Highly selective microbial transformation of major ginsenoside Rb1 to gypenoside LXXV by Esteya vermicola CNU120806

Authors


Correspondence

Chang-Keun Sung, Department of Food Science and Technology, College of Agriculture and Biotechnology, Chungnam National University, Taejon 305-764, Korea. E-mail: sungchangkeun@126.com

Abstract

Aims

This study examined the biotransformation pathway of ginsenoside Rb1 by the fungus Esteya vermicola CNU 120806.

Methods and Results

Ginsenosides Rb1 and Rd were extracted from the root of Panax ginseng. Liquid fermentation and purified enzyme hydrolysis were employed to investigate the biotransformation of ginsenoside Rb1. The metabolites were identified and confirmed using NMR analysis as gypenoside XVII and gypenoside LXXV. A mole yield of 95·4% gypenoside LXXV was obtained by enzymatic conversion (pH 5·0, temperature 50°C). Ginsenoside Rd was used to verify the transformation pathway under the same reaction condition. The product Compound K (mole yield 49·6%) proved a consecutive hydrolyses occurred at the C-3 position of ginsenoside Rb1.

Conclusions

Strain CNU 120806 showed a high degree of specific β-glucosidase activity to convert ginsenosides Rb1 and Rd to gypenoside LXXV and Compound K, respectively. The maximal activity of the purified glucosidase for ginsenosides transformation occurred at 50°C and pH 5·0. Compared with its activity against pNPG (100%), the β-glucosidase exhibited quite lower level of activity against other aryl-glycosides. Enzymatic hydrolysate, gypenoside LXXV and Compound K were produced by consecutive hydrolyses of the terminal and inner glucopyranosyl moieties at the C-3 carbon of ginsenoside Rb1 and Rd, giving the pathway: ginsenoside Rb1→ gypenoside XVII → gypenoside LXXV; ginsenoside Rd→F2→Compound K, but did not hydrolyse the 20-C, β-(1-6)-glucoside of ginsenoside Rb1 and Rd.

Significance and Impact of the Study

The results showed an important practical application on the preparation of gypenoside LXXV. Additionally, this study for the first time provided a high efficient preparation method for gypenoside LXXV without further conversion, which also gives rise to a potential commercial enzyme application.

Introduction

Panax ginseng belongs to the Araliaceae family. The use of Panax ginseng for medical purposes dates back to 5000 years, beginning with ancient traditional Chinese medicine (Himi et al. 1989; Wen et al. 1996). Ginsenoside presents in P. ginseng root, leaf, root hair, rhizome and stem (Wei et al. 2007). Nowadays, commercial interest of ginsenoside is based on the pharmacological activities of ginseng (Attele et al. 1999; Lee et al. 2009), including the positive effects on enhancement of cholesterol biosynthesis (Banza et al. 2007), stimulation of serum protein synthesis, immunomodulatory function (Li et al. 1999; Wang et al. 2000), anti-inflammatory activity, increasing the free radical scavenging activities and reducing body weight (Hu and Kitts 2001; Naama et al. 2007; Park et al. 2004; Ki et al., 2007). Low abundant ginsenosides, with less sugar moieties, are considered to be more effective than common ginsenoside and proposed to be the real active form especially after they are transformed by the bacteria of the small intestine (Tawab et al. 2003). Nevertheless, low abundant ginsenosides are difficult to obtain because of their lower natural abundance in ginseng. More and more researches have been focusing on the microbial conversion of ginseng components, given that the enzyme produced by various micro-organisms possess high specificity and is feasible to scale production (Chen et al. 2008; Yan et al. 2010).

Ginsenoside Rb1 (2), protopanaxadiol-type ginsenoside, has higher content in the various ginseng samples (Ji et al. 2001). Ginsenoside is of dammarane skeleton, sugar moieties attached by a glycosidic bond at C-3 and C-6 of the skeleton. Two main categories of ginsenosides exist, the 20(S)-protopanaxadiol (Fig. 1) and 20(S)-protopanaxatriol classifications. The C-20 position is a tertiary centre within the structure of ginsenosides that frequently acts as the attachment point for glycosyl moieties. Steric considerations suggest that hydrolysis should occur more readily at the C-3 position than at the C-20 position. However, previous studies of fungal biotransformation of ginsenosides have shown high selectivity for hydrolysis firstly at the C-20 position, after which hydrolysis at the C-3 position is observed, leading to sequential biotransformation such as ginsenoside Rb1 (2)→ Rd (4)→ F2 (5)→ CK (6); ginsenoside Rb1 (2)→ Rd (4)→ Rg3 → Rh2 → protopanaxadiol (Zhao et al. 2009a,b; Yan et al. 2010). Among these pathways, Ginsenoside Rd (4), F2 (5) and Compound K (6) were mainly considered as the common intermediates in the biotransformation process. A large number of investigations focused on the conversion of protopanaxadiol ginsenoside because of the pharmacological action of the metabolites. However, gypenoside XVII (3) differs from other intermediates observed during the transformation of ginsenoside in that its formation requires hydrolysis at the C-3 position before the C-20 position. There have been few reports of this type of pathway (An et al. 2010), nor of the formation of the subsequent hydrolysis product gypenoside LXXV (1). Additionally, the studies presented in previous implied that most of the fungal strains were lack of specificity and always had further transformation (Chi and Ji 2005; Kim et al. 2010; Yan et al. 2010).

Figure 1.

Chemical structures of protopanaxadiol-type saponins. glc, β-d-glucopyranosyl; arap, α-l-arabinopyranosyl; araf, α-l-arabinofuranosyl; xyl, β-d-xylopranosyl.

In this study, a novel endoparasitic fungal strain, CNU120806, was utilized to study the biotransformation of ginsenoside Rb1 (2) and first discovered in our laboratory (Wang et al. 2008). It is the first time to report the pathway converting ginsenoside Rb1 (2) to gypenoside LXXV (1) with an intermediate gypenoside XVII (3) by glycosidase derived from nematophagous fungus Esteya vermicola (CNU 120806) without further transformation. Ginsenoside Rd (4) was used to verify the consecutive hydrolysis occurred at C-3 position, and Compound K (6) was obtained as the final product. In addition, gypenosides possessed some bioactivities such as hepatoprotective and antifibrotic activities (Chen et al. 2000) and inhibition on the proliferation or viability of the Hep3B and HA22T cells (Chen et al. 1999). Therefore, it is of greatest interest to propose viability to the preparation for individual gypenoside LXXV (1).

Materials and methods

Materials and special chemical

HPLC-grade acetonitrile was purchased from Merck Co. (Merck, Darmstadt, Germany). Deionized water was purified by Milli-Q system (Millipore, Bedford, MA, USA). Other chemicals were of reagent grade. Standard ginsenoside Rb1, Rd, F2, Rg3, Rh2 and Compound K and ginseng powder were purchased from the Hongjiu Biotech Co., Ltd (Jilin, China). The purity of all these standards was over 98% as indicated by the manufacturer. Stock solution was prepared by adding 0·8–2·0 mg ml−1 of these ginsenosides to methanol prior to HPLC analysis and was stored at −20°C.

The substrates, p-nitrophenyl-β-d-glucopyranosidase (pNPG), pNP-β-l-arabinopyranoside, pNP-α-l-arabinopyranoside, pNP-α-d-galactopyranoside, pNP-β-d-galactopyranoside, pNP-β-l-fucopyranoside, pNP-α-l-fucopyranoside, as well as other disaccharides, were obtained from Sigma (St. Louis, MO, USA). Sephadex G-150 and Sephacryl S-400 HR were obtained from TOSOH Corporation.

Preparation of ginsenoside Rb1 and Rd

The procedure for the preparation of ginsenosides Rb1 and Rd was as follow: 2·5 l of ethanol/water (7:3, v/v) was added to 250 g ginseng powder, and the mixture was firstly refluxed by chloroform for 3 h at 80°C for removing lipid layer and then refluxed by methanol for 8 h at 80°C. The extraction solution was filtered, evaporated and dissolved in water. After that, the extracts solution was applied to an HP-20 resin column eluting with the process as follows: distilled water, ethanol/water (3:7, v/v) and ethanol/water (8:2, v/v). The ethanol/water (8:2, v/v) fraction was evaporated and purified with silica gel column eluting by chloroform/methanol (7:3, v/v). Finally, 515 mg ginsenoside Rb1 (2) and 237 mg ginsenoside Rd (4) were obtained.

Culture of strain CNU 120806

The fungal strain designated E. vermicola CNU 120806 was obtained from the Agricultural Bioscience Biotech Centre, Chungnam National University, Korea. The 28S rRNA gene sequence of strains CNU 120806 has been deposited in the GenBank database. The accession number is EU627684. It was preserved on potato dextrose agar (PDA) slant at 4°C and cultured in potato dextrose broth (PDB, Difco, NJ, USA) plate at 26°C before use.

Enzyme assay

For routine assays and monitoring of purification, the method of β-glucosidase activity assay was same to those optimized by our group previous work (Liu et al. 2010) except the reaction temperature was changed to 50°C. Enzyme activities against different substrates were determined by measuring the amount of glucose released from these substrates.

Purification of beta-glucosidase

The cells harvested from the PDB were rinsed with 0·1 mol l−1 sodium chloride solution. Acetone-dried cells (3 g) of E. vermicola CNU 120806 were suspended in 40 ml of NaOAc/HOAc buffer (pH 5·0, 50 mmol l−1); then, an equal volume of quartz sand was added and cells were grinded. After centrifugation (15 000 g, 30 min), the supernatant was precipitated with ammonium sulphate (80% saturation) to enrich the glucosidase activity. Then, the supernatant was concentrated and subjected to Sephadex G-150 column (2·0 × 100 cm, bed volume: 85 ml) and eluted with NaOAc/HOAc buffer (pH 5·0, 50 mmol l−1). Fractions containing hydrolase activity were pooled and further purified by gel filtration on a Sephacryl S-400 HR column (1·6 × 70 cm) eluting at 0·55 ml min−1 with distilled water. Fractions with β-glucosidase were collected and used as purified enzyme. Operations were performed at 4°C. The purified enzyme was collected, and purity was estimated by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), and the purified enzyme running on PAGE produced a single band when stained with Coomassie Blue.

Enzyme activity optimization

The enzyme activity was optimized by accessing the catalytic ability over a range of pH and temperature. The pH was adjusted from 4·5 to 6·5 with NaOAc-HOAc buffer (pH 5·0, 50 mmol l−1), and temperature was controlled from 30 to 60°C. Three independent experiments were conducted.

Biotransformation pathway

About 150 ml of PDB was weighed exactly to collect spores of strain CNU 120806 by rinsing the potato dextrose agar (PDA) slant and then subcultured for 3 days at 26°C without shaking. After 3 days, 25 mg of ginsenoside Rb1 (2) was dissolved in the culture medium and incubated afterwards on a rotary shaker at 26°C. A constant volume of each sample (mycelium) was withdrawn at different time points (5, 7, 9 and 11 days). The sample was extracted with n-BuOH. The n-BuOH extract was evaporated, and the residue was dissolved in methanol for chromatographic analysis.

The standard reaction mixture of catalytic conversion consisted of 100 μl purified enzyme solution, 75 μl of NaOAc/HOAc buffer (pH 5·0, 50 mmol l−1) and 25 μl of ginsenoside Rb1 (2) solution (2 mg ml−1). After incubated at 50°C on a shaker (150 rev min−1) for 4 h, the reaction solution was subsequently extracted by 200 μl of n-BuOH. The n-BuOH extract was evaporated, and the residue was dissolved in methanol for chromatographic analysis.

Chromatographic analysis of ginsenoside

Ginsenoside Rb1 analysis and its metabolites were performed on the Shimazdu (Japan) HPLC apparatus, with a column of Discovery C18 reversed-phase column (25 cm × 4·6 mm, 5 μm, Sigma-Aldrich Co.) with a C18 guard column (8 × 4 mm, 5 μm, Sigma-Aldrich Co.), and kept at room temperature. The mobile phase consisted of water (A) and ACN (B) was used for the gradient program, at a flow rate of 1 ml min−1, as follows: 0–30 min, 20% B, 30–60 min, 20–45% B, 60–78 min, 45–75% B, 78–80 min, 75–80% B, 80–100 min and 80–100% B. The method was performed with an injection volume of 20 μl and detection wavelength at 203 nm.

Structural identification

The metabolites ①, ② and ③ were prepared by semi-preparation HPLC. Compounds ① and ② were dissolved in pyridine-d5, and the structure was analysed via NMR, using a Varion Inova AS 400 spectrometer (CA, USA).

Compound ③ was analysed by ESI-MS (Finnigan, San Jose, CA, USA).

Results

Identification of micro-organism producing Gypenoside LXXV (1)

Strain CNU 120806 was first isolated in our laboratory from infected nematodes in forest soil samples during a survey of nematophagous fungi in Korea. The strain CNU 120806 showed the ability to produce β-glucosidase-converting ginsenoside Rb1 (2) to gypenoside LXXV (1) in PDB broth. The 28S rRNA gene sequence of strain CNU 120806 was blasted in the NCBI database, and it belonged to the genus Esteya. The accession number is EU627684.

Substrate specificity of β-glucosidase from strain CNU 120806

The survey with various p-nitrophenyl glycosides indicated that this enzyme was greatly specific to β-d-glucoside (Table 1). Compared with its activity against pNPG (100%), the activity against other aryl-glycosides was quite less (pNP-β-d-galactopyranoside of 0·7%, others were not detected).

Table 1. Hydrolytic activity of strain 120806 β-glucosidase on various substrates
SubstrateActivity (%)a
  1. ND, activity is not detected by the analytical method used in this study. Data represent the means of three independent experiments.

  2. a

    Activity expressed relative to activity measured on pNPG (100%, 0·29 mmol min−1 mg−1).

pNPG100 ± 0·38
pNP-β-l-arabinopyranosideND
pNP-α-l-arabinopyranosideND
pNP-α-d-galactopyranosideND
pNP-β-d-galactopyranoside0·7 ± 0·02
pNP-β-l-fucopyranosideND
pNP-α-l-fucopyranosideND
Sophorose94·3 ± 0·22
LactoseND
GentiobioseND

Among disaccharides tests, the enzyme showed high activity to β-glucosidic linkage and no activity to others, such as lactose. To the substrates with β-glucosidic linkage, the relative activity of sophorose (β-(1-2)) was 94·3%, whereas activity against gentiobiose (β-(1-6)) was not detected.

Optimization of enzyme activity from strain CNU 120806

To exert enzyme activity under the optimum conditions, the pH value and temperature of the reaction system were investigated. The optimum pH and temperature were 5 and 50°C, respectively. These results indicated that the enzyme produced by CNU 120806 was stable in the moderate acidic condition.

Biotransformation pathway of ginsenoside Rb1 (2) by strain CNU 120806

Based on the structures of ginsenoside Rb1 (2) and gypenoside LXXV (1), we can theoretically propose two pathways for the biotransformation of ginsenoside Rb1 (2) to gypenoside LXXV (1) (Fig. 2). It was obvious that one is through the intermediate gypenoside XVII (3) by sequentially hydrolysing the two glucoses at C-3 position and the other is by directly hydrolysing the inner glucopyranosyl moieties at the C-3 position. To understand the biotransformation pathway of ginsenoside Rb1 (2) by CNU 120806, we determined the liquid fermentation process of strain CNU 120806 cultured with ginsenoside Rb1 (2). The results revealed that strain CNU120806 converted ginsenoside Rb1 (2) into gypenoside LXXV (1) through the intermediate gypenoside XVII (3) according to the analyses of HPLC method (Fig. S1, Supporting Information). After adding ginsenoside Rb1 (2) to the culture medium of strain CNU120806, the content of ginsenoside Rb1 (2) gradually decreased and gypenoside XVII (3) and gypenoside LXXV (1) gradually increased from 1st day to 11th days. Gypenoside XVII (3) reached its maximum at 5th day and then began to decrease. Meantime, the content of gypenoside LXXV (1) remarkably increased and reached maximum at 11th day. These aforementioned results proved that ginsenoside Rb1 (2) was indirectly converted to gypenoside LXXV (1) with the intermediate Gypenoside XVII (3).

Figure 2.

Biotransformation pathway of ginsenoside Rb1 (2) by the strain CNU 120806.

However, liquid fermentation was a time-consuming process; hence, we turned to use purified enzyme solution to investigate the pathway of ginsenoside Rb1 (2). To verify the hydrolysis occurred at C-3 position of ginsenoside, ginsenoside Rd (4) was also tested under the same reaction condition. Ginsenoside Rb1 (2) and ginsenoside Rd (4) were treated with purified enzyme solution derived from strain CNU 120806 for 30 h. The quantity variations of each individual ginsenoside were shown in Fig. 3 and Fig. 4. After the incubation for 30 h, the yield of gypenoside LXXV (1) was 95·4%. On the other hand, ginsenoside Rd (4) was hydrolysed to Compound K (6) through an intermediate F2 (5). The proposed pathway was shown in Fig. 5. The yield of enzymatic production of Compound K (6) was moderate (49·6%) compared with the product yield (95·4%) of ginsenoside Rb1 (2).

Figure 3.

Product variation during the conversion process of ginsenoside Rb1 (2) by the strain CNU 120806. ■ – Rb1 (2); ● – gypenoside XVII (3); ▲ – gypenoside LXXV (1).

Figure 4.

Product variation during the conversion process of ginsenoside Rd (4) by the strain CNU 120806. ■ – Rd (4); ● – F2 (5); ▲ – Compound K (6).

Figure 5.

Biotransformation pathway of ginsenoside Rd (4) by the strain CNU 120806.

Structural identification

The metabolites of ginsenoside Rb1 (2) by strain CNU 120806 determined by HPLC was also identified by 1H-NMR and 13C-NMR to confirm their authentic structures because the same retention time in HPLC analysis of standard gypenoside LXXV (1) and gypenoside XVII (3) was not enough to establish the product structure. These compounds were prepared and dissolved in pyridine-d5 and analysed by NMR spectroscopy. The metabolites ① and ② were assumed as LXXV (1) and gypenoside XVII (3).

In the NMR spectrum of metabolite ①, three anomeric proton signals were observed at δ 5·12 (1H, d, = 7·2 Hz,), δ 4·95 (1H, d, = 7·2 Hz,) and δ 5·09 (1H, d, = 7·2). Metabolite ② was similar to ①, but lacking one glucose unit. The left corresponding carbon signals were detected at δC 98·1 (C″) and δC 105·3 (C′). The 13 C-NMR: Compound ① aglycone moiety: C1-C30: 39·6, 27·0, 89·3, 40·1, 56·8, 18·9, 35·4, 40·5, 50·5, 37·0, 31·2, 70·7, 50·0, 51·9, 30·9, 27·1, 52·1, 16·5, 16·8, 83·8, 22·9, 36·7, 23·7, 126·1, 131·5, 26·3, 18·2, 28·3, 17·0, 17·5; Sugar moiety C1′-C6′: 105·2, 75·6, 78·3, 72·0, 78·8, 63·3; C1″–C6″: 98·3, 75·2, 79·3, 72·0, 77,4, 69·8; C1‴–C6‴: 105·9, 75·5, 78·3, 71·7, 78·9, 63·0. Compound ② aglycone moiety: C1-C30: 39·6, 26·7, 89·1, 39·8, 56·5, 19·0, 35·3, 39·9, 50·4, 36·7, 31·0, 70·2, 49·7, 51·7, 30·6, 26·9, 51·4, 16·1, 16·5, 83·9, 22·7, 36·5, 23·4, 125·9, 131·4, 25·8, 18·1, 28·3, 16·6, 17·7; Sugar moiety C1″–C6″: 98·1, 75·0, 79·3, 71·8, 77,1, 69·9; C1‴–C6‴: 105·5, 75·4, 78·5, 71·9, 78·5, 63·1. These data were consistent with the values in the study by An et al. (2010).

Furthermore, the metabolite ③ was the final product of ginsenoside Rd (4). Compound ③ was identified by ESI-MS. Fragments m/z 645[M+Na]+, m/z 623[M+H]+, gave a molecular weight of 622; m/z 443 [M-OH-162]+, providing that there existed a terminal glucosyl; m/z 425 [M-OH-162-H2O]+, m/z 407 [M-OH-162-2H2O]+. These data were consistent with the values in the study by Zhou et al. (2006). The mass spectrogram is shown in Fig. 6.

Figure 6.

Electrospray ionization mass spectrogram (positive ion mode) of metabolite ③ ginsenoside Rd (4) by strain CNU120806.

Discussions

Low abundant gypenoside LXXV (1) was the intermediate product in previous reports (Chi and Ji 2005; Kim et al. 2010; Yan et al. 2010). Most microbial transformations of ginsenoside have been focused on C-20 carbon because of the practical existence of large steric hindrance. Therefore, the final products of protopanaxadiol saponins mostly turned to be Compound K (6) and protopanaxadiol. Theoretically, the enzymatic biotransformation of ginsenoside Rb1 (2) into gypenoside LXXV (1) was easier because C-3 has much smaller steric hindrance than that of C-20. However, this type of conversion has seldom been observed before the present study.

It is well known that preparation of low abundant ginsenoside has been widely investigated by many methods including chemical and physical treatment or modification. However, biocatalytic approaches have substantial advantages because of the high selectivity, mild reaction conditions and environmental compatibility. The enzyme of high selectivity was not easily accessible if required certain product. To find out the specific enzyme, the strain CNU 120806 was employed to prepare low abundant gypenoside LXXV (1). After the purification process, the enzyme was characterized using different aryl-glycosides and disaccharides as substrates, as well as pH and temperature tests. Compared with its activity against pNPG (100%), the β-glucosidase exhibited quite lower level of activity against other aryl-glycosides. Furthermore, the activity of sophorose (β-(1-2)) was relatively high (94·3%). Because ginsenoside Rb1 contains four β-glucosidic linkages including a C-20, β-(1-6) and a C-3, β-(1-2) linkage, we can address conversion of Rb1 (2) should produce gypenoside LXXV (1) and gypenoside XVII (3).

Considering the biotransformation processes carried out above, the results of ginsenoside Rd (4) again proved that strain CNU 120806 had the ability to hydrolyse the glucopyranosyl moieties of C-3 carbon of ginsenoside. It is noteworthy that the objective product can only be obtained in the hypha and the substrate residual only existed in the liquid layer in fermentation process. To some extent, it is convenient to purify and prepare the product and as well as recycle the substrate. On the other hand, in the purified enzyme solution, the conversion process was greatly reduced on time course.

Interestingly, in contrast to previous reports (An et al. 2010; Yan et al. 2010) with similar pathway, gypenoside LXXV (1) was the final product of strain CNU120806 with the substrate ginsenoside Rb1 (2). This result is new and differs from the fact that gypenoside LXXV (1) would further hydrolyse to Compound K in An et al.'s (2010) researches. Generally, the β-glucosidase produced in various fungal strains was complex. It is therefore the metabolite converted by different fungi was not supposed to be a specific one in consideration of ginsenoside structure that was multiple identified with various glycoside bonds. In the present study, the enzyme produced by CNU 120806 shows strongly selectivity and successfully simulated the biotransformation process in vitro.

Consequently, it was shown here that consecutive hydrolyses of the terminal and inner glucopyranosyl moieties at the C-3 carbon of ginsenosides Rb1 (2) and Rd (4) proposed the pathways: ginsenoside Rb1 (2)→ gypenoside XVII (3)→ gypenoside LXXV(1); ginsenoside Rd (4)→F2 (5)→Compound K (6), which corresponded with the regioselective hydrolysis. The properties of the enzyme could be a useful tool in the biotransformation of commercial ginsenosides, especially for gypenoside LXXV (1).

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