Catalysis Diversity Of β-D-glucuronidase And Cloning And Expression Of The Enzyme Directed Biosynthesis Of GAMG

Three fungi with the different type of biotransformation of glycyrrhizin were determined by analyzing the products of metabolism, and were identified by morphological observation and gene homology analysis. The catalytic characteristic and kinetics ofβ-D-glucuronidase from the three fungi were also investigated.β-D-glucuronidase from Penicillium sp. Li-3 which could directly biosynthesize Glycyrrhetinic acid monoglucuronide (GAMG) from glycyrrhizin was cloned and expressed. The results showed as follows:According to morphology, phylogenetic analysis and 18SrDNA sequence homology, the results demonstrated that three Fungi were identified as Penicillium purpurogenum Li-3, Aspergillus terreus Li-20 and Aspergillus ustus Li-62. Penicillium purpurogenum Li-3 converted glycyrrhizin to GAMG, Aspergillus terreus Li-20 converted glycyrrhizin to GAMG and GA, and Aspergillus ustus Li-62 converted glycyrrhizin to GA, respectively.Research about the catalytic characteristic and kinetics ofβ-D-glucuronidase from three fungi showed that the optimal reaction temperature ofβ-D-glucuronidase from Penicillium purpurogenum Li-3, Aspergillus terreus Li-20 and Aspergillus ustus Li-62 was 50℃, 45℃and 55℃, respectively. The optimal pH for the catalytic reaction ofβ-D-glucuronidases was 4.2, 5.8 and 6.2, respectively. The Km ofβ-D-glucuronidase from these strains was 0.328μmol·L-1, 3.61 mmol·L–1 and 0.43 mmol·L–1, and the Vmax was 0.0035 mmol·L-1·min-1, 0.034 mmol·L-1·min-1 and 0.106 mmol·L-1·min-1, respectively. For the production of the enzymes, the time of formation was about 48h, 24h and 72h, respectively.Catabolite repression was found when Penicillium purpurogenum Li-3 grew in medium including other carbon sources except of glycyrrhizin, and the inducing strategy ofβ-D-glucuronidase was studied. The results indicated that the enzyme activity was enhanced from 646U/mL to 2356U/mL by adding 20% inducer (1.2% Tween80 and 10% glycyrrhizin) when glucose was completely consumed in the medium including 5g/L of glucose.The genes ofβ-D-glucuronidase (Pgus) were triumphantly cloned by PCR from the genome DNA of Penicillium purpurogenum Li-3. The sequence analysis indicated that the gene of Pgus has 1815 base pairs and encodes 604 amino acids, the molecular weight of subunit was 67.6KDa. The expression plasmids of pET-28a(+)-Pgus and pPIC9K-Pgus were conceived, and then transformed into E.coli BL21(DE3) and Pichia pastoris GS115, respectively. The inclusion body was formed after induced in E.coli BL21(DE3), and the H is+ M u ts- Pichia pastoris with 6~10 gene copy were successfully screened in Pichia pastoris GS115.