Biotransformation of Protopanaxadiol-Type Ginsenosides in Korean Ginseng Extract into Food-Available Compound K by an Extracellular Enzyme from Aspergillus niger
Department of Bioscience and Biotechnology, Konkuk University, Seoul 05029, Republic of KoreaCorrespondence to:
J. Microbiol. Biotechnol. 2020; 30(10): 1560-1567
Published October 28, 2020
Copyright © The Korean Society for Microbiology and Biotechnology.
The root of Korean ginseng (
C-K, one of the most pharmacologically effective minor ginsenosides, has anti-allergic, anti-diabetic, anti-inflammatory, anti-tumor, and hepatoprotective effects . Since C-K is absent in natural ginseng, it can be transformed from the glycoside hydrolysis of major PPDGs like Rb1, Rb2, Rc, and Rd. The production of C-K from PPDGs in ginseng extract has been attempted through fermentation [4-11] and biotransformation using cells  and recombinant , commercial [14-16], and wild-type enzymes . The biotransformation using recombinant enzymes shows the highest yield, selectivity, and productivity for C-K production , however, the produced C-K is debatable on food-safety problems.
The use of enzymes originated from “generally recognized as safe (GRAS)” microorganisms is the proper way to solve food safety problems. GRAS microorganisms typically include lactic acid bacteria and some fungi that have been used for the manufacture of products in the food industry for a long time. Fungi are more suitable for C-K production than lactic acid bacteria because they are easier to grow in cheaper mediums and exhibit higher productivity . Fermentation using GRAS fungi is one of the most popular methods of C-K production. However, this method results in the formation of unnecessary by-products that can cause a problem in the product recovery. Moreover, fermentation exhibits low productivity owing to prolonged cultivation time . Although the productivity of C-K produced by extracellular enzymes from GRAS fungi is higher than that by fermentation, the productivity is still quite low.
In this study, we aimed to overcome the current disadvantages of C-K production such as food safety problems of recombinant enzymes and low productivities of enzymes from GRAS microorganisms. For those purposes, we chose
Materials and Methods
The PPDG standards such as Rb1, Rb2, Rc, Rd, compound Mc-1 (C-Mc-1), compound O (C-O), F2, compound Mc (C-Mc), compound Y (C-Y), and C-K (≥ 98.0% purity) were provided from Ambo Laboratories (Republic of Korea). The PPDG mixture from Korean ginseng (PPDKG) was purchased from Ace EMzyme (Republic of Korea). The extract from 4-year-old Korean ginseng roots was purified using a HP-20 resin for the enrichment of PPDGs. The detailed extraction method was previously described , and the partial purified ginsenosides were used as PPDKG. The
Medium, Culture Conditions, and Enzyme Preparation
The culture medium contained 20 g/l polysaccharide such as CMC, cellulose, ginseng powder, pectin from citrus, sugar beet sludge, or wheat bran, 10 g/l corn steep solid, 2 g/l KH2PO4, 0.3 g/l MnSO4·H2O, 0.3 g/l CaCl2, 5mg/l FeSO4·7H2O, 3.7 mg/l C°Cl2·6H2O, 1.4 mg/l ZnSO4·7H2O, and 1.3 mg/l MnSO4·H2O. The initial pH was 5.0. All fungi including
The culture broth (100 ml) was filtered, and solid ammonium sulfate was added to the filtrate with initially up to 30% and eventually 80% saturation. The precipitate was collected by centrifugation at 4°C at 13,000 ×
Effect of Inducer
The effect of the inducer added during cultivation on the C-K-producing activity of the extracellular enzyme was examined after cultivating the fungus for 6 days within 100 ml of the culture medium using a 500 ml-baffled flask. The inducer was 20 g/l of a polysaccharide such as CMC, cellulose, ginseng powder, pectin from citrus, sugar beet sludge, or wheat bran. The optimal concentration of CMC for obtaining extracellular enzyme with effective C-K-producing activity was determined by varying the concentration from 3 to 20 g/l. The reactions were carried out at 50°C for 6 h in 0.2 M citrate-phosphate buffer (pH 5.5) containing 1.0 mg/ml extracellular enzyme and 0.4 mg/ml of each ginsenoside (Rb1, Rb2, or Rc). The specific C-K-producing activity was defined as the amount of produced C-K/reaction time/amount of reacted enzyme. Thus, total C-K-producing activity referred to be multiplied by the amount of produced enzyme and the C-K-producing activity.
Properties of the Extracellular Enzyme
Unless otherwise noted, the reaction was performed containing 0.4 mg/ml ginsenoside in 0.2 M citrate-phosphate buffer (pH 5.0) at 55°C for 10 min as the optimum conditions. The glycoside-hydrolyzing activity of extracellular enzyme from
The effects of temperature and pH on the glycoside-hydrolyzing activity of ginsenoside Rb1 were investigated by varying the temperature from 40 to 65°C, at pH 5.5 and the pH value from 4.0 to 6.5 at 55°C for 10 min, respectively. The temperature stability of the extracellular enzyme was determined by measuring the residual activity after incubating in 0.2 M citrate-phosphate buffer (pH 5.0) at different temperatures from 40 to 65°C for 24 h. The pH stability was examined at 55°C for 24 h by measuring the residual activity after incubating the extracellular enzyme at different pH values from 4.0 to 6.5. The residual activity was decided after reacting the extracellular enzyme with ginsenoside Rb1 at 55°C and pH 5.0 for 10 min.
Biotransformation of PPDGs into Compound K
To investigate the hydrolyzing pathways and biotransformation of ginsenoside Rb1, Rb2, and Rc into C-K by the extracellular enzyme, the reactions were conducted at 55°C and pH 5.0 with 1.0 mg/ml of each ginsenoside and 2.5 mg/ml extracellular enzyme for 2 or 24 h.
To determine the optimal concentrations of substrate for C-K production, the concentrations of PPDGs in PPDKG were varied at 55°C and pH 5.0 for 12 h from 0.5 to 10.0 mg/ml (from 0.47 to 9.4 mM) with 1.0 mg/ml (1.8 U/ml) of the extracellular enzymes. The optimal concentration of the extracellular enzymes was determined by varying the concentration from 0.5 to 10.0 mg/ml (from 0.9 to 18.0 U/ml) with PPDKG as a substrate, respectively. The time-course reactions for the biotransformation of PPDGs to C-K were performed at 55°C for 18 h in 0.2 M citrate-phosphate buffer (pH 5.0) containing 8.0 mg/ml (14.4 U/ml) of the extracellular enzyme and 6.0 mg/ml (5.6 mM) of PPDGs in PPDKG.
The reaction mixture was extracted by adding the equal volume of n-butanol supplemented with 1.0 mg/ml digoxin as an internal standard. The fraction containing n-butanol was dried, and the residue was dissolved in methanol. The ginsenosides were analyzed using the HPLC system with an octadecylsilica column and a UV detector at 203 nm. The column was eluted at 40°C by ginsenoside solution containing digoxin with a linear gradient of solvents such as acetonitrile/water (v/v) from 30:70 to 60:40 for 20 min, 60:40 to 90:10 for 10 min, 90:10 to 30:70 for 5 min, and at a constant 30:70 for 10 min with a flow rate of 1 ml/min. All ginsenosides, including reagent ginsenosides, biotransformed ginsenosides, and ginsenosides in PPDGs, biotransformed PPDGs, PPDKG, and biotransformed PPDKG, were quantified by the calibration curves using the ginsenoside standards.
Results and discussion
Effect of Inducer Added during Cultivation on the C-K-Producing Activity of the Extracellular Enzyme for Ginsenosides Rb1, Rb2, and Rc
For finding a fungus that produces an effective glycoside-hydrolyzing enzyme, extracellular enzymes from a hundred of fungi were screened by measuring their C-K-producing activities for ginsenosides Rb2 and Rc. The extracellular enzyme from
The polysaccharide as a carbon source in the medium during cultivation of fungus is known to effectively induce the glycoside-hydrolyzing enzyme. For an example, wheat bran is used to induce the enzymatic activity of exoglucanase from
Biochemical Properties of the Extracellular Enzyme for Ginsenosides
The effects of temperature and pH on the glycoside-hydrolyzing activity of the extracellular enzyme were evaluated by the decrease of ginsenoside Rb1 as a substrate. Although the glycoside-hydrolyzing activity was a maximum at 60°C within 10 min, the residual activity significantly decreased after the reaction of the extracellular enzyme above 60°C for 24 h (Fig. S2A). The activity and stability were maximal at pH 5.0 (Fig. S2B). Therefore, the reaction temperature and pH for C-K production were determined as 55°C and 5.0, respectively.
Since the extracellular enzymes of fungi seemed to contain various enzymes, it was difficult to define exactly enzyme involved in the hydrolysis of specific ginsenoside without fractionation and high-purity refining of enzymes. Although enzymes were not defined exactly, the hydrolytic properties of extracellular enzyme for various sugars could be determined using
The glycoside-hydrolyzing activity of the extracellular enzyme for PPDGs as substrates followed the order Rb1 C-Mc1 > C-O > Rc > Rd > Rb2 > C-Mc > F2 > C-Y, but no activity was found for C-K (Table 1). The glycoside-hydrolyzing activity of the extracellular enzyme for major PPDGs followed the order Rb1 > Rc > Rd > Rb2. This order was the same as those for purified enzymes from
Biotransformation of Ginsenoside Rb1, Rb2, and Rc into Compound K by the Extracellular Enzyme
The reactions of time-course for the biotransformation of ginsenoside Rb1, Rb2, and Rc into C-K were conducted at 55°C and pH 5.0. The extracellular enzyme from
The extracellular enzymes from
Glycoside-Hydrolyzing Pathways of PPD-Type Ginsenosides into Compound K by the Extracellular Enzyme
Ginsenosides Rb1, Rb2, and Rc typically have glucose molecules at C3 and C20, however, the outer monosaccharide portions at C20 in the ginsenosides contain different monosaccharides such as glucose, arabinopyranose, and arabinofuranose, respectively. The intermediates of the conversion of Rb1, Rb2, and Rc as substrates into C-K by extracellular enzyme from
The biotransformation pathways of PPDGs into C-K by extracellular enzymes from
Biotransformation of PPD-type ginsenoside mixture from Korean ginseng extract into compound K by the extracellular enzyme
PPDKG contained 47.3% (w/w) PPDGs, including Rb1 (165 mg/g), Rc (100 mg/g), Rb2 (117 mg/g), and Rd (91 mg/g). The optimal concentration of PPDGs in PPDKG for C-K production was decided by varying the concentration from 0.5 to 10.0 mg/ml with 1.0 mg/ml extracellular enzyme. C-K production was reached 100%conversion yields when treating 0.5 mg/ml of PPDGs and the amount of produced compound K was steadily increased with a rise in the concentration of PPDGs. However, the rate of C-K production above 6.0 mg/ml decreased slightly (Fig. S5A). To determine the optimal concentration of the extracellular enzyme, the concentration was varied from 0.5 to 10.0 mg/ml with 6.0 mg/ml PPDGs in PPDKG. C-K production increased as the concentration of extracellular enzyme increased, however, C-K production above 8.0 mg/ml reached a plateau (Fig. S5B). Thus, the optimal concentrations of PPDGs and extracellular enzyme for C-K production were 6.0 mg/ml and 8.0 mg/ml, respectively.
The time-course reactions for the biotransformation of PPDGs in PPDKG into C-K were performed for 18 h under the optimized conditions of 55°C, pH 5.0, 6.0 mg/ml PPDGs, and 8.0 mg/ml extracellular enzyme. The extracellular enzyme converted 6.0 mg/ml (5.6 mM) PPDGs in PPDKG into 2.8 mg/ml (4.5 mM) C-K in 9 h, with a productivity of 313 mg/l/h and a molar conversion of 80% (Fig. 4). The biotransformation of PPDGs into C-K by crude enzymes from food-available wild-type microorganisms is presented in Table 2. The commercial enzyme Cytolase PCL5 converted white ginseng extract into 2.1 mg/ml C-K in 78 h, with a productivity 26.9 mg/l/h, which was the previously highest recorded concentration and productivity . The concentration and productivity of C-K by extracellular enzyme from
In conclusion, the biochemical properties of extracellular enzyme from
Supplementary data for this paper are available on-line only at http://jmb.or.kr.
This study was supported by the Individual Basic Science & Engineering Research Program through the National Research Foundation grant funded by the Ministry of Science and ICT, Republic of Korea (NRF-2017R1D1A1B03033762)
Conflict of Interest
The authors have no financial conflicts of interest to declare.
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