title: β-Glucuronidase from Lactobacillus brevis useful for baicalin hydrolysis belongs to glycoside hydrolase family 30.
abstract: 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.
authors.affiliation: Laboratory of Industrial Microbiology, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto, 606-8502, Japan.
authors.forename: Haruko
authors.initials: H
authors.lastname: Sakurama
authors.forename: Shigenobu
authors.initials: S
authors.lastname: Kishino
authors.forename: Yoshie
authors.initials: Y
authors.lastname: Uchibori
authors.forename: Yasunori
authors.initials: Y
authors.lastname: Yonejima
authors.forename: Hisashi
authors.initials: H
authors.lastname: Ashida
authors.forename: Keiko
authors.initials: K
authors.lastname: Kita
authors.forename: Satomi
authors.initials: S
authors.lastname: Takahashi
authors.forename: Jun
authors.initials: J
authors.lastname: Ogawa
mesh_terms: D000595:Amino Acid Sequence; D001711:Biotransformation; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D004970:Estrone / Q000031:analogs & derivatives / Q000378:metabolism; D044950:Flavanones / Q000378:metabolism*; D005419:Flavonoids / Q000378:metabolism*; D015870:Gene Expression; D005966:Glucuronidase / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D006026:Glycoside Hydrolases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D006868:Hydrolysis; D052196:Levilactobacillus brevis / Q000201:enzymology*; D008969:Molecular Sequence Data; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
chemical_list: D004269:DNA, Bacterial; D044950:Flavanones; D005419:Flavonoids; C015856:estrone-3-glucuronide; D004970:Estrone; C038044:baicalin; C006680:baicalein; D006026:Glycoside Hydrolases; D005966:Glucuronidase