Enhancing the Catalytic Specificity of the β-Glucuronidase <i>At</i>GUS from <i>Aspergillus terreus</i> Li-20 by Site-Directed Mutagenesis on Loop 8

AtGUS, identified in the genome of Aspergillus terreus Li-20, can hydrolyze glycyrrhetinic (GL) into glycyrrhetinic acid monoglucuronide (GAMG) and glycyrrhetinic acid (GA). However, the poor substrate specificity of AtGUS often limits its further application. In this study, the highly conserved Glu416 and Glu507 residues were identified as the catalytic sites of AtGUS, while Glu158, Asp163, and Arg565, which form hydrogen bonds with the substrate, play critical roles in the enzymatic activity, with Arg565 being particularly important. When Arg565 on loop 8 of the surface of the TIM barrel was mutated to glutamate, GAMG became the sole catalytic product, and the yield of GAMG from GL conversion was up to 85%. When the hydrolysis reaction was terminated, the yield of GAMG was 7.32-fold higher than that of the wild enzyme. Molecular docking and dynamic simulation analyses revealed that the affinity for the mutant R565E (Km = 0.192 mmol/L) to the substrate GL was improved, but with reduced catalytic efficiency toward GAMG (kcat = 5.515 ± 0.07 mmol–1 L s–1). This study provides new insights into understanding the catalytic mechanism and new ideas for efficient enzymatic preparation of GAMG.