Sakurama Haruko, Kishino Shigenobu, Uchibori Yoshie, Yonejima Yasunori, Ashida Hisashi, Kita Keiko, Takahashi Satomi, Ogawa Jun
Laboratory of Industrial Microbiology, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto, 606-8502, Japan.
Appl Microbiol Biotechnol. 2014 May;98(9):4021-32. doi: 10.1007/s00253-013-5325-8. Epub 2013 Nov 20.
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.
黄芩苷（黄芩素7 - O -β - D - 葡萄糖醛酸苷）是传统草药中发现的主要黄酮类葡萄糖醛酸苷之一。由于其苷元黄芩素比黄芩苷吸收更快且表现出更有效的特性，β - 葡萄糖醛酸酶（GUS）将黄芩苷转化为黄芩素引起了研究人员的关注。最近，我们发现短乳杆菌凝固亚种可以将黄芩苷转化为黄芩素。因此，我们旨在鉴定和表征来自短乳杆菌凝固亚种的转化酶。首先，我们从短乳杆菌凝固亚种的无细胞提取物中纯化了这种酶并克隆了其基因。令人惊讶的是，该酶被发现是一种属于糖苷水解酶（GH）家族30的GUS（命名为LcGUS30），其氨基酸序列与迄今为止报道的属于GH家族1、2和79的任何GUS几乎没有相似性。然后我们在大肠杆菌中建立了重组LcGUS30的高水平表达和简单纯化系统。对底物特异性的详细分析表明，LcGUS30对糖苷具有严格的特异性，而对苷元没有。有趣的是，LcGUS30更喜欢黄芩苷而不是人内源性甾体激素之一的雌酮3 -（β - D - 葡萄糖醛酸苷）。这些结果表明，短乳杆菌凝固亚种和LcGUS30应该作为构建安全的黄芩苷生物转化系统的有力工具。此外，我们提出这种新型的GUS在GH家族30的亚家族3中形成一个新的组。
