Cloning, expression, and characterization of the β-glucosidase hydrolyzing secoisolariciresinol diglucoside to secoisolariciresinol from Bacteroides uniformis ZL1.
Previously, from the human intestinal flora we isolated the bacterial strain Bacteroides uniformis ZL1, which could convert secoisolariciresinol diglucoside (SDG) to its aglycone secoisolariciresinol (SECO) in vivo. In this study, 24 putative β-glucosidase genes were screened from the genome of B. uniformis ATCC 8492, which were used as templates to design PCR primers for the target genes in B. uniformis ZL1. Fifteen genes (bgl1-bgl15) were amplified from strain ZL1, and among them we identified bgl8 as the gene encoding the SDG-hydrolyzing β-glucosidase. We sequenced the bgl8 gene, cloned it into the expression vector and then transformed Escherichia coli to construct the recombinant bacteria that could synthesize the target β-glucosidase (BuBGL8). We purified and characterized BuBGL8, which showed maximal activity and stability under the culture conditions of pH 6.0 and 30 °C. SDG (2.0 mg/ml) was converted to SECO by both the purified BuBGL8 (0.035 mg/ml) and crude enzyme extract (0.23 mg crude protein/ml) with the efficiency of more than 90 % after 90 min at the reaction conditions. This is, to our knowledge, the first report of using recombinant bacteria to synthesize the SDG-hydrolyzing β-glucosidase, which could be used to produce SECO from SDG conveniently and highly efficiently.
State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, Beijing, 100191, China.
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Tao
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Cai
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Liu
D000595:Amino Acid Sequence; D001439:Bacteroides / Q000201:enzymology* / Q000235:genetics; D002072:Butylene Glycols / Q000378:metabolism*; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005960:Glucosides / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D017705:Lignans / Q000378:metabolism*; D008958:Models, Molecular; D062105:Molecular Docking Simulation; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D013696:Temperature; D001617:beta-Glucosidase / Q000235:genetics* / Q000378:metabolism*
D002072:Butylene Glycols; D004269:DNA, Bacterial; D005960:Glucosides; D017705:Lignans; D011994:Recombinant Proteins; D001617:beta-Glucosidase; C060283:secoisolariciresinol; C090142:secoisolariciresinol diglucoside