A novel β- glucuronidase from Talaromyces pinophilus Li-93 precisely hydrolyzes glycyrrhizin into glycyrrhetinic acid 3-O-mono-β-D-glucuronide

Glycyrrhetinic acid 3-O-mono-β-D-glucuronide (GAMG), which possesses a higher sweetness and stronger pharmacological activity than those of glycyrrhizin (GL), can be obtained by removal of the distal glucuronic acid (GlcA) from GL. In this study, we isolated a β-glucuronidase (TpGUS79A) from the filamentous fungus Talaromyces pinophilus Li-93 that can specifically and precisely convert GL to GAMG without the formation of the by-product glycyrrhetinic acid (GA) from the further hydrolysis of GAMG. First, TpGUS79A was purified and identified through matrixassisted laser desorption ionization-tandem time of flight mass spectrometry (MALDI-TOF-TOF MS) and deglycosylation, indicating that TpGUS79A is a highly N-glycosylated monomeric protein with a molecular mass of around 85 kDa, including around 25 kDa of glycan moiety. The gene for TpGUS79A was then cloned and verified by heterologous expression in Pichia pastoris. TpGUS79A belonged to glycoside hydrolase family 79 (GH79) but shared low amino acid sequence identity ( < 35%) with the available GH79 GUS enzymes. TpGUS79A had strict specificity toward the glycan moiety but poor specificity toward the aglycone moiety. Interestingly, TpGUS79A recognized and hydrolyzed the distal glucuronic bond of GL but could not cleave the glucuronic bond in GAMG. TpGUS79A showed a much higher catalytic efficiency on GL (k_cat/K_m of 11.14 mM^-1 s^-1) than on the artificial substrate pNP β-glucopyranosiduronic acid (k_cat/K_m of 0.01 mM^-1 s^-1), which is different from the case for most GUSs. Homology modeling, substrate docking, and sequence alignment were employed to identify the key residues for substrate recognition. Finally, a fed-batch fermentation in a 150-liter fermentor was established to prepare GAMG through GL hydrolysis by T. pinophilus Li-93. Therefore, TpGUS79A is potentially a powerful biocatalyst for environmentally friendly and cost-effective production of GAMG.