• Journal of Microbiology and Biotechnology
• /
• 제25권1호
• /
• pp.18-25
• /
• 2015

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-L-rhamnosidase 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 Ni²⁺-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.

• Journal of Microbiology and Biotechnology
• /
• 제15권3호
• /
• pp.519-524
• /
• 2005

An ${\alpha}$-L-rhamnosidase (EC 3.2.1.40.), which transforms quercitrin to quercetin, was purified from Fusobacterium K-60, a human intestinal anaerobic bacterium. The specific activity of the purified ${\alpha}$-L-rhamnosidase was 2.89 mol/min/mg protein. ${\alpha}$-L-Rhamnosidase, whose molecular size was 170 kDa by gel filtration, was composed of four subunits ($M_r$ 41,000 Da) with pI and optimal pH values of 5.2 and 5.5-7.0, respectively. The apparent $K_m$ and $V_{max}$ values for p-nitrophenyl-${\alpha}$-L-rhamnopyranoside and quercitrin were determined to be 0.057 mM and 3.4 mol/min/mg, and 0.077 mM and 5.0 mol/min/mg, respectively. This enzyme was strongly inhibited by $Cu^{2+},\;Mn^{2+}$, L-rhamnose, and p-chlormercuriphenylsulfonic acid. These findings suggest that the biochemical properties and substrate specificity of the purified enzyme are different from those of the previously purified ${\alpha}$-L-rhamnosidase. This is the first reported purification of quercitrin-hydrolyzing ${\alpha}$-L-rhamnosidase from intestinal bacteria.

• 한국미생물·생명공학회지
• /
• 제45권4호
• /
• pp.305-310
• /
• 2017

발효식품으로부터 분리한 젖산균 중 ${\alpha}$-rhamnosidase 효소활성을 보유한 젖산균 12점을 선별하였다. 효소활성이 우수한 Weissella confuse 1점, L. pentosus 1점과 효소활성이 우수하였던 4점의 L. plantarum 균주를 사용하여 진세노사이드 Rb1과 Re를 Rg1, Rg5로 각각 생물전환하였다. L. plantarum MBE/L2990 균주를 사용한 생물전환반응에서 Rg1과 Rg5의 함량이 각각 0.58 mg, 0.24 mg 가량 증가하였으며, 대조구로 사용한 L. plantarum KCTC21004 균주에 비해 약 56% 및 42% 우수한 생물전환 효율이었다. L. plantarum MBE/L2990 균주는 한국미생물자원센터에 KCTC18529P로 기탁하였다.

• Journal of Microbiology and Biotechnology
• /
• 제31권3호
• /
• pp.419-428
• /
• 2021

To efficiently recycle GH78 thermostable rhamnosidase (TpeRha) and easily separate it from the reaction mixture and furtherly improve the enzyme properties, the magnetic particle Fe3O4-SiO2-NH2-Cellu-ZIF8 (FSNcZ8) was prepared by modifying Fe3O4-NH2 with tetraethyl silicate (TEOS), microcrystalline cellulose and zinc nitrate hexahydrate. FSNcZ8 displayed better magnetic stability and higher-temperature stability than unmodified Fe3O4-NH2 (FN), and it was used to adsorb and immobilize TpeRha from Thermotoga petrophilea 13995. As for properties, FSNcZ8-TpeRha showed optimal reaction temperature and pH of 90℃ and 5.0, while its highest activity approached 714 U/g. In addition, FSNcZ8-TpeRha had better higher-temperature stability than FN. After incubation at 80℃ for 3 h, the residual enzyme activities of FSNcZ8-TpeRha, FN-TpeRha and free enzyme were 93.5%, 63.32%, and 62.77%, respectively. The organic solvent tolerance and the monosaccharides tolerance of FSNcZ8-TpeRha, compared with free TpeRha, were greatly improved. Using naringin (1 mmol/l) as the substrate, the optimal conversion conditions were as follows: FSNcZ8-TpeRha concentration was 6 U/ml; induction temperature was 80℃; the pH was 5.5; induction time was 30 min, and the yield of products was the same as free enzyme. After repeating the reaction 10 times, the conversion of naringin remained above 80%, showing great improvement of the catalytic efficiency and repeated utilization of the immobilized α-L-rhamnosidase.

• Journal of Microbiology and Biotechnology
• /
• 제25권11호
• /
• pp.1842-1848
• /
• 2015

Cyanidin-3-glucoside (C3G) has been known to be more bioavailable than cyanidin-3-rutinoside (C3R), the most abundant anthocyanin in black raspberry (Rubus occidentalis). The aim of this study was to enhance the bioavailability of anthocyanins in black raspberry by cleaving ʟ-rhamnose in C3R using crude enzyme extracts (CEEs) from Aspergillus usamii KCTC 6956, A. awamori KCTC 60380, A. niger KCCM 11724, A. oryzae KCCM 12698, and A. kawachii KCCM 32819. The enzyme activities of the CEEs were determined by a spectrophotometric method using ρ-nitrophenyl-rhamnopyranoside and ρ-nitrophenyl-glucopyranoside. The CEE from A. usamii had the highest α-ʟ-rhamnosidase activity with 2.73 U/ml at 60℃, followed by those from A. awamori and A. niger. When bioconversion of C3R to C3G in black raspberry was analyzed by HPLC-DAD, the CEEs from A. usamii and A. awamori hydrolyzed 95.7% and 95.6% of C3R to C3G, respectively, after 2 h incubation. The CEEs from A. kawachii and A. oryzae did not convert C3R to C3G in black raspberry.

• 대한약학회:학술대회논문집
• /
• 대한약학회 2000년도 춘계총회 및 학술대회
• /
• pp.154.2-154.2
• /
• 2000

• Journal of Microbiology and Biotechnology
• /
• 제33권11호
• /
• pp.1521-1530
• /
• 2023

An α-L-rhamnosidase gene from Thermoclostridium. stercorarium subsp. thermolacticum DSM 2910 (TstRhaA) was cloned and expressed. The maximum TstRhaA activity of the protein reached 25.2 U/ml, and the molecular mass was approximately 106.6 kDa. The protein was purified 8.0-fold by Ni-TED affinity with an overall recovery of 16.6% and a specific activity of 187.9 U/mg. TstRhaA activity was the highest at 65℃ and pH 6.5. In addition, it exhibited excellent thermal stability, better pH stability, good tolerance to low concentrations of organic reagents, and high catalytic activity for p-nitrophenyl-α-L-rhamnopyranoside (pNPR). Substrate specificity studies showed that TstRhaA exhibited a high specific activity for rutin. At 60℃, pH 6.5, and 0.3 U/ml enzyme dosage, 60 g/l rutin was converted to 45.55 g/l isoquercitrin within 150 min. The molar conversion rate of rutin and the yield of isoquercitrin were 99.8% and 12.22 g/l/h, respectively. The results suggested that TstRhaA could be used for mass production of isoquercitrin.

• Archives of Pharmacal Research
• /
• 제21권1호
• /
• pp.17-23
• /
• 1998

Flavonoid glycosides were metabolized to phenolic acids via aglycones by human intestinal microflora producing ${\alpha}$-rhamnosidase, exo-${\beta}$-glucosidase, endo- ${\beta}$-glucosidase and/or ${\beta}$-glucuronidase. Rutin, hesperidin, naringin and poncirin were transformed to their aglycones by the bacteria producing ${\alpha}$-rhamnosidase and ${\beta}$-glucosidase or endo- ${\beta}$-glucosidase, and baicatin, puerarin and daidzin were transformed to their aglycones by the bacteria producing ${\beta}$glucuronidase, C-glycosidase and ${\beta}$-glycosidase, respectively. Anti-platelet activity and cytotoxicity of the metabolites of flavonoid glycosides by human intestinal bacteria were more effective than those of the parental compounds. 3,4-Dihydroxyphenylacetic acid and 4-hydroxyl-phenylacetic acid were more effective than rutin and quercetin on anti-platelet aggregation activity. 2,4,6-Trihydroxybenzaidehyde, quercetin and ponciretin were more effective than rutin and ponciretin on the cytotoxicity for tumor cell lines. We insist that these flavonoid glycosides should be natural prodrugs.

• 약학회지
• /
• 제35권4호
• /
• pp.271-276
• /
• 1991

The extractability and stability of ginkgoflavonolglycosides under presence of several cellulose preparations were investigated. The enzymes used were macerosin, cellulose C and cellulase NC. The content variation of the glycosides was measured with HPLC method, using caffeic acid as an internal standard. The methanol extract of ginkgo leaf, containing the total flavonolglycosides of 4.46%, was used for the content comparison. By extraction with the enzymes, each or mixed, the peak levels of all the glycosides began to decrease after 1 or 2 hours. After 24 hour extraction, most of the glycosides were degraded to minor components. The flavonolglycosides in ginkgo leaf were also hydrolysed simply by the water extraction. After 24 hour extraction with water at $40^{\circ}C$, the peak levels of major glycosides were distinctly decreased. Rutin was hydrolysed by enzyme treatment or by ginkgo leaf itself. As a result, it was concluded that the commercially available cellulases and the ginkgo leaf itself contain the activities of $\beta$-glycosidase and $\alpha$-rhamnosidase. Kaempferol-3-O-(6'"-O-p-coumaroylglucosyl)-rhamnoside and four other ginkgo flavonolglycosides were not hydrolysed under the same condition.tion.

• Food Science and Biotechnology
• /
• 제18권6호
• /
• pp.1365-1370
• /
• 2009

Aspergillus kawachii extracellular pectinases were screened in liquid cultures with different carbon sources. The fungus grown on citrus pectin or lemon pomace produced at least one of these inducible pectinases: acidic polygalacturonase, pectin lyase, pectin methylesterase, $\alpha$-L-arabinofuranosidase, $\alpha$-1,5-endoarabinase, $\beta$-D-galactosidase/exogalactanase, and $\beta$-1,4-endogalactanase. The lemon-pomace filtrates also contained significant $\alpha$-L-rhamnosidase and $\beta$-D-fucosidase activities. Most of the screened pectinases were active at pH 2.0-2.5, indicating that the A. kawachii enzymes were acidophilic. Under the culture conditions employed we could not detect enzymatic degradation of soybean rhamnogalacturonan. The A. kawachii pectinase-production-related regulatory phenomena of induction-repression resemble those described for other Aspergillus sp.