Enhancing the thermostability of Β-glucuronidase from T. pinophilus enables the biotransformation of glycyrrhizin at elevated temperature

Glycosidases have been widely used in natural products modification by precisely hydrolyzing sugar moieties to modulate the biological activities. However, the low stability of the glycosidases under harsh conditions and the low solubility of substrate have largely impeded the biotransformation efficiency. To solve this problem, in this study, we selected the biotransformation of glycyrrhizin (GL) into glycyrrhetinic acid 3-O-mono-β-D-glucuronide (GAMG) catalyzed by β-glucuronidase from T. pinophilus Li-93 (TpGUS79A-P) as a model reaction. We combined three strategies including random mutagenesis, computation-aided design and rational introduction of “clamp” to TIM-barrel domain to enhance the thermostability of TpGUS79A-P, and one mutant TpGUS79A-P4 was obtained with thermostability at 55 °C increased by 2.9-fold. The enzymatic properties of TpGUS79A-P4 were extensively characterized. Finally, a novel high-temperature biotransformation process of GL into GAMG catalyzed by TpGUS79A-P4 was established at 50 °C, with GAMG concentration increased by 84% than that at 30 °C.