Complete Biotransformation of Protopanaxadiol-Type Ginsenosides to

Feb 22, 2018 - The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.7b06108. ... of compoun...
1 downloads 5 Views 4MB Size
Download PDF
Article pubs.acs.org/JAFC

Cite This: J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Complete Biotransformation of Protopanaxadiol-Type Ginsenosides to 20‑O‑β-Glucopyranosyl-20(S)‑protopanaxadiol Using a Novel and Thermostable β‑Glucosidase Kyung-Chul Shin,† Tae-Hun Kim,† Ji-Hyeon Choi, and Deok-Kun Oh* Department of Bioscience and Biotechnology, Konkuk University, Seoul 05029, South Korea S Supporting Information *

ABSTRACT: The ginsenoside 20-O-β-glucopyranosyl-20(S)-protopanaxadiol, compound K, has attracted much attention in functional food, traditional medicine, and cosmetic industries because of diverse pharmaceutical activities. The effective production of compound K from ginseng extracts has been required. However, an enzyme capable of completely converting all protopanaxadiol (PPD)-type ginsenosides to compound K has not been reported until now. In this study, unlike other enzymes, β-glucosidase from Caldicellulosiruptor bescii was able to hydrolyze sugar moieties such as L-arabinofuranose as well as D-glucose and L-arabinopyranose as the C-20 outer sugar in ginsenosides. Thus, ginsenoside Rc containing L-arabinofuranose can be converted to compound K by only this enzyme. Under the optimized reaction conditions, the enzyme completely converted PPD-type ginsenosides in ginseng extracts to compound K with the highest productivity among the reported results. This is the first report of the enzyme capable of completely converting all PPD-type ginsenosides into compound K. KEYWORDS: ginsenoside, compound K, complete conversion, β-glucosidase, 20-O-β-glucopyranosyl-20(S)-protopanaxadiol

■

treatment,15 fermentation,16 whole-cell reaction,17 and enzymatic conversion18 have been attempted previously. Among these methods, enzymatic conversion showed the highest selectivity, yield, and productivity.19 Thus, the enzymatic production of compound K using β-glucosidases has been attempted.20−23 Thermostable β-glucosidases have been also applied to compound K production and they showed higher productivity for compound K than those of mesophilic βglucosidases.24−27 For the industrial production of compound K, it is economically more viable to use ginseng extract as a substrate than to use purified ginsenoside. So far, no enzymes have been reported to completely convert all PPD-type ginsenosides in ginseng extracts into compound K. Therefore, the complete conversion into compound K has been attempted using cocktails of enzymes.24,25 In this study, we characterized a β-glucosidase from Caldicellulosiruptor bescii, which could completely convert PPD-type ginsenosides in ginseng extracts into compound K and applied it to the production of compound K from ginseng extracts.

INTRODUCTION Ginsenosides are biological and pharmaceutical active components of ginseng (Panax ginseng C. A. Meyer), a commonly used herb.1 Most ginsenosides are classified into protopanaxadiol (PPD)- and protopanaxatriol (PPT)-types, and comprise a nonsugar component with PPD or PPT aglycone and a sugar component,2 which contains 1−4 molecule glycosides such as Dglucose, L-arabinopyranose, L-arabinofuranose, or D-xylose at the C-3 and C-20, and D-glucose, L-rhamnose, and D-xylose at the C6 and/or C-20, respectively (Figure 1). Ginsenosides are named in alphabetical order with a subscript number denoting their polarity in thin-layer chromatography (TLC).3 The abbreviations and full names of ginsenosides are shown in Table S1. Major ginsenosides Rb1, Rb2, Rc, Rd, Rg1, and Re constitute more than 80% of all ginsenosides in wild ginseng.4 Minor ginsenosides F2, Rg3, Rh1, Rh2, and compound K, which are deglycosylated from the major ginsenosides, exhibit higher biological activity than the major ginsenosides because of their smaller molecular size, higher bioavailability, and better permeability across the cell membrane.5 The PPD-type ginsenoside 20-O-β-glucopyranosyl-20(S)protopanaxadiol, known as compound K, is one of the most pharmaceutically active minor ginsenosides.6 It has attracted a lot of interest in recent years because of its diverse pharmaceutical activities such as anti-inflammatory,7 antiallergic,8 antitumor,9 and hepatoprotective10 effects. Compound K prepared by Snailase also showed antidiabetic activity.11 Additionally, compound K has been used as an additive in cosmetic products for skin protection as it can efficiently prevent wrinkles12 and skin damage.13 Compound K is not present in ginseng and can only be obtained by the hydrolysis of ginsenosides. To hydrolyze the sugar moieties in glycosylated ginsenosides, heating,14 acid © XXXX American Chemical Society

■

MATERIALS AND METHODS

Microbial Strains, Plasmids, and Gene Cloning. C. bescii DSM 6725 (DSMZ, Braunschweig, Germany), E. coli ER2566 strain, and pET-28a vector (Takara, Shiga, Japan) were used as the source for the DNA template of the β-glucosidase gene (Genbank accession no. ACM59590), host cells, and expression vector, respectively. The genomic DNA was extracted from C. bescii by a genomic DNA extract kit Received: Revised: Accepted: Published: A

December 27, 2017 February 21, 2018 February 22, 2018 February 22, 2018 DOI: 10.1021/acs.jafc.7b06108 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Article

Journal of Agricultural and Food Chemistry

Figure 1. Chemical structures of protopanaxadiol (PPD)- and protopanaxatriol (PPT)-type ginsenosides. PPD- and PPT-type ginsenosides contain glycosides at C-3 and C-20 and at C-6 and/or C-20, respectively. Abbreviations and full names of each ginsenoside are shown in Table S1. C-, compound; Gyp, gypenoside; Glu, β-D-glucopyranosyl; Arap, α-L-arabinopyranosyl; Xyl, β-D-xylopyranosyl; Araf, α-L-arabinofuranosyl; and Rha, α-Lrhamnopyranosyl. (Qiagen, Hilden, Germany). The β-glucosidase gene from C. bescii was cloned with the one-step isothermal assembly method.28 The DNA fragments of the expression vector and β-glucosidase gene were amplified by polymerase chain reaction (PCR) with the primers shown in the Table S4. Primers were designed with stop codon to express only six histidine residues in N-terminal position of pET-28a vector, which contained nucleotides encoding his-tag on the both N- and C-terminal. The amplified linearized vector and DNA fragments were ligated using a Gibson Assembly Master Mix (New England Biolabs, Herfordshire, U.K.), and the ligated DNA products were transformed into E. coli ER2566. Culture Conditions. The recombinant E. coli expressing βglucosidase from C. bescii was cultivated in a 2-L flask containing 500 mL Luria−Bertani (LB) medium mixed with 100 μg/mL kanamycin at 37 °C with agitation at 200 rpm. When the optical density of the culture reached 0.8 at 600 nm, 1.0 mM isopropyl-β-D-thiogalactopyranoside (IPTG) was added to the culture to induce β-glucosidase expression, and the cells were then incubated with shaking at 150 rpm at 16 °C for further 14 h. Enzyme Purification. Recombinant E. coli cells were harvested from culture broth and washed with 0.85% NaCl, suspended in 50 mM phosphate buffer (pH 7.0) containing 300 mM NaCl and 1 mg/mL lysozyme, and disrupted by sonication on ice for 30 min. The unbroken cells and cell debris were eliminated by centrifugation at 13000 × g for 20 min at 4 °C, and the cell-free supernatant was applied to a His-trap HP affinity chromatography column (GE Healthcare, Piscataway, NJ, U.S.A.). The bound protein was eluted with a linear gradient ranging of 10 to 250 mM imidazole in 50 mM phosphate buffer (pH 7.0) at a flow rate of 1 mL/min. The fractions containing active protein were collected and dialyzed against 50 mM citrate/phosphate buffer (pH 5.5) at 4 °C for 16 h. The dialyzed solution was used as the purified enzyme. The purification step with the column was performed in a cold room at 4 °C with a fast protein liquid chromatography system (Bio-Rad, Hercules, CA, U.S.A.). β-Glucosidase Activity Assay. Unless otherwise stated, the reaction was performed at 80 °C for 10 min in 50 mM citrate/ phosphate buffer (pH 5.5) containing 1 mM aryl-glycoside or 0.4 mM

ginsenoside. The activity of β-glucosidase for aryl-glycosides was determined by measuring the increase in absorbance at 405 nm due to the release of pNP. One unit (U) of β-glucosidase from C. bescii for pNPD-glucopyranose was defined as the amount of enzyme required to liberate 1 μmol of pNP per 1 min. The activity for ginsenoside was determined using a HPLC-system (1100; Agilent, Santa Clara, CA, U.S.A.). One unit (U) of β-glucosidase from C. bescii for ginsenoside was defined as the amount of enzyme required to liberate 1 μmol of ginsenoside Rd from ginsenoside Rb1 per 1 min. The enzyme activity (U/mg) was calculated as follows: U/mg = [(product concentration (mM)/time (min)) × vol (mL)]/amount (mg) of protein. Effects of pH and Temperature on the β-Glucosidase Activity. The effects of pH and temperature on the activity of β-glucosidase from C. bescii for pNP-D-glucopyranose and Rb1 were investigated by varying the pH from 4.5 to 6.5 in 50 mM citrate/phosphate buffer at 85 °C and by varying the temperature from 70 to 90 °C in 50 mM citrate/ phosphate buffer (pH 5.5), respectively. The reactions were performed in 50 mM citrate/phosphate buffer containing 0.005 U/mL βglucosidase (based on the unit definition for pNP) and 1 mM pNP-Dglucopyranose or 4 U/mL β-glucosidase (based on the unit definition for ginsenoside) and 0.4 mM Rb1 for 10 min. The effect of temperature on the stability of β-glucosidase from C. bescii was monitored as a function of incubation time by maintaining the solution of enzymes at five different temperatures (70, 75, 80, 85, and 90 °C) in 50 mM citrate/phosphate buffer (pH 5.5). Samples were withdrawn at regular time intervals and assayed. Biotransformation of PPD-Type Ginsenosides into Compound K. The biotransformation of Rb1, Rb2, and Rc (Ambo institute, Daejeon, South Korea) into compound K by β-glucosidase from C. bescii was performed at 80 °C in 50 mM citrate/phosphate buffer (pH 5.5) containing 10.2 U/mL β-glucosidase and 1 mM ginsenoside for 80, 120, and 180 min, respectively. Preparation of Ginseng Root and Leaf Extracts. Roots and leaves of P. ginseng were separately dried for 48 h at 40 °C in a forced air convection oven. The dried roots or leaves were ground to powder with less than 0.5 mm diameter using a mixer, and then thoroughly blended and homogenized using a mixer mill (Rersch MM400; Haan, Germany). B

DOI: 10.1021/acs.jafc.7b06108 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Article

Journal of Agricultural and Food Chemistry The resultant fine ginseng root or leaf powder was added to 1 L of 80% (v/v) methanol and incubated at 80 °C for 1 h. After cooling, each mixture was filtered through a 0.45 μm filter. The obtained filtrate was evaporated to remove methanol, and the residue was dissolved in 1 L of distilled water. The dissolved solution was adsorbed onto a Diaion HP20 resin, which was then rinsed with distilled water to remove free sugars. The ginsenosides attached to the resin were successively eluted with methanol, and the methanol in the eluent was removed with an evaporator. The residue obtained was dissolved in the same volume of distilled water as the original loading volume. The resultant sugar-free ginseng root and leaf extracts would prevent the Maillard reaction between free sugars and the enzyme, which occur at temperatures above 70 °C. Compound K Production from Ginseng Root and Leaf Extracts. The optimal concentration of β-glucosidase for compound K production from ginseng root and leaf extracts was determined by varying the concentration of β-glucosidase from 241 to 1928 U/mL (from 1 to 8 mg/mL) with 2 mM PPD-type ginsenosides in ginseng root and leaf extracts. To determine the optimal substrate concentration, the concentrations of PPD-type ginsenosides in ginseng root and leaf extracts were varied from 0.5 to 5 mM and from 0.5 to 6 mM, at a constant enzyme concentration of 964 U/mL (4 mg/mL) and 723 U/ mL (3 mg/mL), respectively. The reactions were performed in 50 mM citrate/phosphate buffer (pH 5.5) for 3 h at 80 and 75 °C for ginseng root and leaf extracts, respectively. The time-course reactions for converting PPD-type ginsenosides to compound K were performed in 50 mM citrate/phosphate buffer (pH 5.5) with 964 U/mL (4 mg/mL) of β-glucosidase and 3 and 2 mM of PPD-type ginsenosides in the ginseng root and leaf extracts at 80 and 75 °C for 5 and 8 h, respectively. HPLC Analysis. The reaction was stopped and the product was extracted simultaneously by adding an equal volume of n-butanol with digoxin. Digoxin was used as an internal standard because it had similar sensitivity and different retention time with ginsenosides. The n-butanol fraction was then evaporated until the solution was completely dry, and methanol was added. The ginsenosides were assayed at a wavelength of 203 nm using the HPLC system equipped with a UV detector and a C18 column (4.6 × 150 mm, 5 μm particle size; YMC, Kyoto, Japan). The column was eluted at 37 °C for 45 min at a flow rate of 1 mL/min with the following gradient of acetonitrile/water (v/v): from 30:70 to 60:40 for 20 min; from 60:40 to 90:10 for 10 min; from 90:10 to 30:70 for 5 min; and constant at 30:70 for 10 min. All of the ginsenosides were quantified by the calibration curves using the ginsenoside standards, which were purchased from BTGin (Daejon, Korea).

Figure 2. Thermal inactivation of β-glucosidase from C. bescii for ginsenoside Rb1. The enzyme was incubated at 70 (●), 75 (○), 80 (■), 85 (□), and 90 °C (▲) in 50 mM citrate/phosphate buffer (pH 5.5) for various periods of time. A sample was withdrawn at each time point and assayed in 50 mM citrate/phosphate buffer (pH 5.5) containing 0.4 mM ginsenoside Rb1 and 4 U/mL enzyme at 80 °C for 10 min. Data represent the means of three experiments, and error bars represent the standard deviations.

Table 1. Hydrolytic Activity of β-Glucosidase from C. bescii for PPD-Type Ginsenosides

■

RESULTS AND DISCUSSION Gene Cloning, Expression, and Purification of βGlucosidase from C. bescii. The gene (1,356 bp) encoding β-glucosidase from the hyperthermophile bacterium C. bescii was cloned and expressed in Escherichia coli. The recombinant enzyme was purified as a soluble protein from crude extract using His-trap affinity chromatography. C. bescii β-glucosidase was purified with a final purification of 14-fold, a yield of 21% and a specific activity of 49.7 U/mg for pNP-β-D-glucopyranoside. The expressed enzyme was determined as a single band by SDSPAGE, with a molecular mass of approximately 53 kDa (Figure S1), which was almost consistent with the molecular weight calculated from 452 amino acids with the hexahistidine tag (53 194 Da). The amino acid sequence of C. bescii β-glucosidase exhibited 9, 23, and 24% identities with the characterized compound Kproducing β-glucosidases from Microbacterium esteraromaticum,29 Sulfolobus solfataricus,26 and Sulfolobus acidocaldarius,27 respectively. Effects of pH and Temperature on the Activity of βGlucosidase from C. bescii. The hydrolytic activity of βglucosidase from C. bescii was examined over a pH range from 4.5

a

substratea

specific activityb (U mg−1)

Rb1 Rb2 Rb3 Rc Rd F2 compound O compound Y compound Mc1 compound Mc compound K

20.4 ± 0.14 18.9 ± 0.26 17.5 ± 0.12 0.9 ± 0.04 1.3 ± 0.11 1.9 ± 0.02 1.6 ± 0.05 37.9 ± 0.14 0.5 ± 0.01 0.2 ± 0.04 0.0

Substrate concentration: 0.4 Mm. bU = μmol/min.

to 6.5 at 85 °C using ginsenoside Rb1 as the substrate. The maximum activity was observed at pH 5.5 (Figure S2A). The activity at pH 4.5 or 6.0 was less than 40% of the maximum activity, representing that the enzyme was sensitive to changes in pH. The effect of temperature on the enzyme activity was investigated at pH 5.5, and maximum activity was recorded at 80 °C (Figure S2B). Relative activity was 80% at 70 °C, while it dropped to almost 20% at 90 °C. Similarly, when p-nitrophenol (pNP)-D-glucopyranose was used as a substrate, the activity was maximal at pH 5.5 and 80 °C (Figure S3). The effect of temperature on enzyme stability was investigated by varying the temperature from 75 to 95 °C for various periods of time (Figure 2). β-Glucosidase from C. bescii showed the firstorder kinetics for thermal inactivation, and its half-lives at 70, 75, 80, 85, and 90 °C were 96, 29, 6.2, 0.1, and 0.03 h, respectively. All the reactions, except for the reaction with ginseng leaf extract, were terminated within 5 h, and so, the reactions were performed at 80 °C. The reaction with ginseng leaf extract was performed at 75 °C, as the reaction time (10 h) was longer than the half-life of C

DOI: 10.1021/acs.jafc.7b06108 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Article

Journal of Agricultural and Food Chemistry

Figure 3. HPLC profiles of ginsenosides in experiment for substrate specificity of β-glucosidase from C. bescii. When Rb1 (1), Rb2 (2), Rb3 (3), Rc (4), Rd (5), F2 (6), compound Mc1 (7), compound O (8), compound Mc (9), or compound Y (10) were used as a substrate, the ginsenoside was converted into other ginsenoside(s). Compound K (11) was not converted by β-glucosidase from C. bescii. The reactions were performed at 80 °C for 10 min in 50 mM citrate/phosphate buffer (pH 5.5) containing 0.4 mM ginsenoside. C-, compound.

Figure 4. Biotransformation of PPD-type ginsenosides into compound K. Ginsenosides Rb1, Rb2, and Rc were converted into compound K via ginsenosides Rd and F2. The symbols ●, ⧫, ▲, △, □, and ○ represent Rb1, Rb2, Rc, Rd, F2, and compound K, respectively. Data represent the means of three experiments, and error bars represent the standard deviations.

the enzyme at 80 °C (6.2 h) and shorter than that at 75 °C (29 h). Substrate Specificity of β-Glucosidase from C. bescii. The substrate specificity of β-glucosidase from C. bescii was

investigated using aryl-glycosides and ginsenosides. The specific activity of the enzyme with aryl-glycosides as substrates was in the following order: oNP-β-D-glucopyranoside > pNP-β-Dglucopyranoside > pNP-β-D-galactopyranoside > oNP-β-DD

DOI: 10.1021/acs.jafc.7b06108 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Article

Journal of Agricultural and Food Chemistry

Table 2. Biotransformation of Reagent-Grade Ginsenosides and PPD-Type Ginsenosides in Ginseng Extracts into Compound K by Enzymes substrate reagent grade Rb1

Rb2 Rc ginseng extract ginseng root

ginseng leaf a

microorganism

enzyme

molar yield (%)

productivity (μM/h)

Microbacterium esteraromaticum Paecilomyces bainer Sulfolobus acidocaldarius Caldicellulosiruptor bescii Microbacterium esteraromaticum Caldicellulosiruptor bescii Caldicellulosiruptor bescii

β-glucosidase β-glucosidase β-glucosidase β-glucosidase β-glucosidase β-glucosidase β-Glucosidase

77 84 94 100 23 100 100

738 NCa 283 1000 13 500 400

Aspergillus niger Sulfolobus acidocaldarius Sulfolobus solfataricus Caldicellulosiruptor bescii Caldicellulosiruptor bescii

pectinase β-glucosidase β-glucosidase β-glucosidase β-glucosidase

NC 69 80 100 100

87 80 218 600 250

reference 29 22 27

this study 30

this study this study 31 25 26

this study this study

NC, not calculated.

Figure 5. Production of compound K from PPD-type ginsenosides in ginseng root extract and ginseng leaf extract. The symbols ●, ⧫, ▲, △, □, and ○ represent Rb1, Rb2, Rc, Rd, F2, and compound K, respectively. Data represent the means of three experiments, and error bars represent the standard deviations. HPLC profiles shows the change of ginsenosides before acd after reactions using ginseng root extract and ginseng leaf extract. The numbers 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 represent Re, Rg1, Rf, Rb1, Rc, Rb2, Rd, F1, F2, and compound K, respectively.

bescii was the only compound K-producing β-glucosidase to exhibit activity for pNP-α-L-arabinofuranoside. The specific activity of the enzyme with PPD-type ginsenosides as substrates showed the following order: compound Y > Rb1 > Rb2 > Rb3 > F2 > compound O > Rd > Rc > compound Mc1 > compound Mc; no activity was found for compound K (Table 1). The HPLC profiles of the ginsenosides derived from these PPD-type reagent-grade ginsenosides after the reaction with C. bescii β-glucosidase are shown in Figure 3. The activity of the enzyme for ginsenosides with 4 sugars followed the order Rb1 > Rb2 > Rb3 > Rc, which contained glucose, arabinopyranose, xylose, and arabinofuranose, respectively, at the C-20 outer position. This order for glycosides in ginsenosides was the same

galactopyranoside > pNP-α-L-arabinopyranoside > pNP-β-Dxylopyranoside > oNP-β-D-xylopyranoside > pNP-α-L-arabinofuranoside (Supporting Information, Table S2). β-Glucosidase from C. bescii acted on different linkages in glycosides, including β-1,2-, β-1,4-, α-1,2-, and α-1,4-linkages. When the other compound K-producing β-glucosidases from M. esteraromaticum,29 S. solfataricus,26 and S. acidocaldarius27 were used with aryl-glycosides as substrates, the highest hydrolytic activity was observed for pNP-β-D-glucopyranoside, pNP-β-D-glucopyranoside, and oNP-β-D-galactopyranoside, respectively. The hydrolytic activity of β-glucosidase from C. bescii was the highest for oNP-β-D-glucopyranoside. Furthermore, β-glucosidase from C. E

DOI: 10.1021/acs.jafc.7b06108 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Article

Journal of Agricultural and Food Chemistry

Figure 6. Biotransformation pathway for the conversion of PPD-type ginsenosides Rb1, Rb2, and Rc in ginseng root and leaf extracts into compound K via Rd and F2.

that of β-glucosidase from C. bescii. β-Glucosidase from S. acidocaldarius combined with α-L-arabinofuranosidase from C. saccharolyticus25 also completely converted ginsenoside Rc into compound K, with a productivity almost similar to that of βglucosidase from C. bescii. Thus, β-glucosidase from C. bescii was the only single enzyme that could completely convert Rb1, Rb2, and Rc into compound K, with the highest productivity. Production of Compound K from PPD-Type Ginsenosides in Ginseng Extracts. Ginseng root extract (20%, w/v) and leaf extract (20%, w/v) contained 2.31 and 1.24 mg/mL PPD-type ginsenosides, which constituted 55 and 38% (w/w) of total ginsenosides, respectively (Table S3), and thus, ginseng root extract is more suitable for compound K production than ginseng leaf extract. However, ginseng leaf extract is a cheap substrate for compound K production, as ginseng leaves are discarded during harvesting. The effect of the concentration of C. bescii β-glucosidase on compound K production was studied on ginseng root and leaf extracts with 2 mM PPD-type ginsenosides by varying the concentration of β-glucosidase from 241 to 1926 U/mL (1−8 mg/mL; Figures S4A and S5A). Compound K production increased with increasing enzyme concentration. However, compound K production per enzyme concentration significantly decreased above concentrations of 963 and 722 U/mL for ginseng root and leaf extracts, respectively. Thus, the optimal concentration of the enzyme for the production of compound K from 2 mM PPD-type ginsenosides in ginseng root and leaf extracts was 964 U/mL (4 mg/mL) and 723 U/mL (3 mg/mL), respectively. To investigate the effect of substrate concentration on compound K production, the concentrations of PPD-type

as that for aryl-glycosides, which was pNP-β-D-glucopyranoside > pNP-α-L-arabinopyranoside > pNP-β-D-xylopyranoside > pNPα-L-arabinofuranoside. β-Glucosidase from C. bescii was the only compound K-producing β-glucosidase that converted ginsenoside Rc into Rd by catalyzing the cleavage of the outer arabinofuranose residue at the C-20 position, whereas other compound K-producing β-glucosidases converted ginsenoside Rc into compound Mc by catalyzing the cleavages of two glucoses at the C-3 position, indicating that they could not catalyze the cleavage of arabinofuranose. Biotransformation of Ginsenosides Rb1, Rb2, and Rc into Compound K by β-Glucosidase from C. bescii. No compound K was formed when the reaction was run under the same experimental conditions without enzymes. The biotransformation of the major ginsenosides Rb1, Rb2, and Rc to compound K by β-glucosidase from C. bescii was performed with 1 mM major ginsenoside as a substrate. The enzyme converted Rb1, Rb2, and Rc to compound K via Rd and F2 within 60, 120, and 150 min, with molar yields of 100% and productivities of 1000, 500, and 400 μM/h, respectively (Figure 4). The production of compound K from different reagent-grade major ginsenosides through enzymatic biotransformation is shown in Table 2. The productivity of β-glucosidase from C. bescii for the production of compound K from Rb1 and Rb2 was 1.4- and 38-fold, respectively, higher than that of β-glucosidase from M. esteraromaticum,29 which had the highest productivities reported so far. Furthermore, β-glucosidase from C. bescii was the only enzyme that could produce compound K from ginsenoside Rc. A combination of β-glucosidases from S. acidocaldarius and C. saccharolyticus25 completely converted the ginsenoside Rb2 to compound K. However, its productivity was 1.6-fold lower than F

DOI: 10.1021/acs.jafc.7b06108 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Article

Journal of Agricultural and Food Chemistry ginsenosides in ginseng root and leaf extracts were varied from 0.5 to 6 mM and from 0.5 to 5 mM, respectively. The yield of compound K decreased with increasing substrate concentrations (Figures S4B and S5B). However, the maximum production of compound K was observed at 3 and 2 mM of PPD-type ginsenosides in ginseng root and leaf extracts, respectively, and these concentrations were therefore considered optimal. Under the optimized reaction conditions, β-glucosidase from C. bescii produced 3 mM and 2 mM of compound K from 3 mM and 2 mM PPD-type ginsenosides in ginseng root and leaf extracts, with productivities of 600 and 250 μM/h, respectively. The production was performed by the transformation pathway: Rb1, Rb2, and Rc → Rd → F2 → compound K (Figures 5 and 6). Nevertheless, β-glucosidase from C. bescii was not active on any PPT-type gisnenosides. The enzymatic production of compound K from the different PPD-type ginsenosides in ginseng extracts is shown in Table 2. There are no reports so far on the production of compound K from ginseng leaf extract. β-Glucosidase from C. bescii produced compound K from ginseng leaf extract, however, the productivity from ginseng leaf extract was lower than that from ginseng root extract because ginseng leaf extract contained more PPT-type ginsenosides than PPD-type ones. The compositions of PPTand PPD-type ginsenosides in 20% (w/v) ginseng root extract and leaf extracts are shown in Table S3. It is notable that PPDtype ginsenosides present in discarded ginseng leaf were completely converted to compound K. Until now, a single enzyme has never been capable of completely converting PPDtype ginsenosides into compound K. However, a combination of β-glucosidase from S. acidocaldarius, α-L-arabinofuranosidase from C. saccharolyticus, and β-galactosidase from C. saccharolyticus25 completely converted PPD-type ginsenosides in ginseng root extract into compound K, with a productivity of 231 μM/h. This productivity was still 2.6-fold lower than that achieved by β-glucosidase from C. bescii. Therefore, βglucosidase from C. bescii completely converted PPD-type ginsenosides in ginseng root extract into compound K, with the highest productivity reported thus far. In conclusion, the biochemical properties of β-glucosidase from C. bescii were characterized, and the reaction conditions for producing compound K were optimized. Under the optimized conditions, the enzyme completely converted all the PPD-type ginsenosides in ginseng root extract into compound K, with the highest productivity reported thus far. To the best of our knowledge, β-glucosidase from C. bescii is the first enzyme capable of completely converting all PPD-type ginsenosides into compound K. Moreover, compound K was also produced from PPD-type ginsenosides present in discarded ginseng leaves, with a molar yield of 100%. Our results would provide great contributions to improving the industrial production of compound K by biotransformation.

■

■

analysis of β-glucosidase from C. bescii. Lane M, protein marker; lane A, pellet; lane B, crude extract; and lane C, purified enzyme. Figure S2: Effects of (A) pH and (B) temperature on the activity of β-glucosidase from C. bescii for ginsenoside Rb1. Figure S3: Effects of (A) pH and (B) temperature on the activity of β-glucosidase from C. bescii for pNP-β-D-glucopyranoside. Figure S4: Effects of the concentrations of (A) C. bescii β-glucosidase and (B) PPDtype ginsenosides in ginseng root extract on the production of compound K. Figure S5: Effects of the concentrations of (A) C. bescii β-glucosidase and (B) PPDtype ginsenosides in ginseng leaf extract on the production of compound K (PDF).

AUTHOR INFORMATION

Corresponding Author

*Phone: (822) 454-3118. Fax: (822) 444-5518. E-mail: [email protected]. ORCID

Deok-Kun Oh: 0000-0002-6886-7589 Author Contributions †

K.-C.S. and T.-H.K. equally contributed to this work.

Funding

This study was supported by Konkuk University in 2016. Notes

The authors declare no competing financial interest.

■

REFERENCES

(1) Lu, J. M.; Yao, Q.; Chen, C. Ginseng compounds: an update on their molecular mechanisms and medical applications. Curr. Vasc. Pharmacol. 2009, 7, 293−302. (2) Shin, K. C.; Oh, D. K. Classification of glycosidases that hydrolyze the specific positions and types of sugar moieties in ginsenosides. Crit. Rev. Biotechnol. 2016, 36, 1036−1049. (3) Jia, L.; Zhao, Y. Current evaluation of the millennium phytomedicine–ginseng (I): etymology, pharmacognosy, phytochemistry, market and regulations. Curr. Med. Chem. 2009, 16, 2475−2484. (4) Kim, M. W. K.; Choi, K. J.; Kim, S. C. Distribution of saponin in various sections of Panax ginseng root and changes of its contents according to root age. J. Ginseng. Sci. 1987, 11, 10−16. (5) Kim, M. K.; Lee, J. W.; Lee, K. Y.; Yang, D. C. Microbial conversion of major ginsenoside rb(1) to pharmaceutically active minor ginsenoside rd. J. Microbiol. 2005, 43, 456−462. (6) Akao, T.; Kanaoka, M.; Kobashi, K. Appearance of compound K, a major metabolite of ginsenoside Rb1 by intestinal bacteria, in rat plasma after oral administration–measurement of compound K by enzyme immunoassay. Biol. Pharm. Bull. 1998, 21, 245−249. (7) Park, J. S.; Park, E. M.; Kim, D. H.; Jung, K.; Jung, J. S.; Lee, E. J.; Hyun, J. W.; Kang, J. L.; Kim, H. S. Anti-inflammatory mechanism of ginseng saponins in activated microglia. J. Neuroimmunol. 2009, 209, 40−49. (8) Choo, M. K.; Park, E. K.; Han, M. J.; Kim, D. H. Antiallergic activity of ginseng and its ginsenosides. Planta Med. 2003, 69, 518−522. (9) Choi, K.; Kim, M.; Ryu, J.; Choi, C. Ginsenosides compound K and Rh(2) inhibit tumor necrosis factor-alpha-induced activation of the NFkappaB and JNK pathways in human astroglial cells. Neurosci. Lett. 2007, 421, 37−41. (10) Lee, H. U.; Bae, E. A.; Han, M. J.; Kim, N. J.; Kim, D. H. Hepatoprotective effect of ginsenoside Rb1 and compound K on tertbutyl hydroperoxide-induced liver injury. Liver Int. 2005, 25, 1069− 1073. (11) Li, W.; Zhang, M.; Gu, J.; Meng, Z. J.; Zhao, L. C.; Zheng, Y. N.; Chen, L.; Yang, G. L. Hypoglycemic effect of protopanaxadiol-type ginsenosides and compound K on Type 2 diabetes mice induced by

ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.7b06108. Table S1: Abbreviations of ginsenosides. Table S2: Hydrolytic activity of β-glucosidase from C. bescii for aryl-glycosides. Table S3: Contents of protopanaxatriol (PPT)- and protopanaxadiol (PPD)-type ginsenosides in 20% (w/v) ginseng root extract and leaf extracts. Table S4: Sequences of the primers used. Figure S1: SDS-PAGE G

DOI: 10.1021/acs.jafc.7b06108 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Article

Journal of Agricultural and Food Chemistry high-fat diet combining with streptozotocin via suppression of hepatic gluconeogenesis. Fitoterapia 2012, 83, 192−198. (12) Lim, T. G.; Jeon, A. J.; Yoon, J. H.; Song, D.; Kim, J. E.; Kwon, J. Y.; Kim, J. R.; Kang, N. J.; Park, J. S.; Yeom, M. H.; Oh, D. K.; Lim, Y.; Lee, C. C.; Lee, C. Y.; Lee, K. W. 20-O-beta-D-glucopyranosyl-20(S)protopanaxadiol, a metabolite of ginsenoside Rb1, enhances the production of hyaluronic acid through the activation of ERK and Akt mediated by Src tyrosin kinase in human keratinocytes. Int. J. Mol. Med. 2015, 35, 1388−1394. (13) Shin, D. J.; Kim, J. E.; Lim, T. G.; Jeong, E. H.; Park, G.; Kang, N. J.; Park, J. S.; Yeom, M. H.; Oh, D. K.; Bode, A. M.; Dong, Z.; Lee, H. J.; Lee, K. W. 20-O-beta-D-glucopyranosyl-20(S)-protopanaxadiol suppresses UV-Induced MMP-1 expression through AMPK-mediated mTOR inhibition as a downstream of the PKA-LKB1 pathway. J. Cell. Biochem. 2014, 115, 1702−1711. (14) Kim, W. Y.; Kim, J. M.; Han, S. B.; Lee, S. K.; Kim, N. D.; Park, M. K.; Kim, C. K.; Park, J. H. Steaming of ginseng at high temperature enhances biological activity. J. Nat. Prod. 2000, 63, 1702−1704. (15) Bae, E. A.; Han, M. J.; Kim, E. J.; Kim, D. H. Transformation of ginseng saponins to ginsenoside Rh2 by acids and human intestinal bacteria and biological activities of their transformants. Arch. Pharmacal Res. 2004, 27, 61−67. (16) Zhou, W.; Yan, Q.; Li, J. Y.; Zhang, X. C.; Zhou, P. Biotransformation of Panax notoginseng saponins into ginsenoside compound K production by Paecilomyces bainier sp. 229. J. Appl. Microbiol. 2008, 104, 699−706. (17) Cui, C. H.; Choi, T. E.; Yu, H.; Jin, F.; Lee, S. T.; Kim, S. C.; Im, W. T. Mucilaginibacter composti sp. nov., with ginsenoside converting activity, isolated from compost. J. Microbiol. 2011, 49, 393−398. (18) Shin, K. C.; Lee, H. J.; Oh, D. K. Substrate specificity of betaglucosidase from Gordonia terrae for ginsenosides and its application in the production of ginsenosides Rg(3), Rg(2), and Rh(1) from ginseng root extract. J. Biosci. Bioeng. 2015, 119, 497−504. (19) Park, C. S.; Yoo, M. H.; Noh, K. H.; Oh, D. K. Biotransformation of ginsenosides by hydrolyzing the sugar moieties of ginsenosides using microbial glycosidases. Appl. Microbiol. Biotechnol. 2010, 87, 9−19. (20) Park, S. Y.; Bae, E. A.; Sung, J. H.; Lee, S. K.; Kim, D. H. Purification and characterization of ginsenoside Rb1-metabolizing betaglucosidase from Fusobacterium K-60, a human intestinal anaerobic bacterium. Biosci., Biotechnol., Biochem. 2001, 65, 1163−1169. (21) Cheng, L. Q.; Kim, M. K.; Lee, J. W.; Lee, Y. J.; Yang, D. C. Conversion of major ginsenoside Rb1 to ginsenoside F2 by Caulobacter leidyia. Biotechnol. Lett. 2006, 28, 1121−1127. (22) Yan, Q.; Zhou, W.; Shi, X.; Zhou, P.; Ju, D.; Feng, M. Biotransformation pathways of ginsenoside Rb1 to compound K by βglucosidases in fungus Paecilomyces Bainier sp. 229. Process Biochem. 2010, 45, 1550−1556. (23) Quan, L. H.; Kim, Y. J.; Li, G. H.; Choi, K. T.; Yang, D. C. Microbial transformation of ginsenoside Rb1 to compound K by Lactobacillus paralimentarius. World J. Microbiol. Biotechnol. 2013, 29, 1001−1007. (24) Shin, K. C.; Choi, H. Y.; Seo, M. J.; Oh, D. K. Compound K production from red ginseng extract by beta-glycosidase from Sulfolobus solfataricus supplemented with alpha-L-arabinofuranosidase from Caldicellulosiruptor saccharolyticus. PLoS One 2015, 10, e0145876. (25) Shin, K. C.; Oh, H. J.; Kim, B. J.; Oh, D. K. Complete conversion of major protopanaxadiol ginsenosides to compound K by the combined use of alpha-L-arabinofuranosidase and beta-galactosidase from Caldicellulosiruptor saccharolyticus and beta-glucosidase from Sulfolobus acidocaldarius. J. Biotechnol. 2013, 167, 33−40. (26) Noh, K. H.; Son, J. W.; Kim, H. J.; Oh, D. K. Ginsenoside compound K production from ginseng root extract by a thermostable beta-glycosidase from Sulfolobus solfataricus. Biosci., Biotechnol., Biochem. 2009, 73, 316−321. (27) Noh, K. H.; Oh, D. K. Production of the rare ginsenosides compound K, compound Y, and compound Mc by a thermostable betaglycosidase from Sulfolobus acidocaldarius. Biol. Pharm. Bull. 2009, 32, 1830−1835.

(28) Gibson, D. G.; Young, L.; Chuang, R. Y.; Venter, J. C.; Hutchison, C. A., 3rd; Smith, H. O. Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat. Methods 2009, 6, 343−345. (29) Quan, L. H.; Min, J. W.; Jin, Y.; Wang, C.; Kim, Y. J.; Yang, D. C. Enzymatic biotransformation of ginsenoside Rb1 to compound K by recombinant beta-glucosidase from Microbacterium esteraromaticum. J. Agric. Food Chem. 2012, 60, 3776−3781. (30) Quan, L. H.; Jin, Y.; Wang, C.; Min, J. W.; Kim, Y. J.; Yang, D. C. Enzymatic transformation of the major ginsenoside Rb2 to minor compound Y and compound K by a ginsenoside-hydrolyzing betaglycosidase from Microbacterium esteraromaticum. J. Ind. Microbiol. Biotechnol. 2012, 39, 1557−1562. (31) Kim, B. H.; Lee, S. Y.; Cho, H. J.; You, S. N.; Kim, Y. J.; Park, Y. M.; Lee, J. K.; Baik, M. Y.; Park, C. S.; Ahn, S. C. Biotransformation of Korean Panax ginseng by Pectinex. Biol. Pharm. Bull. 2006, 29, 2472− 2478.

H

DOI: 10.1021/acs.jafc.7b06108 J. Agric. Food Chem. XXXX, XXX, XXX−XXX