β-Glucuronidase from Lactobacillus brevis useful for baicalin hydrolysis belongs to glycoside hydrolase family 30
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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.
KeywordsFlavonoid glucuronides Baicalin β-Glucuronidase (EC 126.96.36.199) Glycoside hydrolase family 30 Lactic acid bacteria Lactobacillus brevis subsp. coagulans
- 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
- Kobashi K, Akao T (1997) Relation of intestinal bacteria to pharmacological effect of glycosides. Biosci Microflora 16:1–7Google Scholar
- 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