Heterologous Expression, Enzymatic Characterization, and Application of β-Glucuronidase from the Thermophilic Archaeon Dictyoglomus

Abstract Background β-Glucuronidase can catalyze the hydrolysis of glycyrrhizic acid to produce high-value derivatives including glycyrrhetic acid monoglucuronide and glycyrrhetic acid, which possess broad application prospects. Nevertheless, the currently reported enzymes generally exhibit low activity, weak hydrolysis efficiency and poor thermostability, which greatly restrict their industrial applications. This study aimed to screen recombinant β-glucuronidase with high activity and high hydrolysis efficiency, and further explore its enzymatic characteristics, catalytic conversion performance on glycyrrhizic acid, and molecular mechanism. This research provides a theoretical basis for the efficient biological preparation of glycyrrhizic acid derivatives and the molecular modification of enzyme. Results Four recombinant expression strains of E. coli Rosetta (DE3)/pET-28a-GUS were constructed in this study to realize the heterologous expression and enzymatic property characterization of four β-glucuronidases. The results showed that the enzyme from E. coli Rosetta (DE3)/pET-28a-DthGUS possessed the highest activity of 123 U/µL, with optimal reaction conditions at pH 5.0 and 85°C. The enzyme tended to be inactivated under pH conditions lower than 4.5. K⁺, Zn²⁺, Cu²⁺ and Ca²⁺ were able to enhance enzyme activity, whereas Fe²⁺ and Fe³⁺ completely inactivated the enzyme. After 8 h of hydrolysis under optimal conditions, the hydrolysis rate of glycyrrhizic acid catalyzed by this enzyme reached 98.7%. Molecular dynamics simulation analysis revealed that Tyr153-mediated π-π stacking and amide-π interaction served as the core forces for DthGUS catalysis toward pNPG, and Trp351-mediated π-alkyl interaction acted as the key mechanism for its catalytic hydrolysis of glycyrrhizic acid. The total binding free energies of DthGUS with pNPG and glycyrrhizic acid were − 73.410 ± 12.935 kJ/mol and − 88.144 ± 20.512 kJ/mol, respectively, which confirmed its favorable binding capacity and catalytic hydrolysis ability for both substrates. Conclusion In conclusion, DthGUS presents prominent advantages of high enzymatic activity, excellent thermostability and efficient glycyrrhizic acid hydrolysis capacity, and can efficiently convert glycyrrhizic acid into target derivatives. This study clarified the core catalytic amino acid residues and molecular mechanism of DthGUS, providing theoretical support and modification targets for the biological preparation of glycyrrhizic acid derivatives and the directed molecular modification of DthGUS.