Hydrolysis of the outer β-(1,2)-d-glucose linkage at the C-3 position of ginsenosides by a commercial β-galactosidase and its use in the production of minor ginsenosides

Article abstract of DOI:10.1080/10242422.2018.1483348

Commercial β-galactosidase from Aspergillus oryzae (SUMILACT L^TM) was used for the bioconversion of the ginsenosides Rb1, Rb2, Rc, Rd, and Rg3 to gypenoside-XVII, compound-O, compound-MC1, F2, and Rh2, respectively. The optimal conditions were

Full text of DOI:10.1080/10242422.2018.1483348

Biocatalysis and Biotransformation
ISSN: 1024-2422 (Print) 1029-2446 (Online) Journal homepage: http://www.tandfonline.com/loi/ibab20
Hydrolysis of the outer β-(1,2)-d-glucose linkage at
the C-3 position of ginsenosides by a commercial
β-galactosidase and its use in the production of
minor ginsenosides
Yeong-Su Kim, Do-Yeon Kim, Dong Wook Kang & Chang-Su Park
To cite this article: Yeong-Su Kim, Do-Yeon Kim, Dong Wook Kang & Chang-Su Park (2018):
Hydrolysis of the outer β-(1,2)-d-glucose linkage at the C-3 position of ginsenosides by a
commercial β-galactosidase and its use in the production of minor ginsenosides, Biocatalysis and
Biotransformation, DOI: 10.1080/10242422.2018.1483348
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BIOCATALYSIS AND BIOTRANSFORMATION
https://doi.org/10.1080/10242422.2018.1483348
RESEARCH ARTICLE
Hydrolysis of the outer b-(1,2)-D-glucose linkage at the C-3 position
of ginsenosides by a commercial b-galactosidase and its use in
the production of minor ginsenosides
Yeong-Su Kima , Do-Yeon Kimb , Dong Wook Kangc
and Chang-Su Parkd
b
aPlant Resource Industry Division, Baekdudaegan National Arboretum, Bonghwa, Republic of Korea; International Ginseng & Herb
c
Research Institute, Geumsan, Republic of Korea; Department of Pharmaceutical Science and Technology, Daegu Catholic University,
d
Gyeongsan, Republic of Korea; Department of Food Science and Technology, Daegu Catholic University, Gyeongsan,
Republic of Korea
ABSTRACT
ARTICLE HISTORY
Commercial b-galactosidase from Aspergillus oryzae (SUMILACT LTM) was used for the bioconver-
sion of the ginsenosides Rb1, Rb2, Rc, Rd, and Rg3 to gypenoside-XVII, compound-O, compound-
MC1, F2, and Rh2, respectively. The optimal conditions were pH 4.5, 50ꢀC, 60 UÁmLÀ1 enzyme,
and 8.0mM substrate. Interestingly, the enzyme hydrolyzed only the outer b-(1,2)-D-glucose
linkage at the C-3 position of ginsenosides. Under optimum conditions, the enzyme completely
converted Rb1, Rb2, Rc, Rd, and Rg3 to gypenoside-XVII, compound-O, compound-MC1, F2, and
Rh2, respectively, with the highest productivity.
Received 10 December 2017
Revised 4 April 2018
Accepted 19 May 2018
KEYWORDS
Gypenoside-XVII; com-
pound-MC1; compound-O;
bioconversion; SUMILACT
L
TM; ginsenoside
Introduction
implicated as the most promising method for the
preparation of minor ginsenosides, via the selective
hydrolysis of the sugar moieties of ginsenosides,
owing to its high specificity, conversion yield, and
productivity (Park et al. 2010).
Ginsenosides are secondary metabolites of ginseng
(the root of Panax ginseng Meyer), which has been
used as a traditional medicine to treat various diseases
for several thousand years. Ginsenosides possess vari-
ous pharmacological functions such as anti-cancer
(Chae et al. 2009; Song et al. 2009), anti-diabetic (Ni
et al. 2010), anti-hypertensive (Stavro et al. 2005), anti-
fatigue (Lee et al. 2005), and anti-oxidative activities
(Liu et al. 2003). There are two classes of ginsenosides:
major and minor. Because of their large size, low solu-
bility, and poor permeability, the human body cannot
directly absorb major ginsenosides. Minor ginseno-
sides, however, have better pharmaceutical activities,
because of their smaller size, higher solubility, and bet-
ter permeability (Karikura et al. 1991; Xu et al. 2003).
For this reason, the efficient conversion of major ginse-
nosides to minor ginsenosides is important for indus-
trial and medical applications.
Previous studies have examined the biotransform-
ation of minor ginsenosides such as Rb2 (Park et al.
2010), Rd (Shin et al. 2003; Son et al. 2008), Rg3 (Ko
et al. 2007; Chang et al. 2009), F2 (Ko et al. 2007), and
compound K (Ko et al. 2007) from major ginsenosides
using microbes or enzymes. However, there are only a
few studies examining the production of other minor
ginsenosides, including gypenoside-XVII, compound-O,
and compound-MC1 (Andreea Neculai et al. 2009; Hou
et al. 2012). These minor ginsenosides possess neuro-
protective effects, and possibly anti-genotoxic effects
in vitro as chemo preventive agents that moderate
cytotoxicity (Meng et al. 2014).
In this study, the ginsenoside Rb1, Rb2, Rc, Rd, and
Rg3 were converted into the minor ginsenosides gype-
noside-XVII, compound-O, compound-MC1, F2, and
Rh2, respectively, using a commercial b-galactosidase
(SUMILACT LTM) with C-3 outer glucose moiety-specific
hydrolyzing activity.
There are several methods to convert major ginse-
nosides to minor ginsenosides, including acid hydroly-
sis (Bae et al. 2004), heat treatment (Kim et al. 2000),
and microbial or enzymatic transformation (Son et al.
2008; Park et al. 2010). Enzymatic hydrolysis has been
CONTACT Chang-Su Park
parkcs@cu.ac.kr
Department of Food Science and Technology, Daegu Catholic University, Hayang-Ro 13-13, Hayang-Eup,
Gyeongsan 38430, Republic of Korea
Supplemental data for this article can be accessed here
ß 2018 Informa UK Limited, trading as Taylor & Francis Group

2
Y.-S. KIM ET AL.
biotransformation of Rb1 to gypenoside-XVII was
observed at pH 4.5 and 50 ꢀC (Figure 1), conditions
that also produce its maximum hydrolytic activity
with pNP-galactopyranoside. The effect of tempera-
ture on enzyme thermostability was investigated
(Figure 2). The half-lives of the enzyme at 30, 40, 50,
60, and 70 ꢀC were 87.6, 62.8, 35.7, 22.7, and 14.1 h,
respectively. The maximal pH of commercial lactase,
b-D-galactosidase, pectinase, hesperidinase, and narin-
ginase for bioconversion of ginsenosides were
reported to be 4.5, 4.5, 5.0, 4.0, and 6.5, respectively
(Ko et al. 2003, 2007; Kim et al. 2006). The maximum
activities of reported commercial glycosidases for bio-
conversion of ginsenosides were observed at 37 ꢀC,
except for pectinase and cellulose, which were max-
imal at 50 ꢀC (Ko et al. 2003, 2007; Kim et al. 2006).
These results indicate that SUMILACT LTM is a thermo-
stable option for ginsenoside production
Materials and methods
Materials
The ginsenosides Rb1, Rb2, Rc, Rd, Rg3, gypenoside-
XVII, compound-O, F2, Rh2, and compound-MC1 were
purchased from Ambo Institute (Daejeon, Korea). The
commercial b-galactosidase from Aspergillus oryzae
(SUMILACT LTM) was purchased from Shin Nihon
Chemical Co. Ltd. (Aichi, Japan).
Enzyme assay
Hydrolytic reactions with SUMILACT LTM were performed
at 50 ꢀC for 30min in 50 mM citrate/phosphate buffer
(pH 4.5) containing 0.5 mM ginsenoside and 6 UÁmLÀ1
enzyme. One unit (U) of enzyme activity was defined as
the amount of enzyme required to liberate 1 lmol of 4-
nitrophenyl-b-D-galactopyranoside per min at 50 ꢀC and
pH 4.5. All assays were performed in triplicate.
Production of gypenoside-XVII from ginsenoside
Rb1 by SUMILACT LTM
(a)
100
To evaluate the effect of the amount of enzyme on gype-
noside-XVII production, reactions were performed in
50 mM citrate/phosphate buffer (pH 4.5) containing 8 mM
ginsenoside Rb1 and 1.2–120 UÁmLÀ1 enzyme at 50 ꢀC for
60 min. Time courses for the production of gypenoside-
XVII, compound-O, compound-MC1, F2, and Rh2 by
SUMILACT LTM were performed at 50 ꢀC in 50mM citrate/
phosphate buffer (pH 4.5) containing 8 mM Rb1, Rb2, Rc,
Rd, or Rg3 and 60 UÁmLÀ1 enzyme at 50 ꢀC.
80
60
40
20
0
20
30
40
50
60
70
80
Temperature (°C)
Analytical methods
120
(b)
Ginsenosides were assayed using a high-performance
liquid chromatography (HPLC) system (Agilent
Technologies, Santa Clara, CA, USA) equipped with a
100
80
60
40
20
0
UV detector at 203 nm using
a
C18 column
(250 Â 4.6 mm, Agilent Technologies). The column
was initially eluted with 20:80 (v/v) acetonitrile:water
as the mobile phase, a gradient of 40:60 for 40 min,
and then a gradient of 80:20 for 80 min. The flow rate
was 1 mLÁminÀ1 and the column temperature
was 37 ꢀC.
2
3
4
5
6
7
8
pH
Results and discussion
SUMILACT LTM bioconversion activity on
ginsenosides
Figure 1. Effects of pH (A) and temperature (B) on gypeno-
side-XVII production from ginsenoside Rb1 by SUMILACT LTM
The reaction was performed in 50 mM citrate/phosphate buffer
(pH 5.5) containing 0.5 mM substrate at 50 ꢀC for 10 min. The
data represent the means of three experiments and the error
bars represent the SD.
.
The maximal hydrolytic activity of commercial
b-galactosidase from A. oryzae (SUMILACT LTM) for the

BIOCATALYSIS AND BIOTRANSFORMATION
3
of 637, 296, 102, and 85 UÁmgÀ1 for pNP-b-D-galactopyra-
noside, pNP-b-D-glucopyranoside, pNP-a-D-glucopyrano-
side, pNP-a-D-galactopyranoside, respectively (Table 1),
and no activity was found for pNP-a-L-arabinopyranose,
Substrate specificity of SUMILACT LTM for
ginsenosides
The hydrolytic activity of SUMILACT LTM with the aryl-
glycosides and ginsenosides (Rb1, Rb2, Rc, Rd, Rg3,
gypenoside-XVII, compound-O, and compound-MC1)
was investigated. The enzyme exhibited specific activities
pNP-a-L-arabinofuranose,
pNP-a-L-rhamnopyranoside,
and pNP-b-D-xylopyranoside. These results indicate
that SUMILACT LTM has narrow substrate specificity for
aryl-glycosides.
The enzyme exhibited specific activities of 124, 92,
71, 74, and 35 UÁmgÀ1 for ginsenoside Rb1, Rb2, Rc,
Rd, and Rg3, respectively, which have two b-D-gluco-
pyranosides at the C-3 position; however, gypenoside-
XVII, compound-O, compound-MC1, which contain
only inner b-(1,2)-D-glucose linkages at their C-3 posi-
tions, were not converted to other ginsenosides (Table
2, Figure 3). These results suggest that SUMILACT LTM
hydrolyzes only outer b-(1,2)-D-glucose linkages at the
C-3 position of ginsenosides, and therefore displays
100
80
60
40
20
10
0
5
10
15
20
25
30
Time (h)
Figure 2. Thermal inactivation of SUMILACT LTM at 30 ꢀC (open
circle), 40 ꢀC (open triangle), 50 ꢀC (open square), 60 ꢀC (closed cir-
cle), and 70 ꢀC (closed triangle). The enzymes were preincubated
at 30–70 ꢀC for varying times. At each time interval, the enzyme
activity was assayed in 50 mM citrate/phosphate buffer (pH 5.5)
containing 0.5 mM substrate at 50 ꢀC for 10 min. The experimental
data for thermal deactivation were fit to a first-order curve, and
the half-lives of the enzyme were calculated using Sigma Plot
10.0 Software. Error bars indicate the SD of triplicate samples.
Table 1. Substrate specificity of SUMILACT LTM for aryl-
glycosidess.
Substrate
Specific activity (UÁmgÀ1
)
pNP-b-D-galactopyranoside
pNP-b-D-glucopyranoside
pNP-a-D-glucopyranoside
pNP-a-D-galactopyranoside
pNP-a-L-arabinopyranose
pNP-a-L-arabinofuranose
pNP-a-L-rhamnopyranoside
pNP-b-D-xylopyranoside
637 18.4
296 2.1
102 9.8
85 1.2
N.D
N.D.
N.D.
N.D.
N.D.: not detected.
Table 2. Substrate specificity of SUMILACT LTM for ginsenosides.
Substrate
Product
Specific activity (UÁmgÀ1
)
Rb1
Rb2
Rc
Rd
Gypenoside-XVII
Compound-O
Compound-MC1
124 2.7
92 1.8
71 1.5
74 2.3
35 0.7
N.D.
F2
Rh2
–
–
–
Rg3
Gypenoside-XVII
Compound-O
Compound-MC1
Figure 3. Schematic representation of the hydrolysis
pathway of ginsenoside Rb1, Rb2, Rc, Rd, and Rg3 by
N.D.
N.D.
SUMILACT LTM
.
N.D.: not detected.

4
Y.-S. KIM ET AL.
narrow
substrate
specificity
for
ginsenosides
from Rb1 plateaued at approximately 60 UÁmLÀ1
(Figure 4). Thus, 60 UÁmLÀ1 was considered the opti-
mum enzyme concentration for gypenoside-XVII pro-
duction. We next assessed gypenoside-XVII production
with 60 UÁmLÀ1 enzyme for 60 min with 0.4–8 mM gin-
senoside Rb1. Regardless of the substrate concentra-
tion, ginsenoside Rb1 was completely converted to
gypenoside-XVII.
(Supplementary Figures S1 and S2). A recombinant
b-glucosidase from Sphingomonas sp. 2F2 (Wang et al.
2011) and Sphingopyxis alaskensis (Shin and Oh 2014)
reported also hydrolyzing outer C-3 glycoside of
ginsenoside. These two recombinant enzymes and
SUMILACT LTM have similar substrate specificity for
ginsenoside, however, the SUMILACT LTM is commer-
cial enzyme. There are some advantages to using
commercial enzyme in production of minor ginseno-
sides for food and cosmetic industry, such as enzyme
safety of environment, enzyme stability, and eco-
nomical production process control without need
for microbial culture and enzyme purification.
Other reported commercial ginsenoside hydrolysis
enzymes, such as Cytolase PCL5, Sumizyme AC,
Multifect Pectinase FE, and Pectinex Ultra AFP, have
broad substrate specificity (Kim et al. 2006). The C-20
outer glucose and arabinose hydrolysis enzymes are
reported to include b-glucosidase (Son et al. 2008),
a-L-arabinofuranosidase, and a-L-arabinopyranosidase
(Lee et al. 2011).
Production of gypenoside-XVII, compound-O, and
compound-MC1 from the ginsenosides Rb1, Rb2,
and Rc by SUMILACT LTM
The optimum conditions for the production of gype-
noside-XVII, compound-O, and compound-MC1 by
SUMILACT LTM were pH 4.5, 50 ꢀC, 60 UÁmLÀ1 enzyme,
and 8.0 mM substrate. Under optimum conditions, the
enzyme completely hydrolyzed 8 mM ginsenoside Rb1,
Rb2, Rc, Rd, and Rg3 to gypenoside-XVII, compound-O,
compound-MC1, F2, and Rh2 within 120, 240, 360,
360, and 540 min, respectively (Figure 5). SUMILACT
LTM produced the minor ginsenosides with conversion
yields of 100% and productivities of 4, 2, 1.3, 1.3, and
0.9 mmolÁLÀ1ÁhÀ1, respectively. b-Glucosidase from S.
alaskensis also hydrolyzes only the outer glucose at
the C-3 position, producing gypenoside-XVII from gin-
senoside Rb1 with a molar conversion yield of 100%
and a productivity of 7.1 mmolÁLÀ1ÁhÀ1 (Shin et al.
2013). From the above results, it is apparent that com-
mercial glycosidases are suitable candidates for minor
ginsenoside production.
SUMILACT LTM reaction conditions for ginsenoside
bioconversion
The effects of enzyme concentration on the produc-
tion of gypenoside-XVII were investigated at 50 ꢀC.
Reactions were performed with 8 mM ginsenoside Rb1
as
a
substrate and enzyme concentrations of
1.2–120 UÁmLÀ1 for 60 min. The substrate concentra-
tion was chosen to accommodate the solubility of gin-
senoside Rb1 in water. Gypenoside-XVII production
100
80
60
40
20
0
Conclusions
In this study, we have optimized the production of
gypenoside-XVII, compound-O, compound-MC1, F2,
and Rh2 from the ginsenosides Rb1, Rb2, Rc, Rd, and
Rg3, respectively, using a commercial b-galactosidase
from A. oryzae (SUMILACT LTM). SUMILACT LTM hydro-
lyzed only the outer b-(1,2)-D-glucose linkage at the
C-3 position of ginsenosides. The optimum condi-
tions for the production of gypenoside-XVII, com-
pound-O, and compound-MC1 by SUMILACT LTM
were pH 4.5, 50 ꢀC, 60 UÁmLÀ1 enzyme, and 8.0 mM
substrate. Under optimum conditions, the enzyme
completely converted the ginsenosides Rb1, Rb2, Rc,
Rd, and Rg3 to gypenoside-XVII, compound-O, com-
pound-MC1, F2, and Rh2, respectively, with high
productivity.
0
20
40
60
80
100
120
140
Enzyme concentration (U/ml)
Figure 4. Effect of enzyme concentration on gypenoside-
XVII production from ginsenoside Rb1. The reaction was
performed in 50 mM citrate/phosphate buffer (pH 5.5) con-
taining 8 mM substrate at 50 ꢀC for 60 min. The data repre-
sent the means of three experiments and the error bars
represent the SD.

BIOCATALYSIS AND BIOTRANSFORMATION
5
(a)
(b)
10
8
10
8
6
6
4
4
2
2
0
0
0
20
40
60
80
100
120
140
160
0
50
100
150
200
250
Time (min)
Time (min)
(c)
(d)
10
8
10
8
6
6
4
4
2
2
0
0
0
100
200
300
400
0
100
200
300
400
Time (min)
Time (min)
(e)
10
8
6
4
2
0
0
100
200
300
400
500
600
Time (min)
Figure 5. Production of (A) gypenoside-XVII (open circle), (B) compound-O (open triangle), (C) compound-MC1 (open square),
F2 (open diamond), and Rh2 (open inverted triangle) from Rb1 (closed circle), Rb2 (closed triangle), Rc (closed square), Rd
(closed diamond), and Rg3 (closed inverted triangle), respectively. The reaction was performed in 50 mM citrate/phosphate
buffer (pH 5.5) containing 8 mM substrate at 50 ꢀC. The data represent the means of three experiments and the error bars
represent the SD.
Disclosure statement
Funding
The authors report no conflicts of interest. The authors alone
are responsible for the content and writing of this article.
This work was supported by the Basic Science Research
Program through the National Research Foundation of Korea

6
Y.-S. KIM ET AL.
(NRF) funded by Ministry of Education under grant number
2015R1D1A1A01059570.
Lee JH, Hyun YJ, Kim DH. 2011. Cloning and characterization
of a-L-arabinofuranosidase and bifunctional a-L-arabinopyr-
anosidase/b-D-galactopyranosidase from Bifidobacterium
longum H-1. J Appl Microbiol. 111:1097–1107.
Liu ZQ, Luo XY, Liu GZ, Chen YP, Wang ZC, Sun YX. 2003. In
vitro study of the relationship between the structure of
ginsenoside and its antioxidative or prooxidative activity
in free radical induced hemolysis of human erythrocytes. J
Agric Food Chem. 51:2555–2558.
ORCID
Yeong-Su Kim
Do-Yeon Kim
Dong Wook Kang
Chang-Su Park
http://orcid.org/0000-0002-9831-2883
http://orcid.org/0000-0001-8007-7347
http://orcid.org/0000-0003-4626-512X
http://orcid.org/0000-0002-0770-5068
Meng X, Wang M, Sun G, Ye J, Zhou Y, Dong X, Wang T, Lu
S, Sun X. 2014. Attenuation of Ab 25-35-induced parallel
autophagic and apoptotic cell death by gypenoside XVII
through the estrogen receptor-dependent activation of
Nrf2/ARE pathways. Toxicol Appl Pharmacol. 279:63–75.
Ni HX, Yu NJ, Yang XH. 2010. The study of ginsenoside on
PPARgamma expression of mononuclear macrophage in
type 2 diabetes. Mol Biol Rep. 37:2975–2979.
Park CS, Yoo MH, Noh KH, Oh DK. 2010. Biotransformation of
ginsenosides by hydrolyzing the sugar moieties of ginse-
nosides using microbial glycosidases. Appl Microbiol
Biotechnol. 87:9–19.
Shin HY, Lee JH, Lee JY, Han YO, Han MJ, Kim DH. 2003.
Purification and characterization of ginsenoside Ra-hydro-
lyzing beta-D-xylosidase from Bifidobacterium breve K-
110, a human intestinal anaerobic bacterium. Biol Pharm
Bull. 26:1170–1173.
Shin KC, Oh DK. 2014. Characterization of a novel recombin-
ant b-glucosidase from Sphingopyxis alaskensis that spe-
cifically hydrolyzes the outer glucose at the C-3 position
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