanced storage stability and further increased kinsenoside yield to 54.51%.

Conclusions:

• Enzyme engineering of DtBGL via semirational design effectively enhances kinsenoside production through reverse hydrolysis.
• The improved catalytic efficiency is attributed to structural alterations optimizing the enzyme's active site and substrate tunnel.
• Enzyme immobilization provides a viable strategy for improving enzyme stability and achieving high yields in sustainable kinsenoside biomanufacturing.