Applied Microbiology and Biotechnology

, Volume 98, Issue 9, pp 4021–4032 | Cite as

β-Glucuronidase from Lactobacillus brevis useful for baicalin hydrolysis belongs to glycoside hydrolase family 30

  • Haruko Sakurama
  • Shigenobu Kishino
  • Yoshie Uchibori
  • Yasunori Yonejima
  • Hisashi Ashida
  • Keiko Kita
  • Satomi Takahashi
  • Jun OgawaEmail author
Biotechnologically relevant enzymes and proteins


Baicalin (baicalein 7-O-β-d-glucuronide) is one of the major flavonoid glucuronides found in traditional herbal medicines. Because its aglycone, baicalein, is absorbed more quickly and shows more effective properties than baicalin, the conversion of baicalin into baicalein by β-glucuronidase (GUS) has drawn the attention of researchers. Recently, we have found that Lactobacillus brevis subsp. coagulans can convert baicalin to baicalein. Therefore, we aimed to identify and characterize the converting enzyme from L. brevis subsp. coagulans. First, we purified this enzyme from the cell-free extracts of L. brevis subsp. coagulans and cloned its gene. Surprisingly, this enzyme was found to be a GUS belonging to glycoside hydrolase (GH) family 30 (designated as LcGUS30), and its amino acid sequence has little similarity with any GUS belonging to GH families 1, 2, and 79 that have been reported so far. We then established a high-level expression and simple purification system of the recombinant LcGUS30 in Escherichia coli. The detailed analysis of the substrate specificity revealed that LcGUS30 has strict specificity toward glycon but not toward aglycones. Interestingly, LcGUS30 prefers baicalin rather than estrone 3-(β-d-glucuronide), one of the human endogenous steroid hormones. These results indicated that L. brevis subsp. coagulans and LcGUS30 should serve as powerful tools for the construction of a safe bioconversion system for baicalin. In addition, we propose that this novel type of GUS forms a new group in subfamily 3 of GH family 30.


Flavonoid glucuronides Baicalin β-Glucuronidase (EC Glycoside hydrolase family 30 Lactic acid bacteria Lactobacillus brevis subsp. coagulans 


  1. Adlercreutz H, Martin F, Pulkkinen M, Dencker H, Rimér U, Sjöberg NO, Tikkanen MJ (1976) Intestinal metabolism of estrogens. J Clin Endocrinol Metab 43:497–505. doi: 10.1210/jcem-43-3-497 PubMedCrossRefGoogle Scholar
  2. Adlercreutz H, Martin F, Järvenpää P, Fotsis T (1979) Steroid absorption and enterohepatic recycling. Contraception 20:201–223. doi: 10.1016/0010-7824(79)90094-5 PubMedCrossRefGoogle Scholar
  3. Altschul SF, Madden TL, Schäffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25:3389–3402. doi: 10.1093/nar/25.17.3389 PubMedCentralPubMedCrossRefGoogle Scholar
  4. Aspeborg H, Coutinho PM, Wang Y, Brumer H 3rd, Henrissat B (2012) Evolution, substrate specificity and subfamily classification of glycoside hydrolase family 5 (GH5). BMC Evol Biol 12:186. doi: 10.1186/1471-2148-12-186 PubMedCentralPubMedCrossRefGoogle Scholar
  5. Bowers LD, Johnson PR (1981) Characterization of immobilized β-glucuronidase in aqueous and mixed solvent systems. Biochim Biophys Acta 661:100–105. doi: 10.1016/0005-2744(81)90087-5 CrossRefGoogle Scholar
  6. Brumshtein B, Greenblatt HM, Butters TD, Shaaltiel Y, Aviezer D, Silman I, Futerman AH, Sussman JL (2007) Crystal structures of complexes of N-butyl- and N-nonyl-deoxynojirimycin bound to acid β-glucosidase: insights into the mechanism of chemical chaperone action in Gaucher disease. J Biol Chem 282:29052–29058. doi: 10.1074/jbc.M705005200 PubMedCrossRefGoogle Scholar
  7. Cantarel BL, Coutinho PM, Rancurel C, Bernard T, Lombard V, Henrissat B (2009) The Carbohydrate-Active EnZymes database (CAZy): an expert resource for glycogenomics. Nucleic Acids Res 37:D233–D238. doi: 10.1093/nar/gkn663 PubMedCentralPubMedCrossRefGoogle Scholar
  8. Cha SK, Ortega B, Kurosu H, Rosenblatt KP, Kuro-O M, Huang CL (2008) Removal of sialic acid involving Klotho causes cell-surface retention of TRPV5 channel via binding to galectin-1. Proc Natl Acad Sci U S A 105:9805–9810. doi: 10.1073/pnas.0803223105 PubMedCentralPubMedCrossRefGoogle Scholar
  9. Finn RD, Mistry J, Tate J, Coggill P, Heger A, Pollington JE, Gavin OL, Gunesekaran P, Ceric G, Forslund K, Holm L, Sonnhammer EL, Eddy SR, Bateman A (2010) The Pfam protein families database. Nucleic Acids Res 38:D211–D222. doi: 10.1093/nar/gkr1065 PubMedCentralPubMedCrossRefGoogle Scholar
  10. Graef V, Furuya E, Nishikaze O (1977) Hydrolysis of steroid glucuronides with beta-glucuronidase preparations from bovine liver, Helix pomatia, and E. coli. Clin Chem 23:532–535PubMedGoogle Scholar
  11. Ikemoto S, Sugimura K, Yoshida N, Yasumoto R, Wada S, Yamamoto K, Kishimoto T (2000) Antitumor effects of Scutellariae radix and its components baicalein, baicalin, and wogonin on bladder cancer cell lines. Urology 55:951–955. doi: 10.1016/S0090-4295(00)00467-2 PubMedCrossRefGoogle Scholar
  12. Inoue H, Nojima H, Okayama H (1990) High efficiently transformation of Escherichia coli with plasmids. Gene 96:23–28. doi: 10.1016/0378-1119(90)90336-P PubMedCrossRefGoogle Scholar
  13. Kim HS, Kim JY, Park MS, Zheng H, Ji GE (2009) Cloning and expression of β-glucuronidase from Lactobacillus brevis in E. coli and application in bioconversion of baicalin and wogonoside. J Microb Biotechnol 19:1650–1655CrossRefGoogle Scholar
  14. Kishi A, Uno K, Matsubara Y, Okuda C, Kishida T (1996) Effect of the oral administration of Lactobacillus brevis subsp. coagulans on interferon-alpha producing capacity in humans. J Am Coll Nutr 15:408–412PubMedCrossRefGoogle Scholar
  15. Kobashi K, Akao T (1997) Relation of intestinal bacteria to pharmacological effect of glycosides. Biosci Microflora 16:1–7Google Scholar
  16. Kubo M, Kimura Y, Odani T, Tani T, Namba K (1981) Studies on Scutellariae radix. Part II: the antibacterial substance. Planta Med 43:194–201PubMedCrossRefGoogle Scholar
  17. Kuhn JG (1998) Pharmacology of irinotecan. Oncology 12:39–42PubMedGoogle Scholar
  18. Lai MY, Hsiu SL, Tsai SY, Hou YC, Chao PD (2003) Comparison of metabolic pharmacokinetics of baicalin and baicalein in rats. J Pharm Pharmacol 55:205–209. doi: 10.1211/002235702522 PubMedCrossRefGoogle Scholar
  19. Liu JJ, Huang TS, Cheng WF, Lu FJ (2003) Baicalein and baicalin are potent inhibitors of angiogenesis: inhibition of endothelial cell proliferation, migration and differentiation. Int J Cancer 106:559–565. doi: 10.1002/ijc.11267 PubMedCrossRefGoogle Scholar
  20. Middleton E Jr, Kandaswami C, Theoharides TC (2000) The effects of plant flavonoids on mammalian cells: implications for inflammation, heart disease, and cancer. Pharmacol Rev 52:673–751PubMedGoogle Scholar
  21. Rémy-Martin A, Prost O, Nicollier M, Burnod J, Adessi GL (1983) Estrone sulfate concentrations in plasma of normal individuals, postmenopausal women with breast cancer, and men with cirrhosis. Clin Chem 29:86–89PubMedGoogle Scholar
  22. Roy AB (1970) Enzymological aspects of steroid conjugation. In: Bernstein S, Soloman S (eds) Chemical and biological aspects of steroid conjunction. Springer, New York, pp 74–130CrossRefGoogle Scholar
  23. Russell WM, Klaenhammer TR (2001) Identification and cloning of gusA, encoding a new β-glucuronidase from Lactobacillus gasseri ADH. Appl Environ Microbiol 67:1253–1261. doi: 10.1128/AEM.67.3.1253-1261.2001 PubMedCentralPubMedCrossRefGoogle Scholar
  24. Sasaki K, Taura F, Shoyama Y, Morimoto S (2000) Molecular characterization of a novel β-glucuronidase from Scutellaria baicalensis Georgi. J Biol Chem 275:27466–27472. doi: 10.1074/jbc.M004674200 PubMedGoogle Scholar
  25. Shen YC, Chiou WF, Chou YC, Chen CF (2003) Mechanisms in mediating the anti-inflammatory effects of baicalin and baicalein in human leukocytes. Eur J Pharmacol 465:171–181. doi: 10.1016/S0014-2999(03)01378-5 Google Scholar
  26. Simons AL, Renouf M, Hendrich S, Murphy PA (2005) Human gut microbial degradation of flavonoids: structure-function relationships. J Agric Food Chem 53:4258–4263. doi: 10.1021/jf0500177 PubMedCrossRefGoogle Scholar
  27. St John FJ, González JM, Pozharski E (2010) Consolidation of glycosyl hydrolase family 30: a dual domain 4/7 hydrolase family consisting of two structurally distinct groups. FEBS Lett 584:4435–4441. doi: 10.1016/j.febslet.2010.09.051 PubMedCrossRefGoogle Scholar
  28. Tohyama O, Imura A, Iwano A, Freund JN, Henrissat B, Fujimori T, Nabeshima Y (2004) Klotho is a novel β-glucuronidase capable of hydrolyzing steroid β-glucuronides. J Biol Chem 279:9777–9784. doi: 10.1074/jbc.M312392200 PubMedCrossRefGoogle Scholar
  29. Wallace BD, Wang H, Lane KT, Scott JE, Orans J, Koo JS, Venkatesh M, Jobin C, Yeh LA, Mani S, Redinbo MR (2010) Alleviating cancer drug toxicity by inhibiting a bacterial enzyme. Science 330:831–835. doi: 10.1126/science.1191175 PubMedCentralPubMedCrossRefGoogle Scholar
  30. Walle T (2004) Absorption and metabolism of flavonoids. Free Radic Biol Med 36:829–837. doi: 10.1016/j.freeradbiomed.2004.01.002 PubMedCrossRefGoogle Scholar
  31. Wilson KJ, Hughes SG, Jefferson RA (1992) The Escherichia coli gus operon: induction and expression of the gus operon in E. coli and the occurrence and use of GUS in other bacteria. In: Gallagher SR (ed) GUS protocols. Using the GUS gene as reporter of gene expression. Academic, San Diego, pp 7–22Google Scholar
  32. Xing J, Chen X, Zhong D (2005) Absorption and enterohepatic circulation of baicalin in rats. Life Sci 78:140–146. doi: 10.1016/j.lfs.2005.04.072 PubMedCrossRefGoogle Scholar
  33. Yim JS, Kim YS, Moon SK, Cho KH, Bae HS, Kim JJ, Park EK, Kim DH (2004) Metabolic activities of ginsenoside Rb1, baicalin, glycyrrhizin and geniposide to their bioactive compounds by human intestinal microflora. Biol Pharm Bull 27:1580–1593. doi: 10.1248/bpb.27.1580 PubMedCrossRefGoogle Scholar
  34. Zhang C, Zhang Y, Chen J, Liang X (2005) Purification and characterization of baicalin-β-d-glucuronidase hydrolyzing baicalin to baicalein from fresh roots of Scutellaria viscidula Bge. Process Biochem 40:1911–1915. doi: 10.1016/j.procbio.2004.07.003 CrossRefGoogle Scholar
  35. Zhao Y, Li H, Gao Z, Gong Y, Xu H (2006) Effects of flavonoids extracted from Scutellaria baicalensis Georgi on hemin-nitrite-H2O2 induced liver injury. Eur J Pharmacol 536:192–199. doi: 10.1016/j.ejphar.2006.02.045 PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2013

Authors and Affiliations

  • Haruko Sakurama
    • 1
  • Shigenobu Kishino
    • 2
  • Yoshie Uchibori
    • 2
    • 3
  • Yasunori Yonejima
    • 3
  • Hisashi Ashida
    • 4
  • Keiko Kita
    • 5
  • Satomi Takahashi
    • 1
  • Jun Ogawa
    • 2
    Email author
  1. 1.Laboratory of Industrial Microbiology, Graduate School of AgricultureKyoto UniversityKyotoJapan
  2. 2.Laboratory of Fermentation Physiology and Applied Microbiology, Division of Applied Life Sciences, Graduate School of AgricultureKyoto UniversityKyotoJapan
  3. 3.Research and Development DepartmentNitto Pharmaceutical Industries, Ltd.MukoJapan
  4. 4.The Faculty of Biology-Oriented Science and TechnologyKinki UniversityKinokawaJapan
  5. 5.Laboratory of Molecular Microbiology, Division of Applied Life Sciences, Graduate School of AgricultureKyoto UniversityUjiJapan

Personalised recommendations