Enhanced production of β-glucuronidase from Penicillium purpurogenum Li-3 by optimizing fermentation and downstream processes

Abstract

β-Glucuronidase from Penicillium purpurogenum Li-3 (PGUS) can efficiently hydrolyze glycyrrhizin into the more valuable glycyrrhetic acid monoglucuronide. However, a low productivity of PGUS and the lack of an effective separation strategy have significantly limited its industrial applications. Therefore, the production of PGUS has been improved by optimizing both the fermentation and purification strategies. A two-stage fermentation strategy was developed where PGUS was first grown with glucose and then PGUS was produced in the presence of glycyrrhizin as an inducer. By using this strategy, the biomass was increased 1.5 times and the PGUS activity increased 5.4 times compared to that when glycyrrhizin was used as the sole carbon source. The amount of PGUS produced was increased another 16.6% when the fermentation was expanded to a 15-L fermenter. An effective protocol was also established to purify the PGUS using a sequential combination of hydrophobic, strong anion-exchange and gel filtration chromatography. This protocol had a recovery yield of 6% and gave PGUS that was 39 times purer than the crude PGUS. The purified PGUS had a specific activity of 350 U·mg⁻¹.

Graphical abstract

Keywords

β-glucuronidase / glycyrrhetic acid monoglucuronide / cell disruption / purification / chromatography

References

[1] Makino T, Okajima K, Uebayashi R, Ohtake N, Inoue K, Mizukami H. 3-Monoglucuronyl-glycyrrhretinic acid is a substrate of organic anion transporters expressed in tubular epithelial cells and plays important roles in licorice-induced pseudoaldosteronism by inhibiting 11 beta-hydroxysteroid dehydrogenase 2. Journal of Pharmacology and Experimental Therapeutics, 2012, 342(2): 297–304

[2] Seki H, Ohyama K, Sawai S, Mizutani M, Ohnishi T, Sudo H, Akashi T, Aoki T, Saito K, Muranaka T. Licorice beta-amyrin 11-oxidase, a cytochrome P450 with a key role in the biosynthesis of the triterpene sweetener glycyrrhizin. Proceedings of the National Academy of Sciences of the United States of America, 2008, 105(37): 14204–14209

[3] Akao T. Differences in the metabolism of glycyrrhizin, glycyrrhetic acid and glycyrrhetic acid monoglucuronide by human intestinal flora. Biological & Pharmaceutical Bulletin, 2000, 23(12): 1418–1423

[4] Matsui S, Matsumoto H, Sonoda Y, Ando K, Aizu-Yokota E, Sato T, Kasahara T. Glycyrrhizin and related compounds down-regulate production of inflammatory chemokines IL-8 and eotaxin 1 in a human lung fibroblast cell line. International Immunopharmacology, 2004, 4(13): 1633–1644

[5] Doll R, Hill I D, Hutton C, Underwood D J V II. Clinical trial of a triterpenoid liquorice compound in gastric and duodenal ulcer. Lancet, 1962, 280(7260): 793–796

[6] Pompei R, Flore O, Marccialis M A, Pani A, Loddo B. Glycyrrhizic acid inhibits virus growth and inactivates virus-particles. Nature, 1979, 281(5733): 689–690

[7] Shiota G, Harada K, Ishida M, Tomie Y, Okubo M, Katayama S, Ito H, Kawasaki H. Inhibition of hepatocellular carcinoma by glycyrrhizin in diethylnitrosamine-treated mice. Carcinogenesis, 1999, 20(1): 59–63

[8] Isbrucker R A, Burdock G A. Risk and safety assessment on the consumption of Licorice root (Glycyrrhiza sp.), its extract and powder as a food ingredient, with emphasis on the pharmacology and toxicology of glycyrrhizin. Regulatory Toxicology and Pharmacology, 2006, 46(3): 167–192

[9] Feng S J, Li C, Xu X L, Wang X Y. Screening strains for directed biosynthesis of beta-D-mono-glucuronide-glycyrrhizin and kinetics of enzyme production. Journal of Molecular Catalysis. B, Enzymatic, 2006, 43(1−4): 63–67

[10] Zou S P, Zhou J J, Kaleem I, Xie L P, Liu G Y, Li C. Preparative enrichment and separation of glycyrrhetinic acid monoglucuronide from fermentation broths with macroporous resins. Separation Science and Technology, 2012, 47(7): 1055–1062

[11] Zou S P, Liu G Y, Kaleem I, Li C. Purification and characterization of a highly selective glycyrrhizin-hydrolyzing beta-glucuronidase from Penicillium purpurogenum Li-3. Process Biochemistry, 2013, 48(2): 358–363

[12] Qi F, Kaleem I, Lv B, Guo X, Li C. Enhancement of recombinant beta-D-glucuronidase production under low-shear modeled microgravity in Pichia pastoris. Journal of Chemical Technology and Biotechnology (Oxford, Oxfordshire), 2011, 86(4): 505–511

[13] Mizutani K, Kuramoto T, Tamura Y, Ohtake N, Doi S, Nakaura M, Tanaka O. Sweetness of glycyrrhetic acid 3-O-beta-D-monoglucuronide and the related glycosides. Bioscience, Biotechnology, and Biochemistry, 1994, 58(3): 554–555

[14] Park H Y, Park S H, Yoon H K, Han M J, Kim D H. Anti-allergic activity of 18 beta-glycyrrhetinic acid-3-O-beta-D-glucuronide. Archives of Pharmacal Research, 2004, 27(1): 57–60

[15] Maitraie D, Hung C F, Tu H Y, Liou Y T, Wei B L, Yang S C, Wang J P, Lin C N. Synthesis, anti-inflammatory, and antioxidant activities of 18 beta-glycyrrhetinic acid derivatives as chemical mediators and xanthine oxidase inhibitors. Bioorganic & Medicinal Chemistry, 2009, 17(7): 2785–2792

[16] Zou S, Guo S, Kaleem I, Li C. Purification, characterization and comparison of Penicillium purpurogenum beta-glucuronidases expressed in Escherichia coli and Pichia pastoris. Journal of Chemical Technology and Biotechnology (Oxford, Oxfordshire), 2013, 88(10): 1913–1919

[17] Song X, Jiang Z, Li L, Wu H. Immobilization of β-glucuronidase in lysozyme-induced biosilica particles to improve its stability. Frontiers of Chemical Science and Engineering, 2014, 8(3): 353–361

[18] Kim D H, Jin Y H, Jung E A, Han M J, Kobashi K. Purification and characterization of beta-glucuronidase from Escherichia coli HGU-3, a human intestinal bacterium. Biological & Pharmaceutical Bulletin, 1995, 18(9): 1184–1188

[19] Akao T. Competition in the metabolism of glycyrrhizin with glycyrrhetic acid mono-glucuronide by mixed Eubacterium sp GLH and Ruminococcus sp PO1-3. Biological & Pharmaceutical Bulletin, 2000, 23(2): 149–154

[20] Amin H A S, El-Menoufy H A, El-Mehalawy A A, Mostafa E S. Biosynthesis of glycyrrhetinic acid 3-O-mono-beta-D-glucuronide by free and immobilized Aspergillus terreus beta-D-glucuronidase. Journal of Molecular Catalysis. B, Enzymatic, 2011, 69(1-2): 54–59

[21] Kuramoto T, Ito Y, Oda M, Tamura Y, Kitahata S. Microbial-production of glycyrrhetic acid 3-O-mono-beta-D-glucuronide from glycyrrhizin by Cryptococcus-magnus MG-27. Bioscience, Biotechnology, and Biochemistry, 1994, 58(3): 455–458

[22] Lu D Q, Li H, Dai Y, Ouyang P K. Biocatalytic properties of a novel crude glycyrrhizin hydrolase from the liver of the domestic duck. Journal of Molecular Catalysis. B, Enzymatic, 2006, 43(1−4): 148–152

[23] Bradford M M. Rapid and sensitive method for quantitation of microgram quantities of protein utilizing principle of protein-dye binding. Analytical Biochemistry, 1976, 72(1−2): 248–254

[24] Papagianni M. Fungal morphology and metabolite production in submerged mycelial processes. Biotechnology Advances, 2004, 22(3): 189–259

[25] Posch A E, Herwig C, Spadiut O. Science-based bioprocess design for filamentous fungi. Trends in Biotechnology, 2013, 31(1): 37–44

[26] Choonia H S, Saptarshi S D, Lele S S. Release of intracellular beta-galactosidase from Lactobacillus acidophilus and L-asparaginase from Pectobacterium carotovorum by high-pressure homogenization. Chemical Engineering Communications, 2013, 200(11): 1415–1424

[27] Liu Y, Pietzsch M, Ulrich J. Purification of L-asparaginase II by crystallization. Frontiers of Chemical Science and Engineering, 2013, 7(1): 37–42

[28] Park H Y, Kim N Y, Han M J, Bae E A, Kim D H. Purification and characterization of two novel beta-D-glucuronidases converting glycyrrhizin to 18 beta-glycyrrhetinic acid-3-O-beta-D-glucuronide from Streptococcus LJ-22. Journal of Microbiology and Biotechnology, 2005, 15(4): 792–799

[29] Kuroyama H, Tsutsui N, Hashimoto Y, Tsumuraya Y. Purification and characterization of a beta-glucuronidase from Aspergillus niger. Carbohydrate Research, 2001, 333(1): 27–39

[30] Sakurama H, Kishino S, Uchibori Y, Yonejima Y, Ashida H, Kita K, Takahashi S, Ogawa J. beta-Glucuronidase from Lactobacillus brevis useful for baicalin hydrolysis belongs to glycoside hydrolase family 30. Applied Microbiology and Biotechnology, 2014, 98(9): 4021–4032

[31] Nguyen Q D, Rezessy-Szabo J M, Bhat M K, Hoschke A. Purification and some properties of beta-fructofuranosidase from Aspergillus niger IMI303386. Process Biochemistry, 2005, 40(7): 2461–2466

Acknowledgments

This work was financially supported by the National Science Foundation of China (Grant. Nos. 21506011 and 21425624), and China Postdoctoral Science Foundation funded project (No. 2015M570038).

RIGHTS & PERMISSIONS

2014 Higher Education Press and Springer-Verlag Berlin Heidelberg