Metabolism of Rutin and Poncirin by Human Intestinal Microbiota and Cloning of Their Metabolizing α-L-Rhamnosidase from Bifidobacterium dentium

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 alpha-L-rhamnosidase activities on the p-nitrophenyl-alpha-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, alpha-L-rhamnosidase-producing bacteria were isolated from the specimens, and the alpha-L-rhamnosidase gene was cloned from a selected organism, Bifidobacterium dentium, and expressed in E. coli. The cloned alpha-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 Ni2+-NTA and Q-HP column chromatography. The specific activity of the purified alpha-L-rhamnosidase was 23.3 mu mol/min/mg. Of the tested natural product constituents, the cloned alpha-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 alpha-L-rhamnosidase, a flavonoid rhamnoglycoside-metabolizing enzyme, from bifidobacteria. Based on these findings, the alpha-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.