Skip to main content
SpringerLink
Log in

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

  • Biotechnologically relevant enzymes and proteins
  • Published:
Applied Microbiology and Biotechnology Aims and scope Submit manuscript

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.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

References

  • 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):6–11

    Article  CAS  Google Scholar 

  • Akao T, Akao T, Kobashi K (1987) Glycyrrhizin β-D-glucuronidase of Eubacterium sp. from human intestinal flora. Chem Pharm Bull 35(2):705–710

    Article  CAS  Google Scholar 

  • Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ (1990) Basic local alignment search tool. J Mol Biol 215(3):403–410

    Article  CAS  Google Scholar 

  • Baltina LA (2003) Chemical modification of glycyrrhizic acid as a route to new bioactive compounds for medicine. Curr Med Chem 10:155–171

    Article  CAS  Google Scholar 

  • 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 31:708–711

    Article  CAS  Google Scholar 

  • 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):2545–2556

    Article  CAS  Google Scholar 

  • 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):1911–1917

    Article  CAS  Google Scholar 

  • Hattori M, Sakamoto T, Kobashi K, Namba T (1983) Metabolism of glycyrrhizin by human intestinal flora. Planta Med 48(05):38–42

    Article  CAS  Google Scholar 

  • 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):8163–8173

    Article  CAS  Google Scholar 

  • 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):167–192

    Article  CAS  Google Scholar 

  • 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):22627–22633

    CAS  PubMed  Google Scholar 

  • 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 81:245–252

    Article  CAS  Google Scholar 

  • 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):172–177

    Article  CAS  Google Scholar 

  • 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):1650–1655

    Article  CAS  Google Scholar 

  • Koppel N, Rekdal VM, Balskus EP (2017) Chemical transformation of xenobiotics by the human gut microbiota. Science 356(6344), eaag2770

    Article  Google Scholar 

  • Li JY, Cao HY, Liu P, Cheng GH, Sun MY (2014) Glycyrrhizic acid in the treatment of liver diseases: literature review. BioMed Res Int 2014

  • 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):e30998

    Article  CAS  Google Scholar 

  • 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 341:447–454

    Article  CAS  Google Scholar 

  • 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):148–152

    Article  CAS  Google Scholar 

  • 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):1876–1887

    Article  CAS  Google Scholar 

  • 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 8:4171–4174

    CAS  Google Scholar 

  • Nose M, Ito M, Kamimura K, Shimizu M, Ogihara Y (1994) A comparison of the antihepatotoxic activity between glycyrrhizin and glycyrrhetinic acid. Planta Med 60:136–139

    Article  CAS  Google Scholar 

  • 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):792–799

    CAS  Google Scholar 

  • 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):967–977

    Article  CAS  Google Scholar 

  • 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):481–485

    Article  CAS  Google Scholar 

  • 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):4021–4032

    Article  CAS  Google Scholar 

  • 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-00869

  • 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):27466–27472

    CAS  PubMed  Google Scholar 

  • 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):123–129

    Article  CAS  Google Scholar 

  • Wallace BD, Redinbo MR (2013) The human microbiome is a source of therapeutic drug targets. Curr Opin Chem Biol 17(3):379–384

    Article  CAS  Google Scholar 

  • 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):831–835

    Article  CAS  Google Scholar 

  • 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):1238–1249

    Article  CAS  Google Scholar 

  • 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):781–788

    Article  Google Scholar 

  • 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):68345–68350

    Article  CAS  Google Scholar 

  • Yang W, Wei B, Yan R (2018) Amoxapine demonstrates incomplete inhibition of β-glucuronidase activity from human gut microbiota. SLAS Discovery 23(1):76–83

    CAS  PubMed  Google Scholar 

  • 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, 002

  • 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):358–363

    Article  CAS  Google Scholar 

Download references

Funding

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).

Author information

Authors and Affiliations

  1. State Key Laboratory of Quality Research in Chinese Medicine, Institute of Chinese Medical Sciences, University of Macau, Taipa, Macao, China

    Bin Wei, Pan-Pan Wang, Zhi-Xiang Yan & Ru Yan

  2. Zhuhai UM Science & Technology Research Institute, Zhuhai, 519080, China

    Ru Yan

Authors
  1. Bin Wei
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Pan-Pan Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Zhi-Xiang Yan
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Ru Yan
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Ru Yan.

Ethics declarations

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.

Electronic supplementary material

ESM 1

(PDF 1275 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Wei, B., Wang, PP., Yan, ZX. et al. Characteristics and molecular determinants of a highly selective and efficient glycyrrhizin-hydrolyzing β-glucuronidase from Staphylococcus pasteuri 3I10. Appl Microbiol Biotechnol 102, 9193–9205 (2018). https://doi.org/10.1007/s00253-018-9285-x

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00253-018-9285-x

Keywords

Search

Navigation