A novel and thermostable β-glucuronidase from Thermobacillus composti precisely hydrolyzes glycyrrhizin into glycyrrhetinic acid

β-Glucuronidase (GUS) catalyzes the hydrolysis of the glucuronide group in glycyrrhizin (GL) to produce glycyrrhetinic acid (GA). However, the industrial application of β-glucuronidases (GUSs) is often limited by their poor stability, low hydrolytic activity, and particularly insufficient substrate specificity. In this work, a novel and thermostable β-glucuronidase from Thermobacillus composti ( Tc GUS) was identified through gene mining. Evolutionary relationship and sequence analyses revealed that Tc GUS belongs to the family of typical GUS enzymes. Tc GUS demonstrated specificity and efficiently transformed GL to generate GA, exhibiting maximum activity at 35 °C and pH 6.5, with a half-life of 5 days at 50 °C, indicating excellent thermostability. The K m and V max values of Tc GUS were 3.20 mM and 151.18 μM·mg⁻¹·min⁻¹, respectively, reflecting its high substrate selectivity and biotransformation efficiency. Therefore, Tc GUS is a promising biocatalyst for the synthesis of glycyrrhetinic acid.