(335n) Diversity of Glycyrrhizin Biotransformation By â-Glucoronidases From Fungi

Authors: 
Li, C., Beijing Institute of Technology


                                                  Diversity of Glycyrrhizin Biotransformation by β-Glucoronidases from Fungi

Chun Li

School of Life Science, Beijing Institute of Technology, Beijing, 100081, P.R. China

Glycyrrhizin (GL), a triterpenoid saponin glycoside and the main constituent of licorice extract (Glycyrrhiza glabra), is a kind of natural edulcorant and sweeter as well as an important ingredient of traditional Chinese medicine. Generally GL can be hydrolyzed into β-D-mono-glucuronide- glycyrrhizin (GAMG) and glycyrrhetic acid, which have higher merits than GL due to its physiological and commercial values. In our previous work, the three β-glucuronidases from Penicillium purpurogenum Li-3, Aspergillus terreus Li-20 and Aspergillus ustus Li-62 could transform glycyrrhizin into GAMG, GAMG and GA, and GA, respectively. The three genes were cloned and also named pgus, atgus and augus, and not only their alignment results showed significant differences in their N-terminal and C-terminal, but the properties of these enzymes also showed obvious differences.

Wherein a novel β-glucuronidase from Penicillium purpurogenum (PGUS) could specially hydrolyze the distal β-D-glucuronic acid residue from glycyrrhizin into GAMG directly, but PGUS-E which pgus gene was expressed by E.coli BL21(DE3), PGUS-P expressed by Pichia pastoris and PGUS-C expressed by CHO cells could sequentially catalyze to release non-reducing terminal β-D-glucuronic acid residues from glycyrrhizin or GAMG into GA. So there was probably predicted a few difference of structures between PGUS and the others. Here we firstly reported the crystal structure of PGUS-E from fungus Penicillium purpurogenum. And the crystal structure of PGUS-E has been solved to 3.1 Ǻ in space group c2221. The model of PGUS-E from basically consisted of two dimer in the asymmetric unit which was composed of three domains including the (a/b)8 TIM-barrel motif, a sandwich domain and a typical carbohydrate recognition domain. Furthermore we confirmed that E414 and E505 were the key residues essentially affecting the enzyme activity by site-directed mutagenesis and enzymatic activity assays. These findings also could provide a solid foundation for developing substrate (aglycone) specificity of β-glucuronidases from glycoside hydrolase Family 2.

In addition, the strategies of GAMG production with high efficiency by different types of PGUS were systematically investigated and usefully developed in an ionic liquid/buffer biphasic system and in scale-up fermentation.