Bioconversion of Ginsenoside Rb1 to the Pharmaceutical Ginsenoside Compound K using Aspergillus usamii KCTC 6954
Bioconversion of Ginsenoside Rb1 to the Pharmaceutical Ginsenoside Compound K using Aspergillus usamii KCTC 6954
Microbiology and Biotechnology Letters. 2014. Dec, 42(4): 347-353
Copyright © 2014, The Korean Society for Microbiology and Biotechnology
  • Received : July 25, 2014
  • Accepted : October 10, 2014
  • Published : December 28, 2014
Export by style
Cited by
About the Authors
미나 조
지은 정
현주 윤
경훈 장
희숙 지
기태 김
현동 백

β-Glucosidase from Aspergillus usamii KCTC 6954 was used to convert ginsenoside Rb1 to compound K, which has a high bio-functional activity. The enzymatic activities during culturing for 15 days were determined using ρ-nitrophenyl-β-glucopyranoside. The growth rate of the strain and the enzymatic activity were maximized after 6 days (IU; 175.93 μM ml -1 min -1 ). The activities were maximized at 60℃ in pH 6.0. During culturing, Rb1 was converted to Rd after 9 d and then finally converted to compound K at 15 d. In the enzymatic reaction, Rb1 was converted to the ginsenoside Rd within 1 h of reaction time and compound K could be detected after 8 h. As a result, this study demonstrates that Rb1→Rd→F2→compound K is the main metabolic pathway catalyzed by β-glucosidase and that β-glucosidase is a feasible option for the development of specific bioconversion processes to obtain minor ginsenosides such as Rd and compound K.
Ginseng has been used as a traditional medicine in Asian countries for a long time. Ginsenoside, i.e., ginseng saponin, is responsible for the biological and pharmacological activities of ginseng species such as Panax ginseng Meyer, Panax quinquefolius L., Panax notoginseng , and Panax japonicus . Ginsenosides, glycosides with steroids or triterpenes as the aglycon, account for 2-10% in ginseng root [4] .
Ginsenosides can be classified into three different groups according to the chemical structure of the aglycon: protopanaxadiol (PPD), protopanaxatriol (PPT), and oleanane saponin (OA). In addition, PPD, PPT, and OA are subdivided into Rb1, Rd, Re, Rc, and so on, depending on the type, number, and sites of attachment of their sugar moieties [23] . Currently, more than 60 types of ginsenosides have been reported in the roots of Panax ginseng Meyer [15] . However, six major ginsenosides (Rb1, Rb2, Rc, Rd, Re, and Rg1) comprise 90% (w/w) of the total ginsenosides in ginseng [12] , and the rest is composed of minor ginsenosides. Despite their small numbers, minor ginsenosides including compound K have been known to have various physiological activities such as anticanceric [17 , 26] , antioxidative [13] , antifungal [26] , and antiinflammatory [18] effects as well as high bioavailability compared to major ginsenosides [20] .
Minor ginsenosides can be produced by hydrolysis of the sugar moieties from the major ginsenosides. Deglycosylation methods such as heating [11] , acid treatment [8] , and enzymatic conversion [7] have been developed for producing minor ginsenosides from major ginsenosides. However, chemical methods produce side reactions such as epimerization, hydration, and hydroxylation. Comparatively, enzymatic conversion is an efficient and environmentfriendly method [25] . However, the production of minor ginsenosides by biological techniques has not been practically studied for industrial applications such as pharmaceutical or functional food materials.
Aspergillus niger is used in food fermentation industry, especially since their products have been recognized as ‘generally recognized as safe’ (GRAS) by the FDA. Aspergillus usamii belongs to the A . niger group. The strain has been used to make Asian traditional fermented food such as doenjang meju [9] because these strains have various hydrolyzing enzymes such as amylase and protease. Recently, many researchers have studied on production of extracellular enzymes such as glucanase and glucoamylase [21] . In particular, β-glucosidase from A . niger have been already studied recently [3] , but that of A . usamii has not been studied yet. In study on industrial enzymatic treatments, there is a limit that the application of purified enzymes at a large scale is uneconomic due to its high cost for industrial production of minor ginsenosides.
Therefore, the objective of this study is to investigate the enzymatic conversion of ginsenosides Rb1 to compound K by using enzymes, particularly β-glucosidase, produced by using Aspergillus usamii KCTC 6954 as basic information for industrial production.
Materials and Methods
- Microorganisms
A . usamii KCTC 6954 was purchased from the Korean Collection for Type Cultures (KCTC; Daejeon, Korea). The fungus was grown on potato dextrose agar at 30℃ for 4 days and the stock culture was maintained at 4℃. Broth medium was composed of glucose [10 g/l], peptone [5 g/l], malt extract [3 g/l], and yeast extract [3 g/l]), and the medium was inoculated with 5% (v/v) of 4-day-culture. The culture broth was incubated at 200 rpm at 30℃ for 15 days. Glass beads were added into the culture to prevent lumping of the mycelia. During culturing, a broth was sampled at 3-day-intervals for 15 days for detection of β-glucosidase activity. In addition, the cell growth was determined at 600 nm with a spectrophotometer (Optizen 2120 UV plus, Mecasys Co. Ltd., Korea).
- Materials
Ginseng harvested in Young-dong, Korea was purchased from a local market in Seoul, Korea. Stems and leaves of the ginseng were collected for the extraction and then dried in an oven (OF12GW, Jeio-Tech Co., Seoul, Korea) at 60℃ for 10 h until a moisture content of 4 to 5% was reached (w/w). The dried stems and leaves were ground to a particle size of less than 10 mm using a high speed mixer (Blender 7012S, Waring, Torrington, CT, USA) and were stored at 4℃ until used.
Ginsenoside Rb1, Rd, F2, Rh2, and compound K were purchased from LKT Laboratories, Inc. (St. Paul, MI, USA). ρ-nitrophenyl-β-glucopyranoside (ρNPG), ρ-nitrophenol (ρNP), and β-glucosidase from almond were purchased from Sigma Aldrich (St. Louis, MO, USA). Peptone, yeast extract, and malt extract were purchased from BD Company (Miller, Becton Dickinson and Co., Sparks, MD, USA). Glucose was purchased from Merck (Darmstadt, Germany). All solvents used in chromatography were of HPLC grade, and other chemicals were of analytical reagent grade.
- Assay of β-glucosidase activity
Culture broth sampled was centrifuged at 14,000 × g for 10 min at 4℃, and the supernatant was used as crude enzyme. Enzyme activity of culture broth was evaluated by a colorimetric method using ρNPG as the substrate [19] . The reaction mixture, which contained 1 ml of 5 mM ρNPG and 100 μl of crude enzyme solution, was reacted at 50℃ for 10 min. The reaction was terminated by adding 0.5 M sodium carbohydrate (1 ml), and the absorbance of the released ρ-nitrophenyl (ρNP) was determined at 400 nm and the amount was calculated by using a standard calibration curve.
- Effects of temperature and pH on enzyme activity
The temperatures were regulated at 30, 40, 50, 60, 70, 80, and 90℃ to detect an optimal temperature [28] . The effect of pH on enzyme activity was tested at optimum temperature (60℃) with different buffers (50 mM). The pH value were controlled at the pH range of 3.0 to 9.0 by using citrate buffer (pH 3.0), acetate buffer (pH 4.0 and 5.0), sodium phosphate buffer (pH 6.0, 7.0, and 8.0), and Tris buffer (pH 9.0), respectively. Enzymatic activities on various temperatures and pH’s were assayed by the methods mentioned above.
- Bioconversion of the ginsenoside Rb1
Enzymatic conversion of the ginsenoside Rb1 was performed at 60℃ for 48 h. The culture supernatant (100 μl) obtained from centrifugation at 14,000 × g for 10 min at 4℃ was reacted with 0.1 mM ginsenoside Rb1 (100 μl) on a heating block [2] . The reaction mixture was extracted twice with 400 μl of water-saturated n-butanol. The water-saturated n-butanol fraction was lyophilized to obtain the crude saponin fraction. Crude saponin was dissolved in 50 μl of methanol, and then analyzed by thin layer chromatography (TLC) and high performance liquid chromatography (HPLC) [10] .
- TLC and HPLC analysis for determination of ginsenosides
TLC was performed on Silica gel 60F 254 plates and the developing solvent was composed of chloroform, methanol, and water (65:35:10 v/v/v). The spots were detected by spraying them with 0.2% ρ-anisaldehyde and heating them under a lamp flame. Ginsenoside Rb1 and converted ginsenosides were compared with the ginsenoside standards (Rb1, Rd, F2, compound K, and Rh2) [6 , 10] .
HPLC was performed using an Agilent 1100 system (Agilent Technologies, Palo Alto, CA, USA) with a UV detection wavelength of 203 nm. The column was a reversed phase column (Eclipse XDB-C18; 4.6 × 160 mm, particle size; 5 μm, Agilent Techonlogies, USA), and the injection volume was 20 μl. The mobile phase utilized gradient conditions with solvents A (CH 3 CN:H 2 O = 100:0) and B (CH 3 CN:H 2 O = 14:86). The solvent A and B ratios were as follows: 20% A (0 min); 20% A (5 min); 30% A (10 min); 30% A (15 min); 60% A (20 min); 60% A (23 min); and 0% A (25 min), with a 1.2 ml/min flow rate [2 , 10] .
- Statistical analysis
All experiments were performed in triplicate. The data was analyzed by using SPSS 18 (Chicago, IL, USA). The mean values were determined by one-way analysis of variance (ANOVA) followed by Duncan’s multiple range test ( p < 0.05).
Results and Discussion
- Production of crude enzyme fromA.usamiiKCTC 6954
Aspergillus species are known to be useful source of β-glucosidase production [24] , and A . usamii KCTC 6954 was the most efficient producer of β-glucosidase among the microorganisms investigated in our previous study (data not shown). Changes in the cell growth and β-glucosidase activity of A . usamii KCTC 6954 at 30℃ were observed under aerobic conditions ( Fig. 1 ). The growth of A . usamii KCTC 6954 sharply increased for 8 days, and then slowed for the subsequent 8 days. β-Glucosidase activity also sharply increased for 6 days (175.93 μM ml -1 min -1 ) at a middle of logarithmic phase. In this phase, it presumed that the cells capable of transforming the primary carbon source into biosynthetic precursor and Lu et al . presented the similar results of enzyme production in this study [16] . After 9 days of culturing, β-glucosidase activity was a little decreased, not significantly ( p < 0.05), at this high level for the duration of the experiment.
PPT Slide
Lager Image
Cell growth and β-glucosidase activity of A. usamii KCTC 6954. ●, β-glucosidase activity (μM ml-1 min-1); ○, growth curve. Enzymatic activities were assayed by the absorbance of the released ρ-nitrophenyl (ρNP) at 400 nm and the cell numbers were detected by the absorbance at 600 nm.
- Effect of pH and temperature on β-glucosidase activity
The effect of pH on β-glucosidase activity was surveyed at the pH range of 3.0 to 9.0. High levels of β-glucosidase activity were detected (151.10 μM ml -1 min -1 ) in the pH range. However, enzymatic activity was lost at pH 7.0 (4.11 μM ml -1 min -1 , Fig. 2A ). Our results showed that the optimal temperature for β-glucosidase activity was 60℃ (179.92 μM ml -1 min -1 ). The enzyme activity was maintained at 70℃ (171.87 μM ml -1 min -1 ), but it sharply decreased to 82.92 μM ml -1 min -1 at 80℃ ( Fig. 2B ). These results are similar to those of the previous studies, which identified the optimal temperature of A . niger and A . oryzae to be 60℃, and that of A . usamii D5 to be 55℃ [1 , 10 , 22] .
PPT Slide
Lager Image
Effect of (A) pH and (B) temperature on β-glucosidase activity. All data were calculated as relative activities. The pH conditions were prepared with citrate buffer (pH 3.0), acetate buffer (pH 4.0-5.0), sodium phosphate buffer (pH 6.0-8.0), and Tris buffer (pH 9.0). β-glucosidase activities were assayed after reaction for 10 min at each temperature.
- Detection of bioconversion of the ginsenoside Rb1 during culturing ofA.usamiiKCTC 6954
Comparison of the enzymatic conversion of the ginsenoside Rb 1 and the growth of A . usamii KCTC 6954 is shown in Fig. 3 . The enzymatic transformation of the ginsenoside Rb1 was performed at 60℃ for 48 h and detected by TLC ( Fig. 3A ) and HPLC ( Fig. 3B ). The enzymes of the 6-day-cultures were unable to convert the ginsenoside Rb1. However, the enzymes of the 9-day-culture converted almost all the ginsenoside Rb1 to Rd; their retention times (RT) were 7.667 min and 10.741 min, respectively. All the ginsenoside Rb1 was converted to Rd, F2 (RT, 17.019 min), and compound K (RT, 21.933 min) by the 12-day-culture. The enzymes of the 15-day-culture were completely converted the ginsenoside Rb1 to compound K. It has been known that Aspergillus sp. g48p strain from the traditional Chinese preparation Koji can hydrolyze PPD ginsenoside [27] , and A . niger can convert the ginsenoside R f to PPT [14] . By these data, it was shown that A . usamii also can bio-convert major ginsenosides during culturing.
PPT Slide
Lager Image
Analysis of conversion patterns of the ginsenoside Rb1 during culturing of A. usamii KCTC 6954 with (A) TLC and (B) HPLC analysis. TLC was done by using silica gel 60F254 as a stationary phase and chloroform:methanol:water (=65:35:10 (v/v/v)) as a mobile phase (S: standard). In HPLC, Eclipse XDB-C18 column was used and a mobile phase was utilized with gradient conditions with CH3CN:H2O (100:0) and CH3CN:H2O (14:86). 1, Rb1; 2, Rd; 3, F2; 4, compound K (CK).
- Reaction time of ginsenoside Rb1 conversion
The reaction time required for the enzymatic conversion of the ginsenoside Rb1 was investigated using the supernatant of the 15-day-culture. The reaction was performed at 60℃ for 48 h, and the ginsenoside pattern was analyzed by TLC ( Fig. 4A ) and HPLC ( Fig. 4B ). In the absence of a reaction time, the ginsenoside Rb1 (RT, 5.551 min) and a small quantity of Rd (RT, 10.088 min) were detected. However, all the ginsenoside Rb1 was converted to Rd following the 1-h reaction. The compound K (RT, 21.933 min) was observed after a reaction of time of 8 h, and all the ginsenosides were converted to compound K after the 48-h reaction.
PPT Slide
Lager Image
Conversion pattern of the ginsenoside Rb1 during enzymatic reaction time. (A) TLC analysis. S: standard; C: Rb1+ medium. (B) HPLC analysis. TLC was done by using silica gel 60F254 as a stationary phase and chloroform:methanol: water (=65:35:10 (v/v/v)) as a mobile phase (S: standard). In HPLC, Eclipse XDB-C18 column was used and a mobile phase was utilized with gradient conditions with CH3CN:H2O (100:0) and CH3CN:H2O (14:86). 1, Rb1; 2, Rd; 3, F2; 4, compound K (CK); 5, Rh2.
In conclusion, the results of HPLC demonstrated that ginsenoside Rb1→Rd→F2→compound K ( Fig. 5 ) is the main metabolic pathway catalyzed by A . usamii KCTC 6954 enzymes indicating that this result is similar to the pathway in Actinosynnema mirum [5] . Specifically, β-glucosidase from A . usamii KCTC 6954 hydrolyzes two different glucosidic linkages of ginsenoside Rb1. That is, the former is to hydrolyze the β-1,6-glucosidic linkage of C-20 in ginsenoside Rb1 to produce ginsenoside Rd and the latter is to hydrolyze the β-1,2-glucosidic linkage of C-3 in ginsenoside Rd to produce ginsenoside F2. Furthermore, F2 can be deglucosylated to compound K by β-glucosidase. Importantly, it demonstrates that β-glucosidase from A . usamii KCTC 6954 is a feasible option for the development of specific bioconversion processes to obtain minor ginsenosides such as Rd, F2, and compound K which are more potent bio-functional materials for human health than major ginsenosides such as Rb1 are.
PPT Slide
Lager Image
Pathway of bioconversion of Rb1 to compound K.
In our further research, β-glucosidase of A . usamii related with a bioconversion of Rb1 will be purified and its characterization and optimization for activity will be determined in more detail. These results suggest that A . usamii strains can be practically applied to develop new functional materials in a pharmaceutical as well as a functional food industry as basic information.
This research was supported by Technology Development Program (Grant# 610002-03-02-SB220) through the Ministry for Food, Agriculture, Forestry and Fisheries (Republic of Korea) and by Priority Research Centers Program (Grant# 2009-0093824) through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology.
Barbagallo RN , Spagna G , Palmeri R , Restuccia C , Giudici P 2004 Selection, characterization and comparison of β-glucosidase from mould and yeasts employable for enological applications Enzyme Microb. Technol. 35 58 - 66    DOI : 10.1016/j.enzmictec.2004.03.005
Chang KH , Jee HS , Lee NK , Park SH , Lee NW , Paik HD 2009 Optimization of the enzymatic production of 20(S)-ginsenoside Rg3 from white ginseng extract using response surface methodology New Biotechnol. 26 181 - 186    DOI : 10.1016/j.nbt.2009.08.011
Chang KH , Jo MN , Kim KT , Paik HD 2012 Purification and characterization of a ginsenoside Rb1-hydrolyzing β-glucosidase from Aspergillus niger KCCM 11239 Int. J. Mol. Sci. 13 12140 - 12152    DOI : 10.3390/ijms130912140
Choi JE , Nam KY , Li X , Kim BY , Cho HS , Hwang KB 2010 Change of chemical components and ginsenoside contents of different root parts of ginsengs with processing method Korean J. Med. Crop Sci. 18 118 - 125
Cui CH , Kim SC , Im WT 2013 Characterization of the ginsenoside-transforming recombinant beta-glucosidase from Actinosynnema mirum and bioconversion of major ginsenosides into minor ginsenosides Appl. Microbiol. Biotechnol. 97 649 - 659    DOI : 10.1007/s00253-012-4324-5
Fuzzati N 2004 Analysis methods of ginsenosides J. Chromatogr. B 812 119 - 133    DOI : 10.1016/S1570-0232(04)00645-2
Hongwei L , Xin L , Xiaohui Q , Ying H , Dacheng H , Yu C 2006 Purification and characterization of novel stable ginsenoside Rb1-hydrolyzing β-glucosidase from China white jade snail Process Biochem. 41 1974 - 1980    DOI : 10.1016/j.procbio.2006.04.011
In JG , Lee BS , Kim EJ , Park MH , Yang DC 2006 Increase of functional saponin by acid treatment and temperature of red ginseng extract Korean J. Plant Res. 19 139 - 143
Kim JW , Doo HS , Kwon TH , Kim YS , Shin DH 2011 Quality characteristics of Doenjang Meju fermentation with Aspergillus species and Bacillus subtilis during fermentation Korean J. Food Preserv. 18 397 - 406    DOI : 10.11002/kjfp.2011.18.3.397
Kim SK , Kwak YS , Kim SW , Hwang SY , Ko YS , Yoo CM 1998 Improved method for the preparation of crude ginseng saponin J. Ginseng Res. 22 155 - 160
Kim WY , Kim JM , Han SB , Lee SK , Kim ND , Park MK 2000 Steaming of ginseng at high temperature enhances biological activity J. Nat. Prod. 63 1702 - 1704    DOI : 10.1021/np990152b
Li WK , Gu CG , Zhang HJ , Awang DVC , Fitzloff JF , Fong HHS 2000 Use of high performance liquid chromatography tandem mass spectrometry to distinguish Panax ginseng C.A Meyer (Asian ginseng) and Panax quinquefolius. L. (America ginseng) Anal. Chem. 72 5417 - 5422    DOI : 10.1021/ac000650l
Lim SI , Cho CW , Choi UK , Kim YC 2010 Antioxidant activity and ginsenoside pattern of fermented white ginseng J. Ginseng Res. 34 168 - 174    DOI : 10.5142/jgr.2010.34.3.168
Liu L , Gu LJ , Zhang DL , Wang Z , Wang CY , Li Z 2010 Microbial conversion of rare ginsenoside Rf to 20(S)-protopanaxatriol by Aspergillus niger Biosci. Biotechnol. Biochem. 74 96 - 100    DOI : 10.1271/bbb.90596
Liu L , Zhu XM , Wang QJ , Zhang DL , Fang ZM , Wang CY 2010 Enzymatic preparation of 20(S, R)-protopanaxadiol by transformation of 20(S, R)-Rg3 from black ginseng Phytochemistry 71 1514 - 1520    DOI : 10.1016/j.phytochem.2010.05.007
Lu J , Weerasiri RR , Liu Y , Wang W , Ji S , Lee I 2013 Enzyme production by the mixed fungal culture with nano-shear pretreated biomass and lignocellulose hydrolysis Biotechnol. Bioeng. 110 2123 - 2130    DOI : 10.1002/bit.24883
Park D , Bae DW , Jeon JH , Lee J , Oh N , Yang G 2011 Immunopotentiation and antitumor effects of a ginsenoside Rg3-fortified red ginseng preparation in mice bearing H460 lung cancer cells Environ. Toxicol. Pharm. 31 397 - 405    DOI : 10.1016/j.etap.2011.01.008
Park HJ , Jung DH , Joo H , Kang NS , Jang SA , Lee JG 2010 The comparative study of anti-allergic and anti-inflammatory effects by fermented red ginseng and red ginseng Korean J. Plant Res. 23 415 - 422
Pyo YH , Lee TC , Lee YC 2005 Enrichment of bioactive isoflavones in soymilk fermented with β-glucosidase-producing lactic acid bacteria Food Res. Int. 38 551 - 559    DOI : 10.1016/j.foodres.2004.11.008
Ryu JS , Lee HJ , Bae SH , Kim SY , Park Y , Suh HJ 2013 The bioavailability of red ginseng extract fermented by Phellinus linteus J. Ginseng Res. 37 108 - 16    DOI : 10.5142/jgr.2013.37.108
Shi H , Yin X , Wu M , Tang C , Zhang H , Li J 2012 Cloning and bioinformatics analysis of an endoglucanase gene (Aucel12A) from Aspergillus usamii and its functional expression in Pichia pastoris J. Ind. Microbiol. Biotechnol. 39 347 - 357    DOI : 10.1007/s10295-011-1039-z
So JH , Do HJ , Rhee IK 2010 Purification and characterization of β-glucosidase from Aspergillus usamii D5 capable of hydrolyzing isoflavone glycosides in soybean and astragali radix J. Korean Soc. Appl. Biol. Chem. 53 626 - 633    DOI : 10.3839/jksabc.2010.095
Sua JH , Xua JH , Lu WY , Lin GQ 2006 Enzymatic transformation of ginsenoside Rg3 to Rh2 using newly isolated Fusarium proliferatum ECU2042 J. Mol. Catal. B-enzyme. 38 113 - 118    DOI : 10.1016/j.molcatb.2005.12.004
Singhania RR , Sukumaran RK , Rajasree KP , Mathew A , Gottumukkala L , Pandey A 2011 Properties of a major β-glucosidase-BGL1 from Aspergillus niger NII-08121 expressed differentially in response to carbon sources Process Biochem. 46 1521 - 1524    DOI : 10.1016/j.procbio.2011.04.006
Wang YT , Li XW , Jin HY , Yu Y , You JY 2007 Degradation of ginsenosides in root of Panax ginseng C. A. Mey. by highpressure microwave-assisted extraction J. Chin. Univ. 28 2264 - 2269
Xu LL , Han T , Wu JZ , Zhang QY , Zhang H , Huang BK 2009 Comparative research of chemical constituents, antifungal and antitumor properties of ether extracts of Panax ginseng and its endophytic fungus Phytomedicine 16 609 - 616    DOI : 10.1016/j.phymed.2009.03.014
Yu H , Liu Q , Zhang C , Lu M , Fu Y , Im WT 2009 A new ginsenosidase from Aspergillus strain hydrolyzing 20-O-multi-glycoside of PPD ginsenoside Process Biochem. 44 772 - 775    DOI : 10.1016/j.procbio.2009.02.005
Zhang C , Li D , Yu H , Zhang B , Jin F 2007 Purification and characterization of piceid-β-glucosidase from Aspergillus oryzae Process Biochem. 42 83 - 88    DOI : 10.1016/j.procbio.2006.07.019