Characterization of a novel ginsenoside-hydrolyzing α-L-arabinofuranosidase, AbfA, from Rhodanobacterginsenosidimutans Gsoil 3054T
Applied Microbiology and Biotechnology
Dong-Shan An, Chang-Hao Cui, Bong Hyun Sung,Hee-Chan Yang, Sun Chang Kim, Sung-Taik Lee, Wan-Taek Im,Song-Gun Kim
Dong-Shan An, Bong Hyun Sung,Song-Gun Kim
Biological Resource Center, Korea Research Institute of Bioscience & Biotechnology, Daejeon 305-806, Republic of Korea
Bong Hyun Sung
Industrial Biotechnology & Bioenergy Research Center, Korea Research Institute of Bioscience & Biotechnology, Daejeon 305-806, Republic of Korea
Chang-Hao Cui,Sun Chang Kim, Sung-Taik Lee
Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Daejeon 305-701, Republic of Korea
Hee-Chan Yang
M.O.S. Co. Ltd, 452-28, Daheung-dong, Jung-gu,Daejeon 301-803, Republic of Korea
Wan-Taek Im
KAIST Institute for Biocentry, Korea Research Institute of Bioscience & Biotechnology, Daejeon 305-701, Republic of Korea
Correspondence to Song-Gun Kim
Phone: +82 42 8604627
Fax:+82 42 8604625
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Or to Wan-Taek Im
Phone: +82 42 8695617
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Preparation and chemical identification of the ginsenosides C-Mc1 and C-Mc
The ginsenosides C-Mc1 and C-Mc were obtained by biotransformation of ginsenoside Rc using a recombinant β-glucosidase, BgpA, from Terrabcter sp. Gsoil3082 (An et al. 2010). Recombinant BgpA preferentially hydrolyzes the outer glucose at position C3, followed by the inner glucose at position C3, and finally the outer glucose at position C20 of ginsenoside Rb1 (1). Because ginsenoside Rc has a terminal non-reducing α-L-arabinofuranosyl moiety at the outer position of C20, it was speculated that BgpA hydrolyzed only the outer and inner glucose moieties attached at position C3 of Rc to generate C-Mc1 and C-Mc, respectively.
For the reaction, two mixtures of 300 ml each were prepared: one contained 1 mg/ml of Rc and 0.1 mg/ml of BgpA, and the other contained 1 mg/ml of Rc and 1.0 mg/ml of BgpA. Both reaction mixtures were incubated at 37°C until complete conversion of Rc into metabolite 1 or 2; reaction progress was monitored by TLC. The reaction mixture was extracted twice with water-saturated n-butanol and then evaporated in vacuo. From the reaction mixtures, 0.21g of metabolite 1 and 0.15g of metabolite 2 were obtained. To further purify the metabolites for nuclear magnetic resonance (NMR) analysis, 100 mg each of metabolite 1 and 2 were purified using a recycling preparative HPLC system, as previously described (An et al. 2010). Finally 42 mg of metabolite 1 and 33 mg of metabolite 2 were obtained and subjected to NMR and mass spectrometry (MS) analysis. NMR spectral data were recorded on a Varian Unity 500 NMR spectrometer in pyridine. Electrospray ionization mass spectra (ESI-MS) were measured on a WatersQuattro Premier XE tandemmass spectrometer.
Structural Identification of C-Mc1 and C-Mc
A protonated molecular ion peak corresponding to metabolite 1 was observed at m/z 917 (C47H81O17) in the ESI-MS spectrum of metabolite 1. The 1H NMR spectroscopic data of metabolite 1 (Table S2) showed two anomeric proton signals at 4.92 (1H, d, J = 7.8 Hz), and 5.12 (1H, d, J = 7.8 Hz), belonging to β-glucose, and one anomeric proton signal at 5.63 (1H, d, J = 1.6 Hz), attributed to α-arabinofuranoside. The presence of three anomeric protons indicated that one glucose unit was removed from the starting material, ginsenoside Rc. 13C NMR chemical shifts of metabolite 1 showed closely related signals to Mb (C-Mc1) based on published data (Bae et al. 2002). Thus, this compound was confirmed as C-Mc1 (Fig. 3). The 1H and 13C NMR data of metabolite 2 (Table S2) were very similar to those of C-Mc1, except that they lacked signals of a glucose unit and the migrating 13C chemical shift of C3 from 88.0 to 78.0. These differences suggested that the glucose at position C3 in C-Mc1 was hydrolyzed and that a free hydroxyl group was present in the structure of metabolite 2. This was confirmed by the detection of a protonated molecular ion peak at m/z 755 (C41H71O12) by ESI-MS and by comparison with the values reported in literature (Bae et al. 2002). Accordingly, the structure of metabolite 2 was confirmed as C-Mc(Fig. 3).
Fig. S1 Time-course of ginsenoside Rc bioconversion by BgpA at an enzyme concentration of 1 mg/ml. Metabolites were analyzed by TLC. Lanes: STD, standards; 0 to 24 h, reaction times
Table S1Purification scheme of AbfA
Purification steps / Total protein / Specific activity / Total activity / Purification / Yield(mg) / (U/mg) / (Ua) / (Fold) / (%)
Crude extract / 1260.0 / 0.5 / 669 / 1.0 / 100
Amylose / 20.5 / 23.0 / 471 / 43.3 / 70
Mono Q / 8.2 / 52.0 / 424 / 98.0 / 63
Mono Q (MBPepitope removed) / 4.2 / 56.0 / 235 / 105.5 / 35
aOne unit(U) ofα-L-arabinofuranosidase was defined as the of enzyme liberating 1μmol/min of p-nitrophenol
Table S21H-and 13C-NMR Data of ginsenoside Rc metabolite 1 and 2
Position / Metabolite 1 / Metabolite 2δ(C) / δ(H) / δ(C) / δ(H)
CH2(1) / 39.2 / 39.4
CH2(2) / 26.6 / 28.3
H-C (3) / 88.8 / 3.35 (dd, J=11.5, 4.4) / 78.0 / 3.39 (dd, J=10.5, 5.0)
C(4) / 39.7 / 39.5
H-C (5) / 56.4 / 56.3
CH2 (6) / 18.5 / 18.7
CH2 (7) / 35.1 / 35.1
C(8) / 40.0 / 40.0
H-C(9) / 50.2 / 50.3
H-C (10) / 37.0 / 37.3
CH2 (11) / 30.7 / 30.7
H-C (12) / 70.2 / 3.95 (ddd-like) / 70.2 / 3.95 (ddd-like)
H-C (13) / 49.5 / 49.5
C(14) / 51.4 / 51.4
CH2 (15) / 30.8 / 30.8
CH2 (16) / 26.8 / 26.6
H-C (17) / 51.7 / 51.6
Me (18) / 16.3 / 0.94 (s) / 16.3 / 0.91 (s)
Me (19) / 16.0 / 0.79 (s) / 16.0 / 0.87 (s)
C(20) / 83.3 / 83.3
Me (21) / 22.4 / 1.62 (s) / 22.3 / 1.62 (s)
CH2 (22) / 36.1 / 36.2
CH2 (23) / 23.2 / 23.1
H-C (24) / 126.0 / 5.30 ( t, J=7.1) / 126.0 / 5.30 ( t, J=7.1)
C(25) / 131.0 / 131.0
Me (26) / 25.8 / 1.60 (s) / 25.8 / 1.60 (s)
Me (27) / 17.8 / 1.64 (s) / 17.8 / 1.64 (s)
Me (28) / 28.1 / 1.28 (s) / 28.7 / 1.20 (s)
Me (29) / 16.8 / 0.97 (s) / 16.3 / 1.01 (s)
Me (30) / 17.4 / 0.94 (s) / 17.4 / 0.98 (s)
H-C(1') / 106.9 / 4.92 (d, J=7.8)
H-C(2') / 75.8
H-C(3') / 78.8
H-C(4') / 72.0
H-C(5) / 78.4
CH2(6') / 63.1
H-C(1'') / 98.1 / 5.12 (d, J=7.8) / 98.1 / 5.12 (d, J=7.8)
H-C(2'') / 75.0 / 75.0
H-C(3'') / 78.9 / 79.3
H-C(4'') / 72.1 / 72.1
H-C(5'') / 76.5 / 76.5
CH2(6'') / 68.5 / 68.5
H-C(1''') / 110.1 / 5.63 (d, J=1.6) / 110.1 / 5.64 (d, J=1.6)
H-C(2''') / 83.4 / 83.4
H-C(3''') / 79.2 / 78.8
H-C(4''') / 86.1 / 86.0
CH2 (5''') / 62.7 / 62.6
1H-and 13C-NMR spectra were recorded in pyridine at 500 MHz.δin ppm, J in Hz; multiplicities (shown in parentheses) are abbreviated as follows: s, singlet; d, doublet; t triplet
References
An D-S, Cui C-H, Lee H-G, Wang L, Kim SC, Lee S-T, Jin F, Yu H, Chin Y-W, Lee H-K, Im W-T, Kim S-G(2010) Identification and characterization of a novel Terrabacter ginsenosidimutans sp. nov. -glucosidase that transforms ginsenoside Rb1 into the rare gypenoside XVII and gypenoside LXXV.Appl Environ Microbiol76: 5827-5836
Bae EA, Choo MK, Park EK, Park SY, Shin HY, Kim DH (2002) Metabolism of ginsenoside Rc by human intestinal bacteria and its related antiallergic activity. Biol Pharm Bull 25: 743-747
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