(583au) Directed Evolution of â-Glucuronidase for Improved Substrate Specificity in Glycyrrhizin Transformation

A noval β-glucuronidase from Penicillium purpurogenum (PGUS) could catalyze glycosidic bond to release non-reducing terminal β-glucuronic acid residues from glycyrrhizin (GL) into b-mono-glucuronide-glycyrrhizin (GAMG) directly. Previously, we cloned the gene pgus and overexpressed it in Escherichia coli BL21 for high production of β-glucuronidase(PGUS-E). Although a large amount of PGUS-E was produced, the substrate specificity was much lower than that of the corresponding wild strain, the main product was Glycyrrhetinic Acid (GA).

In order to Study Substrate binding specificity and catalysis of glycyrrhizin (GL) transformation, here the PGUS-E was subjected to three rounds of direction evolution. Random mutagenesis and screening were used to search for β-glucuronidase with substrate-specificity (Scb) of GAMG production. In addition, λRed recombinant was applied to knockout the uidA gene  from the E. coli origami (DE3) for reduce the background of GAMG transformation.

Different concentrations of MnCl₂ were tested to estimate the appropriate mutation rate. A mutagenic library(`3000 clones ) was constructed by error-prone PCR.  Pgus mutant A8 and 51 were screened, the substrate-specificity (S_cb) of GAMG production were improved to 41.4% and 51.2%, respectively. Structural and functional of wild-type , A8 and 51 enzymes were finished, it reveal that the key mutant sites were located on sugar combining domain, some acidic amino acid were replaced by neutral amino acids. We predict that sugar combining domain has a significant influence of the substrate specificity of these enzymes, but further study is necessary to support our conclusion.