Advertisement

Applied Microbiology and Biotechnology

, Volume 102, Issue 21, pp 9193–9205 | Cite as

Characteristics and molecular determinants of a highly selective and efficient glycyrrhizin-hydrolyzing β-glucuronidase from Staphylococcus pasteuri 3I10

  • Bin Wei
  • Pan-Pan Wang
  • Zhi-Xiang Yan
  • Ru Yan
Biotechnologically relevant enzymes and proteins

Abstract

Glycyrrhizin (GL), the principal sweet-tasting bioactive ingredient of licorice (root of Glycyrrhiza glabra), shows poor oral absorption and gut microbial transformation of GL to glycyrrhetinic acid (GA) plays a major role for its multiple pharmacological effects. Co-administration of GL-hydrolyzing bacteria appears to be a feasible strategy to enhance GA exposure. This study reported a gut bacterial strain Staphylococcus pasteuri 3I10 which exhibited moderate p-nitrophenyl-β-D-glucuronide (PNPG)-hydrolyzing activity but low GL deglucuronidation activity in its crude lysate. The gus gene encoding S. pasteuri 3I10 β-glucuronidase was successfully cloned and overexpressed in Escherichia coli BL21(DE3). The purified β-glucuronidase (SpasGUS) was 71 kDa and showed optimal pH and temperature at 6.0 and 50 °C, respectively. Comparing to E. coli β-glucuronidase (EcoGUS), SpasGUS displayed lower velocity and affinity to PNPG hydrolysis (Vmax 16.1 ± 0.9 vs 140.0 ± 4.1 μmolmin−1 mg−1; Km 469.4 ± 73.4 vs 268.0 ± 25.8 μM), but could selectively convert GL to GA at much higher efficiency (Vmax 0.41 ± 0.011 vs 0.005 ± 0.002 μmolmin−1 mg−1; Km 116.9 ± 15.4 vs 53.4 ± 34.8 μM). Molecular docking studies suggested SpasGUS formed hydrogen bond interactions with the glucuronic acids at Asn414, Glu415 and Leu450, and Val159, Tyr475, Ala368, and Phe367 provided a hydrophobic environment for enhanced activity. Two special substrate interaction loops near the binding pocket of SpasGUS (loop 1 β-glucuronidase) may account for the selective and efficient bioconversion of GL to GA, predicting that loop 1 β-glucuronidases show high possibility in processing GL than mini-loop 1 and loop 2 β-glucuronidases. These findings support potential applications of SpasGUS in cleaving GL to facilitate GA production in vivo or in pharmaceutical industry.

Keywords

Staphylococcus pasteuri Bacterial β-glucuronidase Glycyrrhizin Glycyrrhetinic acid Deglucuronidation Homology modeling 

Notes

Funding information

This work was financially supported by the National Natural Science Foundation (Ref. no: 81473281), University of Macau (MYRG2015-00220-ICMS-QRCM)), and the Science and Technology Development fund of Macao SAR (043/2011/A2, 029/2015/A1).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors.

Supplementary material

253_2018_9285_MOESM1_ESM.pdf (1.2 mb)
ESM 1 (PDF 1275 kb)

References

  1. Akao T (2000) Hasty effect on the metabolism of glycyrrhizin by Eubacterium sp. GLH with Ruminococcus sp. PO1-3 and Clostridium innocuum ES24-06 of human intestinal bacteria. Biol Pharm Bull 23(1):6–11CrossRefGoogle Scholar
  2. Akao T, Akao T, Kobashi K (1987) Glycyrrhizin β-D-glucuronidase of Eubacterium sp. from human intestinal flora. Chem Pharm Bull 35(2):705–710CrossRefGoogle Scholar
  3. Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ (1990) Basic local alignment search tool. J Mol Biol 215(3):403–410CrossRefGoogle Scholar
  4. Baltina LA (2003) Chemical modification of glycyrrhizic acid as a route to new bioactive compounds for medicine. Curr Med Chem 10:155–171CrossRefGoogle Scholar
  5. Chubachi A, Wakui H, Asakura K, Nishimura S, Nakamoto Y, Miura AB (1992) Acute renal failure following hypokalemic rhabdomyolysis due to chronic glycyrrhizic acid administration. Intern Med 31:708–711CrossRefGoogle Scholar
  6. Eichenbaum G, Hsu CP, Subrahmanyam V, Chen J, Scicinski J, Galemmo JR, Tuman RW, Johnson DL (2012) Oral coadministration of β-glucuronidase to increase exposure of extensively glucuronidated drugs that undergo enterohepatic recirculation. J Pharm Sci 101(7):2545–2556CrossRefGoogle Scholar
  7. Guerreiro LR, Carreiro EP, Fernandes L, Cardote TA, Moreira R, Caldeira AT, Guedes RC, Burke AJ (2013) Five-membered iminocyclitol α-glucosidase inhibitors: synthetic, biological screening and in silico studies. Bioorg Med Chem 21(7):1911–1917CrossRefGoogle Scholar
  8. Hattori M, Sakamoto T, Kobashi K, Namba T (1983) Metabolism of glycyrrhizin by human intestinal flora. Planta Med 48(05):38–42CrossRefGoogle Scholar
  9. Ho CY, Ludovici DW, Maharoof US, Mei J, Sechler JL, Tuman RW, Strobel ED, Andraka L, Yen HK, Leo G, Li J, Almond H, Lu H, DeVine A, Tominovich RM, Baker J, Emanuel S, Gruninger RH, Middleton SA, Johnson DL, Galemmo RA (2005) (6, 7-Dimethoxy-2, 4-dihydroindeno [1, 2-c] pyrazol-3-yl) phenylamines: platelet-derived growth factor receptor tyrosine kinase inhibitors with broad antiproliferative activity against tumor cells. J Med Chem 48(26):8163–8173CrossRefGoogle Scholar
  10. Isbrucker RA, Burdock GA (2006) 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. Regul Toxicol Pharmacol 46(3):167–192CrossRefGoogle Scholar
  11. Islam MR, Grubb JH, Sly WS (1993) C-terminal processing of human beta-glucuronidase. The propeptide is required for full expression of catalytic activity, intracellular retention, and proper phosphorylation. J Biol Chem 268(30):22627–22633PubMedGoogle Scholar
  12. Khan KM, Rahim F, Wadood A, Kosar N, Taha M, Lalani S, Khan A, Fakhri MI, Junaid M, Rehman W, Khan M, Perveen S, Sajid M, Choudhary M (2014) Synthesis and molecular docking studies of potent α-glucosidase inhibitors based on biscoumarin skeleton. Eur J Med Chem 81:245–252CrossRefGoogle Scholar
  13. Kim DH, Hong SW, Kim BT, Bae EA, Park HY, Han MJ (2000) Biotransformation of glycyrrhizin by human intestinal bacteria and its relation to biological activities. Arch Pharm Res 23(2):172–177CrossRefGoogle Scholar
  14. Kim HS, Kim JY, Park MS, Zheng H, Ji GE (2009) Cloning and expression of beta-glucuronidase from Lactobacillus brevis in E. coli and application in the bioconversion of baicalin and wogonoside. J Microbiol Biotechnol 19(12):1650–1655CrossRefGoogle Scholar
  15. Koppel N, Rekdal VM, Balskus EP (2017) Chemical transformation of xenobiotics by the human gut microbiota. Science 356(6344), eaag2770CrossRefGoogle Scholar
  16. Li JY, Cao HY, Liu P, Cheng GH, Sun MY (2014) Glycyrrhizic acid in the treatment of liver diseases: literature review. BioMed Res Int 2014Google Scholar
  17. Liu Y, Huangfu J, Qi F, Kaleem I, Wenwen E, Li C (2012) Effects of a non-conservative sequence on the properties of β-glucuronidase from Aspergillus terreus Li-20. PLoS One 7(2):e30998CrossRefGoogle Scholar
  18. LoGuidice A, Wallace BD, Bendel L, Redinbo MR, Boelsterli UA (2012) Pharmacologic targeting of bacterial β-glucuronidase alleviates nonsteroidal anti-inflammatory drug-induced enteropathy in mice. J Pharmacol Exp Ther 341:447–454CrossRefGoogle Scholar
  19. Lu DQ, Li H, Dai Y, Ouyang PK (2006) Biocatalytic properties of a novel crude glycyrrhizin hydrolase from the liver of the domestic duck. J Mol Catal B Enzym 43(1):148–152CrossRefGoogle Scholar
  20. McIntosh FM, Maison N, Holtrop G, Young P, Stevens VJ, Ince J, Johnstone AM, Lobley GE, Flint HJ, Louis P (2012) Phylogenetic distribution of genes encoding β-glucuronidase activity in human colonic bacteria and the impact of diet on faecal glycosidase activities. Environ Microbiol 14(8):1876–1887CrossRefGoogle Scholar
  21. Nakano N, Kato H, Suzuki H, Nakano N, Yano S, Kanaoka M (1980) Enzyme immunoassay of glycyrrhetinic acid and glycrrhizin II. Measurement of glycyrrhetinic acid and glycyrrhizin in serum. Jpn Pharmacol Ther 8:4171–4174Google Scholar
  22. Nose M, Ito M, Kamimura K, Shimizu M, Ogihara Y (1994) A comparison of the antihepatotoxic activity between glycyrrhizin and glycyrrhetinic acid. Planta Med 60:136–139CrossRefGoogle Scholar
  23. Park HY, Kim NY, Myung JH, Bae EA, Kim DH (2005) Purification and characterization of two novel β-d-glucuronidases converting glycyrrhizin to 18β-glycyrrhetinic acid-3-O-β-d-glucuronide from Streptococcus LJ-22. J Microbiol Biotechnol 15(4):792–799Google Scholar
  24. Pollet RM, D’Agostino EH, Walton WG, Xu Y, Little MS, Biernat KA, Pellock SJ, Patterson LM, Creekmore BJ, Isenberg HN, Bahethi RR, Bhatt AP, Liu J, Gharaibeh RZ, Bahethi RR (2017) An atlas of β-glucuronidases in the human intestinal microbiome. Structure 25(7):967–977CrossRefGoogle Scholar
  25. Sakuma K, Kitahara M, Kibe R, Sakamoto M, Benno Y (2006) Clostridium glycyrrhizinilyticum sp.nov., a glycyrrhizin-hydrolysing bacterium isolated from human faeces. Microbiol Immunol 50(7):481–485CrossRefGoogle Scholar
  26. Sakurama H, Kishino S, Uchibori Y, Yonejima Y, Ashida H, Kita K, Takahashi S, Ogawa J (2014) β-Glucuronidase from Lactobacillus brevis useful for baicalin hydrolysis belongs to glycoside hydrolase family 30. Appl Microbiol Biotechnol 98(9):4021–4032CrossRefGoogle Scholar
  27. Sánchez E, Donat E, Ribes-Koninckx C, Fernández-Murga L, Sanz, Y (2013). Duodenal-mucosal bacteria associated with celiac disease in children. Appl Environ Microbiol AEM-00869Google Scholar
  28. Sasaki K, Taura F, Shoyama Y, Morimoto S (2000) Molecular characterization of a novel β-glucuronidase from Scutellaria baicalensis Georgi. J Biol Chem 275(35):27466–27472PubMedGoogle Scholar
  29. Savini V, Catavitello C, Bianco A, Balbinot A, D’antonio D (2009) Epidemiology, pathogenicity and emerging resistances in Staphylococcus pasteuri: from mammals and lampreys, to man. Recent Pat Anti-Infect Drug Discovery 4(2):123–129CrossRefGoogle Scholar
  30. Wallace BD, Redinbo MR (2013) The human microbiome is a source of therapeutic drug targets. Curr Opin Chem Biol 17(3):379–384CrossRefGoogle Scholar
  31. Wallace BD, Wang H, Lane KT, Scott JE, Orans J, Koo JS, Venkatesh M, Jobin C, Yeh L, MaNi S, Redinbo MR (2010) Alleviating cancer drug toxicity by inhibiting a bacterial enzyme. Science 330(6005):831–835CrossRefGoogle Scholar
  32. Wallace BD, Roberts AB, Pollet RM, Ingle JD, Biernat KA, Pellock SJ, Venkatesh MK, Guthrie L, O’Neal SK, Robinson SJ, Dollinger M, Figueroa E, McShane SR, Cohen RD, Jin J, Frye SV, Zamboni WC, Charles PR, Mani S, Kelly L, Redinbo MR (2015) Structure and inhibition of microbiome beta-glucuronidases essential to the alleviation of cancer drug toxicity. Chem Biol 22(9):1238–1249CrossRefGoogle Scholar
  33. Wang C, Guo XX, Wang XY, Qi F, Feng SJ, Li C, Zhou XH (2013) Isolation and characterization of three fungi with the potential of transforming glycyrrhizin. World J Microb Biotechnol 29(5):781–788CrossRefGoogle Scholar
  34. Wang X, Liu Y, Wang C, Feng X, Li C (2015) Properties and structures of β-glucuronidases with different transformation types of glycyrrhizin. RSC Adv 5(84):68345–68350CrossRefGoogle Scholar
  35. Yang W, Wei B, Yan R (2018) Amoxapine demonstrates incomplete inhibition of β-glucuronidase activity from human gut microbiota. SLAS Discovery 23(1):76–83PubMedGoogle Scholar
  36. Zhou Q, Dou T, Qu M, Weng Z, Wu D, Hou J (2017) Bioconversion of baicalin to baicalein with recombinant β-glucuronidase in Escherichia coli. J of Dalian Medical University 2, 002Google Scholar
  37. Zou S, Liu G, Kaleem I, Li C (2013) Purification and characterization of a highly selective glycyrrhizin-hydrolyzing β-glucuronidase from Penicillium purpurogenum Li-3. Process Biochem 48(2):358–363CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  1. 1.State Key Laboratory of Quality Research in Chinese Medicine, Institute of Chinese Medical SciencesUniversity of MacauTaipaChina
  2. 2.Zhuhai UM Science & Technology Research InstituteZhuhaiChina

Personalised recommendations