[Semi-rational modification for improving bond selectivity of recombinant β-glucuronidase]

Area of Science:

• Enzymology
• Protein Engineering
• Biocatalysis

Background:

• Recombinant β-glucuronidase (PGUS-E) is crucial for specific glycosidic bond hydrolysis.
• Improving the bond selectivity of PGUS-E is essential for its applications.
• Understanding the structure-function relationship is key to enzyme engineering.

Purpose of the Study:

• To enhance the bond selectivity of recombinant β-glucuronidase (PGUS-E) using structure-guided mutagenesis.
• To identify specific amino acid residues influencing PGUS-E's bond selectivity.
• To characterize the biochemical properties and activity of engineered PGUS-E variants.

Main Methods:

• Structure-guided identification of key residues (R329, T369, N467) for bond selectivity.
• Saturation mutagenesis at identified key sites.
• Combined selection using thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC).
• Biochemical characterization of enzyme activity, pH, and temperature profiles.

Main Results:

• Two positive mutants, R329K and T369V, were successfully generated.
• Mutant R329K showed a 26.9% increase in bond selectivity.
• Mutant T369V exhibited a 34.3% increase in bond selectivity.
• Biochemical properties (pH, temperature optima) remained unchanged, but activity decreased slightly.

Conclusions:

• Residues R329 and T369 are critical for determining the bond selectivity and activity of PGUS-E.
• Structure-guided saturation mutagenesis is an effective strategy for enhancing enzyme bond selectivity.
• This study provides valuable insights into the structure-function relationship of PGUS-E, supporting its biotechnological applications.