title: Metabolism of rutin and poncirin by human intestinal microbiota and cloning of their metabolizing α-L-rhamnosidase from Bifidobacterium dentium.
abstract: To understand the metabolism of flavonoid rhamnoglycosides by human intestinal microbiota, we measured the metabolic activity of rutin and poncirin (distributed in many functional foods and herbal medicine) by 100 human stool specimens. The average α-Lrhamnosidase activities on the p-nitrophenyl-α-L-rhamnopyranoside, rutin, and poncirin subtrates were 0.10 ± 0.07, 0.25 ± 0.08, and 0.15 ± 0.09 pmol/min/mg, respectively. To investigate the enzymatic properties, α-L-rhamnosidase-producing bacteria were isolated from the specimens, and the α-L-rhamnosidase gene was cloned from a selected organism, Bifidobacterium dentium, and expressed in E. coli. The cloned α-L-rhamnosidase gene contained a 2,673 bp sequcence encoding 890 amino acid residues. The cloned gene was expressed using the pET 26b(+) vector in E. coli BL21, and the expressed enzyme was purified using Ni(2+)-NTA and Q-HP column chromatography. The specific activity of the purified α-L-rhamnosidase was 23.3 μmol/min/mg. Of the tested natural product constituents, the cloned α-L-rhamnosidase hydrolyzed rutin most potently, followed by poncirin, naringin, and ginsenoside Re. However, it was unable to hydrolyze quercitrin. This is the first report describing the cloning, expression, and characterization of α-L-rhamnosidase, a flavonoid rhamnoglycosidemetabolizing enzyme, from bifidobacteria. Based on these findings, the α-L-rhamnosidase of intestinal bacteria such as B. dentium seem to be more effective in hydrolyzing (1-->6) bonds than (1-->2) bonds of rhamnoglycosides, and may play an important role in the metabolism and pharmacological effect of rhamnoglycosides.
authors.affiliation: Department of Life and Nanopharmaceutical Sciences, College of Pharmacy, Kyung Hee University, Seoul 130-701, Republic of Korea.
authors.forename: Seo-Hyeon
authors.initials: SH
authors.lastname: Bang
authors.forename: Yang-Jin
authors.initials: YJ
authors.lastname: Hyun
authors.forename: Juwon
authors.initials: J
authors.lastname: Shim
authors.forename: Sung-Woon
authors.initials: SW
authors.lastname: Hong
authors.forename: Dong-Hyun
authors.initials: DH
authors.lastname: Kim
mesh_terms: D001483:Base Sequence; D001644:Bifidobacterium / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D003001:Cloning, Molecular; D019976:Cloning, Organism; D004926:Escherichia coli / Q000235:genetics; D044950:Flavanones / Q000378:metabolism; D005419:Flavonoids / Q000378:metabolism*; D036145:Ginsenosides / Q000378:metabolism; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D006801:Humans; D007422:Intestines / Q000382:microbiology*; D064307:Microbiota; D008969:Molecular Sequence Data; D009571:Nitrilotriacetic Acid / Q000031:analogs & derivatives / Q000378:metabolism; D009942:Organometallic Compounds / Q000378:metabolism; D011794:Quercetin / Q000031:analogs & derivatives / Q000378:metabolism; D012431:Rutin / Q000096:biosynthesis / Q000378:metabolism*; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D013379:Substrate Specificity
chemical_list: D044950:Flavanones; D005419:Flavonoids; D036145:Ginsenosides; D009942:Organometallic Compounds; C012526:quercitrin; C088321:nickel nitrilotriacetic acid; C049864:ginsenoside Re; D012431:Rutin; C119641:poncirin; D011794:Quercetin; D006026:Glycoside Hydrolases; C022525:alpha-L-rhamnosidase; D009571:Nitrilotriacetic Acid; C005274:naringin