Enhancement of active compound, genipin, from Gardeniae Fructus using immobilized glycosyl hydrolase family 3 β-glucosidase from Lactobacillus antri

Article abstract of DOI:10.1186/s13568-017-0360-y

Geniposide is an iridoid glycoside, which is abundant in Gardeniae Fructus. Despite the various pharmaceutical effects of geniposide on a human body, its hydrolysis into a smaller molecule, genipin, by β-glucosidase produced by bacteria in the intestines is particularly important to improve geniposide uptake into the body. Since geniposide is much more abundant in Gardeniae Fructus than its aglycone genipin, we herein transformed geniposide into genipin using purified recombinant β-glucosidase from Lactobacillus antri (rBGLa), which was expressed in Escherichia coli to enhance the genipin content. Purified rBGLa was characterized using p-nitrophenyl β-d-glucopyranoside, and the optimal temperature and pH for its β-glucosidase activity were found to be 45?°C and 6.0. When the enzyme was immobilized, rBGLa was active at higher temperatures than the free enzyme, and we confirmed that its stability upon changes in pH and temperature was highly improved. Using 0.5?μg/mL free rBGLa, single compound of 0.4?mM geniposide was efficiently converted into genipin within 2?h, and the immobilized rBGLa also successfully transformed geniposide in a hot-water extract of Gardeniae Fructus into the aglycone, which makes it applicable to the food and pharmaceutical industries.

Full text of DOI:10.1186/s13568-017-0360-y

Kim et al. AMB Expr (2017) 7:64
DOI 10.1186/s13568-017-0360-y
Open Access
ORIGINAL ARTICLE
Enhancement of active compound,
genipin, from Gardeniae Fructus using
immobilized glycosyl hydrolase family 3
β-glucosidase from Lactobacillus antri
*
Young Soo Kim, Chung‑Jo Lee and Jin Yeul Ma
Abstract
Geniposide is an iridoid glycoside, which is abundant in Gardeniae Fructus. Despite the various pharmaceutical
effects of geniposide on a human body, its hydrolysis into a smaller molecule, genipin, by β‑glucosidase produced by
bacteria in the intestines is particularly important to improve geniposide uptake into the body. Since geniposide is
much more abundant in Gardeniae Fructus than its aglycone genipin, we herein transformed geniposide into genipin
using purified recombinant β‑glucosidase from Lactobacillus antri (rBGLa), which was expressed in Escherichia coli to
enhance the genipin content. Purified rBGLa was characterized using p‑nitrophenyl β‑d‑glucopyranoside, and the
optimal temperature and pH for its β‑glucosidase activity were found to be 45 °C and 6.0. When the enzyme was
immobilized, rBGLa was active at higher temperatures than the free enzyme, and we confirmed that its stability upon
changes in pH and temperature was highly improved. Using 0.5 μg/mL free rBGLa, single compound of 0.4 mM geni‑
poside was efficiently converted into genipin within 2 h, and the immobilized rBGLa also successfully transformed
geniposide in a hot‑water extract of Gardeniae Fructus into the aglycone, which makes it applicable to the food and
pharmaceutical industries.
Keywords: Glycosyl hydrolase family 3 β‑glucosidase, Gardeniae Fructus, Geniposide, Genipin, Lactobacillus antri,
Enzyme immobilization
Introduction
hydrolase families including β-glucosidase, which are
Orally administered herbal medicine containing flavo- responsible for decomposing substrates having various
noid components is generally absorbed into the blood sugar moieties according to each kind (Ioku et al. 1998;
vessels from the gastrointestinal tract and shows various Nemeth et al. 2003). Lactic acid bacteria, especially Lac-
effects on the human body (Kumar and Pandey 2013). In tobacillus, are well-known probiotic bacteria present in
this process, the glycoside components containing the the intestines that have been studied regarding probiotic
sugar structure are degraded into the non-glycosylated applications, including food fermentation and deglyco-
aglycone form by intestinal bacteria in the gastrointes- sylation of plant-derived glycosides using β-glucosidase
tinal tract, and the degraded smaller molecules of agly- from Lactobacillus (Ku et al. 2016; Urso et al. 2006; Xiu-
cones improve the absorption rate of these glycosides dong et al. 2016).
and exhibit higher efficacy than the glycone form (Hol-
Gardeniae Fructus is the dried mature fruit of Garde-
lman et al. 1999; ilakarathna and Rupasinghe 2013; nia jasminoides Ellis that has been used as a traditional
Yim et al. 2004). Intestinal bacteria have various glycoside medicine for anti-inflammatory (Koo et al. 2006), hepato-
protective (Kim et al. 2013), antioxidant (Pham et al.
2
000) and anti-angiogenic effects (Koo et al. 2004a). Gen-
*Correspondence: jyma@kiom.re.kr
iposide, which is one of the iridoid glycosides, is a major
component of Gardeniae Fructus, and when it is orally
Korean Medicine Application Center, Korea Institute of Oriental Medicine,
Cheomdan‑ro 70, Dong‑gu, Daegu 41062, Republic of Korea
©
The Author(s) 2017. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License
(http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium,
provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license,
and indicate if changes were made.

Kim et al. AMB Expr (2017) 7:64
Page 2 of 8
administered, it is hydrolyzed into the aglycone genipin was introduced into the pETDuet-1 vector (Novagen,
by β-glucosidase in intestinal bacteria. After absorption USA) between BamHI and NdeI sites by using the In-
into the human body, geniposide and genipin exhibit Fusion HD Cloning Kit (Takara, Kusatsu, Japan), and the
various pharmaceutical activities, including anti-inflam- resulting plasmid, named pET-BGLa, was transformed
matory (Koo et al. 2004b), antioxidant (Wang et al. 2015), into E. coli BL21(DE3) (Novagen).
choleretic (Shoda et al. 2004), and anti-cancer effects
(
Kim et al. 2005). Despite these various effects of genipin, Enzyme expression and purification of recombinant
Gardeniae Fructus contains it at a very low concentration, β‑glucosidase
whereas it has abundant geniposide, the β-glycoside- Escherichia coli BL21(DE3) harboring pET-BGLa was
linked compound from which genipin is derived (Gong cultivated at 25 °C in Luria–Bertani medium con-
et al. 2014). A study in which the homogenate did not taining 50 μg/mL ampicillin until OD
reached 0.5
00
6
degrade geniposide despite containing β-glucosidase in to express rBGLa by adding 0.2 mM isopropyl-β-ꢀ-
rat liver suggested that the hydrolysis of geniposide relies thiogalactopyranoside (IPTG) and was additionally incu-
on the β-glucosidase of intestinal bacteria (Akao et al. bated for 8 h at the same temperature. e culture was
1
994). erefore, to improve the genipin content in Gar- harvested by centrifugation at 4000g for 20 min, and
deniae Fructus, an enzyme-catalyzed approach convert- then washed with buffer containing 50 mM Na HPO
2
4
ing geniposide into genipin could be very useful.
and 0.3 M NaCl (pH 7.0). Cells were then resuspended in
In this study, we cloned and expressed recombinant the same buffer and disrupted by ultrasonication (Omni-
β-glucosidase (EC 3.2.1.21) from Lactobacillus antri Ruptor 4000; Omni International, Kennesaw, GA, USA).
(
rBGLa) belonging to glycoside hydrolase family 3 in Cell debris was removed by centrifugation at 14,000g and
Escherichia coli (E. coli), following the improvement 4 °C for 30 min. After the binding to TALON metal affin-
of rBGLa activity and stability by enzyme immobiliza- ity resins (Takara), non-specific binding proteins were
tion in calcium-alginate carrier. Using the immobilized removed by washing with a buffer containing 50 mM
rBGLa, the geniposide in Gardeniae Fructus extract was Na HPO , 0.3 M NaCl, and 30 mM imidazole (pH 7.0).
2
4
efficiently hydrolyzed into genipin, which indicates the Finally, (His) -tagged rBGLa was purified by the elution
6
potential of rBGLa for industrial applications to pro- buffer containing 50 mM Na HPO , 0.3 M NaCl, and
2
4
duce valuable aglycones by transforming plant-derived 250 mM imidazole (pH 7.0), which was confirmed by
glycosides.
12% sodium dodecyl sulfate polyacrylamide gel electro-
phoresis (SDS-PAGE).
Materials and methods
Hot‑water extraction of Gardeniae Fructus
Enzyme characterization
Gardeniae Fructus, a dried mature fruit of Gardenia jas- e specific activity of free and immobilized rBGLa was
minoides Ellis, was purchased from Yeongcheon Orien- evaluated using pNPG as a substrate in 25 mM sodium
tal Herbal Market (Yeongcheon, Republic of Korea) and phosphate buffer (pH 6.0) at 45 °C. e p-nitrophenol
deposited in the herb bank of the Korean Institute of Ori- (pNP) liberated by BGLa was measured immediately
ental Medicine. An extract was prepared by extracting at 405 nm using a microplate reader (GloMax-Multi
5
1
0 g of Gardeniae Fructus in 1000 mL of distilled water at Microplate Multimode Reader; Promega, Madison, WI,
15 °C for 3 h (Gyeongseo Extractor Cosmos-600, Gyeo- USA). Specific activity was defined as μmol pNP gener-
ngseo, Republic of Korea). After filtration through test- ated/min/mg BGLa. e optimum temperature of BGLa
ing sieves (150 μm; Retsch, Germany), Gardeniae Fructus was studied in 25 mM sodium phosphate buffer (pH 6.0)
extract was freeze-dried and kept in a desiccator at 4 °C. containing 5 mM pNPG at various temperatures rang-

e dried extract powder was stored at −20 °C until use.
ing from 25 to 60 °C, and the thermal stability was deter-
mined by measuring residual BGLa activity after 2 h of
incubation at pH 6.0 and each temperature. e effect of
Bacterial strains, plasmid, and gene cloning

e glycosyl hydrolase family 3 β-glucosidase gene was pH on BGLa activity at the optimum temperature was
amplified by polymerase chain reaction (PCR) with examined in the same buffer at various pH levels in the
KOD FX-Neo DNA polymerase (Toyobo, Osaka, Japan), range from 3.0 to 10.0. e pH stability was evaluated
genomic DNA of L. antri DSM 16041 as a template, after 2 h of incubation at optimum temperature and each
and the following primers (Cosmogentech, Republic of pH. e stability of immobilized enzyme was assayed
Korea): forward (5′-ACC ACA GCC AGG ATC CGA after washing the enzyme beads with the reaction buffer
TGG TAA AAA TTG ATT TGC AG-3′) and reverse (5′- after 2 h of incubation.
TTG AGA TCT GCC ATA TGT TAC TTT AAG GCC
e effects of cations and chemical reagents on rBGLa
CGC AGA AA-3′) primers. e amplified PCR product activity were investigated. rBGLa activity was evaluated

Kim et al. AMB Expr (2017) 7:64
Page 3 of 8
at 45 °C in 25 mM Na HPO (pH 6.0) additionally con- Results
2
4
taining 10 mM NaCl, KCl, MgCl , CaCl , sodium dode- Expression and purification of rBGLa
2
2
cyl sulfate (SDS), Triton X-100, glycerol, imidazole. e e β-glucosidase gene from L. antri DSM 16041 which
effects were expressed as relative activities compared is homologous to glycosyl hydrolase family 3 consists of
to that of rBGLa in the absence of cations and chemical 2208 bp and encodes 735 amino acids (UniProtKB acces-
reagents.
sion number: C8P9L9). e gene was inserted into pET-
Duet-1 vector between the BamHI and NdeI sites, which
is located on the downstream of nucleotides encoding
Immobilization of rBGLa
Purified rBGLa was immobilized by the method using the (His) -tag for affinity purification. e rBGLa was
6
alginate carrier (Busto et al. 1995). rBGLa solution was expressed by induction with 0.2 mM IPTG and at a low
mixed with 3% sodium alginate, then the rBGLa-alginate temperature, 25 °C, to improve the solubility of the tar-
mixture was added dropwise into 0.3 M CaCl with con- get protein. After cell disruption, rBGLa was bound to
2
tinuous stirring at room temperature. Immobilized gel the (His) -tag affinity resin by applying the cell lysate into
6
beads formed were cured for 2 h. After the gel beads were the column, and washed with buffer containing 30 mM
washed with 0.03 M CaCl solution, they were stored at imidazole to remove the non-specific binding proteins in
2
4
°C in 25 mM Na HPO solution (pH 6.0).
E. coli. rBGLa was finally eluted in the buffer containing
2
4
2
50 mM imidazole. roughout the purification process,
Enzymatic conversion of geniposide and hot‑water extract
of Gardeniae Fructus
the results exhibited 1.8-fold increase of rBGLa purity
and 33.5% recovery yield (Table 1). e molecular weight
Biotransformation of geniposide was performed by react- of rBGLa, predicted to be approximately 81.0 kDa, was
ing 0.5 mM geniposide with 0.5 μg/mL BGLa enzyme confirmed by SDS-PAGE (Fig. 1).
at 45 °C in 25 mM sodium phosphate buffer (pH 6.0).
Samples for High-performance liquid chromatography Enzymatic characterization of free rBGLa
(
HPLC) analysis were prepared by withdrawing them at e effects of pH and temperature on β-glucosidase were
regular intervals for 4 h. Freeze-dried hot-water extract investigated (Fig. 2). Purified rBGLa was active over the
of Gardeniae Fructus was dissolved in 25 mM sodium pH range of 4.0–7.0 at 45 °C, and in particular had more
phosphate buffer (pH 6.0) to a concentration of 2 mg/mL, than 50% of its maximum activity at a moderately acidic
and an equal volume of 100 μg/mL free BGLa solution in pH of between 5.0 and 7.0 (Fig. 2a). e optimal pH of
the same buffer or appropriate amount of immobilized rBGLa activity was 6.0 in 25 mM sodium phosphate
rBGLa was added to Gardeniae Fructus extract solution. buffer, which is close to the pH at which Lactobacillus
Samples were also withdrawn during 4-h reaction hourly.
grows optimally (pH 6.2–6.5). e enzyme activity was
always more than 50% of its maximum level between pH
5.0 and pH 7.0; however, there was no activity at pH 3 or
HPLC analysis
Biotransformation samples with a volume of 10 μL, with at alkaline pH levels of more than 8.0. After 4 h of incu-
both geniposide and Gardeniae Fructus extract, were ana- bation over a pH between 3.0 and 10.0 at 45 °C, the result
lyzed by HPLC (CM5000; Hitachi, Tokyo, Japan) using a revealed that free rBGLa has low pH stability in the over-
Geminin C18 column (5 µm, 250 × 4.6 mm; Phenom- all pH range, which exhibited 42% of the original activity
enex, Torrance, CA, USA) under an oven temperature at pH 5.0 (Fig. 2a).
of 40 °C. A mobile phase containing 1% (v/v) acetic acid
rBGLa showed β-glucosidase activity over the tempera-
in water (A) and acetonitrile (B) was used with the fol- ture range of 25–55 °C at pH 6.0, and the optimal tem-
lowing gradient at a flow rate of 1.0 mL/min: (A) 85–35% perature for this activity was 45 °C (Fig. 2b). e enzyme
(
(
0–40 min), 35–0% (40–45 min), 0% (45–50 min), 0–85% also had more than 85% activity over the temperature
50–55 min), and 85% (55–70 min). e bioconversion of range of 40–50 °C and less than 50% activity at temper-
geniposide and Gardeniae Fructus extract was detected atures below 30 °C and above 55 °C compared with the
at a UV wavelength of 240 nm. Geniposide (Wako Pure maximum activity. No enzyme activity was observed at
Chemical Industries, Ltd., Osaka, Japan) and genipin 60 °C. ermal stability of free rBGLa exhibited a maxi-
(
Sigma, St. Louis, MO, USA) were used as standards.
mum at 25 °C, which was 57.8% of the original activity.
Table 1 Purification process of rBGLa
Purification steps
Total activity (U, μmol/min) Total protein (mg) Specific activity (U/mg) Purification (fold) Recovery (%)
Crude cell lysate (soluble)
Affinity chromatography
4708
1578
122.3
11.1
38.5
1
100
142.0
3.7
33.5

Kim et al. AMB Expr (2017) 7:64
Page 4 of 8
Fig. 1 SDS‑PAGE analysis through the procedure of (His) ‑tag purification. Lane M molecular weight markers; Lane 1 total soluble fraction of non‑
6
induced cell lysate; Lane 2 total soluble fraction of IPTG‑induced cell lysate; Lane 3 the fraction passed through resin; Lane 4 the fraction with elution
buffer containing 250 mM imidazole
As the temperature rose, the thermal stability gradually
decreased, which was completely lost above 55 °C.

e effect of the cations and chemical reagents on the
BGLa activity was investigated (Table 2). rBLGa activ-
ity decreases in the presence of all cations examined at
1
0 mM. e activity was inhibited about 18% by mono-
+
+
valent cations (Na and K ), whereas the enzyme inhi-
bition was increased in the presence of the divalent
2
+
2+
cations (Mg and Ca ), especially 89.6% of inhibition
2
+
was observed on Ca . On the detergents having differ-
ent properties the activity of the rBGLa was investigated.
Enzyme activity was inactivated in the reaction buffer
containing 10 mM SDS (99.4% inhibition), whereas the
activity was preserved well in the presence of 10 mM
Triton X-100. In addition, the imidazole used in the
Table 2 Effect of cations and chemical reagents on rBGLa
activity
Cation or chemical reagent
Relative activity (%)
Cations
Control
100.0
81.7
82.4
68.3
10.4
+
Na
+
K
Fig. 2 Effects of pH (a) and temperature (b) on the the activity and
stability of free rBGLa. The enzyme activity was examined using pNPG
as a substrate. Reaction samples were withdrawn at regular intervals,
and OD405 was measured immediately using a microplate reader. The
effects of pH and temperature were investigated at 45 °C for various
pHs between 3.0 and 10.0. The effect of temperature (25–60 °C)
was investigated in the reaction buffer (pH 6.0). The stability was
calculated as the ratio of residual activity after 2 h of incubation to the
initial activity
Mg2
+
Ca2
+
Chemical reagents
SDS
0.6
93.9
90.1
62.5
Triton X‑100
Glycerol
Imidazole

Kim et al. AMB Expr (2017) 7:64
Page 5 of 8
purification process inhibited 37.5% of rBGLa activity, which revealed that the immobilization carrier is unsta-
2
+
which is similar to Mg (31.7%).
ble under these conditions.
e activity of immobilized rBGLa increased gradu-
ally with increasing temperature up to 50 °C, and the

Enzymatic characterization of immobilized rBGLa
Purified rBGLa was entrapped using the cross-linking optimum temperature was shifted from 45 to 50 °C by
method in calcium alginate. e effects of pH and tem- immobilization (Fig. 3b). While the activity of free rBGLa
perature on this immobilized rBGLa were investigated decreased rapidly at 55 °C and was inactivated at 60 °C,
(
Fig. 3). In the pH range of 5.0–7.0, the immobilized the results indicated that the immobilized enzyme main-
rBGLa displayed an activity pattern similar to that of the tained more than 79% of its maximum activity at 55 and
free enzyme, which also has maximum activity at pH 6.0. 60 °C. rBGLa was thermostable at 25–55 °C when immo-
However the immobilized enzyme showed higher activity bilized, while the maximum thermal stability of free
than the free enzyme at pH 4.0, and in particular, rBGLa enzyme was about 60% at 25 °C. However, despite the
exhibited β-glucosidase activity under alkaline conditions presence of immobilized enzyme activity at 60 °C, it was
above pH 8.0 when immobilized, even though it was not completely inactivated after 2 h of incubation.
active in the free form (Fig. 3a). e results of pH stability
testing indicated that immobilized rBGLa maintained its Biotransformation of geniposide and hot‑water extract
activity at approximately 100% of the original level after of Gardeniae Fructus
2
h of incubation at the pH range of 5.0–6.0, whereas the To investigate rBGLa activity on its substrate containing
free enzyme showed pH stability of 33–42%. Above pH a β-ꢀ-glucopyranoside structure that has various effects
7
, alginate beads were dissolved in the reaction buffer, on the human body, the enzyme activity was determined
using a reaction with geniposide and Gardeniae Fructus,
and then quantifying the concentrations of both genipo-
side and genipin by HPLC analysis. Before this analysis,
we confirmed that geniposide and genipin in aqueous
solution could be directly analyzed without organic sol-
vent extraction as they exhibited solubility at 0.5 mM.

e transformation efficiency of geniposide (0.5 mM)
into its aglycone, genipin, at 0.5 μg/mL rBGLa was over
5
0% within 1 h, and 80% of the glucosidic linkages in gen-
iposide were hydrolyzed in a 2-h reaction (Fig. 4).
For the enzymatic conversion of a component of the
herbal extract, Gardeniae Fructus containing geniposide
as a functional marker compound was extracted in hot
water at 115 °C, and the powder was prepared by filtering
and freeze-drying the extract. Gardeniae Fructus extract
Fig. 3 Effects of pH (a) and temperature (b) on the activity and sta‑
bility of immobilized rBGLa. The enzyme activity was examined using
pNPG as a substrate. Reaction samples were withdrawn at regular
intervals, and OD405 was measured immediately using a microplate
reader. The effects of pH and temperature were investigated at 45 °C
for various pHs between 3.0 and 10.0. The effect of temperature
Fig. 4 Time‑course analysis of geniposide (closed circle) bioconver‑
sion into genipin (closed square) by rBGLa. The enzyme solution
containing 0.5 μg/mL rBGLa was reacted with 0.5 mM geniposide in
25 mM sodium phosphate buffer (pH 6.0) at 45 °C. The reaction sam‑
ples were withdrawn at regular intervals for 4 h and analyzed by HPLC
(25–60 °C) was investigated in the reaction buffer (pH 6.0). The stabil‑
ity was calculated as the ratio of residual activity after 2 h of incuba‑
tion to the initial activity

Kim et al. AMB Expr (2017) 7:64
Page 6 of 8
was analyzed by HPLC, showing that 1.6 mg/mL extract properties and evaluate the efficiency of conversion of
contained 11.9% geniposide (w/w), which corresponds geniposide into its corresponding aglycone, genipin.
to approximately 0.5 mM, whereas genipin was present
rBGLa linked with a (His) -tag at the N-terminus was
6
in negligible amounts (0.3%) (Fig. 5a). rough the enzy- purified using immobilized metal affinity chromatog-
matic reaction of free rBGLa with Gardeniae Fructus raphy. Non-specific binding proteins were adequately
extract, we confirmed that the geniposide in the extract at removed by washing with 30 mM imidazole solution
the same concentration as the single component reaction and rBGLa was finally eluted in the buffer contain-
was also converted by reacting with 0.5 μg/mL rBGLa. ing 250 mM imidazole. To confirm the reaction condi-

e geniposide in the extract was completely hydrolyzed tions with the maximum activity of purified rBGLa,
after a 4-h reaction, and the concentration of genipin was pNPG was used to analyze the enzyme characteristics,
increased in a manner corresponding to the decrease of such as optimum temperature and pH. As a result, free
geniposide content, which is similar to the single genipo- rBGLa showed high activity in the range of 40–50 °C
side reaction. Finally, we confirmed that the geniposide in and the optimum temperature was 45 °C. In addition,
the extract was also successfully converted into genipin the pH was found to be optimum at 6.0 and the activ-
by reacting with immobilized enzyme (Fig. 5b).
ity decreased rapidly at pH levels other than 6.0. is
suggests that β-glucosidase from L. antri also plays a
role in digesting food containing plant-derived glyco-
Discussion
In this study, recombinant β-glucosidase from L. antri sides in the large intestine, similar to the colon, which
was expressed and purified after cloning of the gene into is the part of the body where Lactobacillus commonly
the pETDuet-1 vector to characterize the enzymatic grows (Baati et al. 2000). As rBGLa was unstable upon
changes in both temperature and pH, efforts to maintain
the enzyme activity were required. To overcome its low
stability, enzyme immobilization using an alginate car-
rier was carried out. e result showed that there was
no pH shift in the maximum activity of the immobilized
enzyme reported in previous studies (Arica et al. 1998;
Erginer et al. 2000). However, the enzyme activity was
improved at pH 4.0 and above 8.0 and the stability of
the immobilized enzyme was highly improved at pH 5.0
and 6.0, suggesting that the alginate matrix protects the
enzyme from structural denaturation by changes in pH
(
Keerti et al. 2014; Su et al. 2010). is result revealed
that immobilization of rBGLa is effective at pH 5.0–6.0
despite the instability of the alginate carrier dissolved
at a pH above 7.0 and enzyme activity at pH less than
4
.0. e investigation of the effect of temperature on
the immobilized enzyme indicated that the enzyme was
active in the range of 25–60 °C and the optimal tempera-
ture of rBGLa was shifted to 50 °C when immobilized.
In addition, enzyme stability was highly improved by
immobilization, which maintained its original activity
up to 55 °C after 2 h of incubation. ese results may
indicate that the alginate matrix preserves the three-
dimensional structure of rBGLa despite a temperature
increase to 55 °C (Keerti et al. 2014). e properties of
immobilized rBGLa enable the conversion efficiency
to be improved by preserving its activity and reuse of
entrapped enzyme by simple recovery, which has a cost
advantage (Mohamad et al. 2015; Sheldon 2007). Espe-
cially, for the bioconversion of food or drug substance
enzyme immobilization is advantageous in eliminating
the enzyme corresponding to contaminant in the final
product after the reaction (Mohamad et al. 2015).
Fig. 5 HPLC analysis for the bioconversion of Gardeniae Fructus
hot‑water extract using rBGLa. The enzymatic reaction of Gardeniae
Fructus hot‑water extract was performed in the reaction buffer at pH
6
.0 and 45 °C. a Gardeniae Fructus extract (1.6 mg/mL) was reacted
with free rBGLa (0.5 μg/mL), and the changes in geniposide and geni‑
pin concentration were monitored. b The transition of geniposide by
immobilized rBGLa was monitored after 4‑h reaction at 240 nm by
HPLC, and was compared using standard geniposide and genipin

Kim et al. AMB Expr (2017) 7:64
Page 7 of 8
Geniposide is generally known to be metabolized into aglycone content using rBGLa on various activities
genipin by bacterial enzymes in the intestine or liver including antioxidative, anti-inflammatory, and antitu-
(
Akao et al. 1994). In this study, we investigated whether mor effects.
geniposide can be effectively converted into genipin by
purified rBGLa, which has biological activities includ-
ing antioxidant, anti-inflammatory and anti-angiogenic
effects. In the reaction of geniposide with rBGLa at 45 °C
and pH 6.0, it was shown that genipin was generated
upon a decrease in geniposide (at 0.4 mM, correspond-
ing to 80% of the initial concentration), after 2 h of incu-
bation. It demonstrated that β-glucosidase from L. antri
can convert geniposide into genipin effectively by break-
ing the glucosidic linkage in the substrate.
Abbreviations
rBGLa: recombinant β‑glucosidase from Lactobacillus antri; pNPG: p‑nitrophe‑
nyl β‑d‑glucopyranoside; E. coli: Escherichia coli; PCR: polymerase chain reac‑
tion; IPTG: isopropyl‑β‑d‑thiogalactopyranoside; SDS‑PAGE: sodium dodecyl
sulfate polyacrylamide gel electrophoresis; pNP: p‑nitrophenol; SDS: sodium
dodecyl sulfate; HPLC: high‑performance liquid chromatography.
Authors’ contributions
The experiments were designed by JYM, and performed by YSK and CJL.
CJL analyzed the data, and YSK wrote this manuscript. All authors read and
approved the final manuscript.
Based on this result, we converted geniposide in plant
extract using rBGLa. As an herb for enzymatic transfor-
mation, we selected Gardeniae Fructus, which contains
a large quantity of geniposide as an index component
for its quality control. HPLC analysis of the hot-water
extract of Gardeniae Fructus showed that it contained
a high level of geniposide, namely, 11.9% (w/w), while
there was little genipin [0.3% (w/w)] in the extract in the
previous report (Gong et al. 2014). As shown in Fig. 5a,
Competing interests
The authors declare that they have no competing interests.
Availability of data and materials
The datasets supporting the conclusions of this article are included within the
article.
Ethics approval and consent to participate
No animal or human subjects were used in this work.
Funding
1
0
.6 mg/mL Gardeniae Fructus contained approximately
.5 mM geniposide, and the geniposide in the extract
This work was supported by Grant K17281 from the Korea Institute of Oriental
Medicine (KIOM), provided by the Ministry of Science, ICT and Future Planning
(MISP), Republic of Korea.
was hydrolyzed by free rBGLa to a similar pattern as
single compound reaction (Fig. 4). e result may imply
that there is no rBGLa inhibitors for geniposide conver-
sion in the extract. is reveals that the rBGLa can also
increase the genipin content by converting the genipo-
side present in herbal extracts, which contains vari-
ous components, without enzyme inhibition. Based on
these results, it may be expected that the use of herbal
extracts containing geniposide transformed by the
enzyme β-glucosidase from L. antri or fermented by the
strain can improve the absorption rate and biological
efficacy by direct action on the human body as the stud-
ies on bioconversion of various ginsenosides from gin-
seng using β-glucosidase (Hong et al. 2012; Quan et al.
Received: 30 January 2017 Accepted: 2 March 2017
References
Akao T, Kobashi K, Aburada M (1994) Enzymic studies on the animal and intes‑
tinal bacterial metabolism of geniposide. Biol Pharm Bull 17:1573–1576
Arica MY, Alaeddinoğlu NG, Hasirci V (1998) Immobilization of glucoamylase
onto activated pHEMA/EGDMA microspheres: properties and application
to a packed‑bed reactor. Enzyme Microb Technol 22:152–157
Baati L, Fabre‑Gea C, Auriol D, Blanc PJ (2000) Study of the cryotolerance of
Lactobacillus acidophilus: effect of culture and freezing conditions on the
viability and cellular protein levels. Int J Food Microbiol 59:241–247
Busto MD, Ortega N, Perez‑Mateos M (1995) Studies on microbial β‑d‑glucosidase
immobilized in alginate gel beads. Process Biochem 30:421–426
Chakraborty S, Rusli H, Nath A, Sikder J, Bhattacharjee C, Curcio S, Drioli E
2
012). e reaction result of Gardeniae Fructus extract
(
2016) Immobilized biocatalytic process development and potential
application in membrane separation: a review. Crit Rev Biotechnol
6:43–58
with immobilized rBGLa exhibited that the geniposide
in the herb can be successfully converted into genipin
by immobilized enzyme (Fig. 5b). In the industrial pro-
cess for producing high content of genipin in the garde-
nia extract, enzymatic conversion is more advantageous
when the enzyme was immobilized, which can improve
the productivity by introducing continuous reaction
system and reduce the production cost by reusing bio-
catalyst and reacting the extract at a relatively higher
temperature compared to optimum temperature of free
enzyme after hot water extraction of Gardeniae Fruc-
tus, which saves energy consumption required for cool-
ing the bioreactor (Chakraborty et al. 2016; Javed et al.
3
Erginer R, Toppare L, Alkan S, Bakir U (2000) Immobilization of invertase in
functionalized copolymermatrices. React Funct Polym 45:227–233
Gong G, Zheng Z, Liu H, Wang L, Diao J, Wang P, Zhao G (2014) Purification
and characterization of a beta‑glucosidase from Aspergillus niger and
its application in the hydrolysis of geniposide to genipin. J Microbiol
Biotechnol 24:788–794
Hollman PC, Bijsman MN, van Gameren Y, Cnossen EP, de Vries JH, Katan MB
(1999) The sugar moiety is a major determinant of the absorption of
dietary flavonoid glycosides in man. Free Radic Res 31:569–573
Hong H, Cui CH, Kim JK, Jin FX, Kim SC, Im WT (2012) Enzymatic biotransfor‑
mation of ginsenoside Rb1 and gypenoside XVII into ginsenosides Rd
and F2 by gecombinant beta‑glucosidase from Flavobacterium johnso-
niae. J Ginseng Res 36:418–424
Ioku K, Pongpiriyadacha Y, Konishi Y, Takei Y, Nakatani N, Terao J (1998) Beta‑
glucosidase activity in the rat small intestine toward quercetin monoglu‑
cosides. Biosci Biotechnol Biochem 62:1428–1431
2
016). We will investigate further the effect of increased

Kim et al. AMB Expr (2017) 7:64
Page 8 of 8
Javed MR, Buthe A, Rashid MH, Wang P (2016) Cost‑efficient entrapment of
beta‑glucosidase in nanoscale latex and silicone polymeric thin films for
use as stable biocatalysts. Food Chem 190:1078–1085
Keerti Gupta A, Kumar V, Dubey A, Verma AK (2014) Kinetic characterization
and effect of immobilized thermostable beta‑glucosidase in alginate gel
beads on sugarcane juice. ISRN Biochem 2014:178498
reactions of crocin with linoleic acid and crocin with oxygen. J Agric Food
Chem 48:1455–1461
Quan LH, Min JW, Jin Y, Wang C, Kim YJ, Yang DC (2012) Enzymatic biotrans‑
formation of ginsenoside Rb1 to compound K by recombinant beta‑
glucosidase from Microbacterium esteraromaticum. J Agric Food Chem
60:3776–3781
Kim BC, Kim HG, Lee SA, Lim S, Park EH, Kim SJ, Lim CJ (2005) Genipin‑induced
apoptosis in hepatoma cells is mediated by reactive oxygen species/c‑
Jun NH2‑terminal kinase‑dependent activation of mitochondrial path‑
way. Biochem Pharmacol 70:1398–1407
Kim J, Kim HY, Lee SM (2013) Protective effects of geniposide and genipin
against hepatic ischemia/reperfusion injury in mice. Biomol Ther (Seoul)
Sheldon RA (2007) Cross‑linked enzyme aggregates (CLEAs): stable and recy‑
clable biocatalysts. Biochem Soc Trans 35:1583–1587
Shoda J, Miura T, Utsunomiya H, Oda K, Yamamoto M, Kano M, Ikegami T,
Tanaka N, Akita H, Ito K, Suzuki H, Sugiyama Y (2004) Genipin enhances
Mrp2 (Abcc2)‑mediated bile formation and organic anion transport in rat
liver. Hepatology 39:167–178
2
1:132–137
Su E, Xia T, Gao L, Dai Q, Zhang Z (2010) Immobilization of β‑glucosidase
and its aroma‑increasing effect on tea beverage. Food Bioprod Process
88:83–89
Koo HJ, Lee S, Shin KH, Kim BC, Lim CJ, Park EH (2004a) Geniposide, an anti‑
angiogenic compound from the fruits of Gardenia jasminoides. Planta
Med 70:467–469
Koo HJ, Song YS, Kim HJ, Lee YH, Hong SM, Kim SJ, Kim BC, Jin C, Lim CJ, Park
EH (2004b) Antiinflammatory effects of genipin, an active principle of
gardenia. Eur J Pharmacol 495:201–208
Koo HJ, Lim KH, Jung HJ, Park EH (2006) Anti‑inflammatory evaluation of gar‑
denia extract, geniposide and genipin. J Ethnopharmacol 103:496–500
Ku S, You HJ, Park MS, Ji GE (2016) Whole‑cell biocatalysis for producing
ginsenoside Rd from Rb1 using Lactobacillus rhamnosus GG. J Microbiol
Biotechnol 26:1206–1215
Thilakarathna SH, Rupasinghe HP (2013) Flavonoid bioavailability and
attempts for bioavailability enhancement. Nutrients 5:3367–3387
Urso R, Rantsiou K, Cantoni C, Comi G, Cocolin L (2006) Technological char‑
acterization of a bacteriocin‑producing Lactobacillus sakei and its use in
fermented sausages production. Int J Food Microbiol 110:232–239
Wang J, Zhang Y, Liu R, Li X, Cui Y, Qu L (2015) Geniposide protects against
acute alcohol‑induced liver injury in mice via up‑regulating the
expression of the main antioxidant enzymes. Can J Physiol Pharmacol
93:261–267
Kumar S, Pandey AK (2013) Chemistry and biological activities of flavonoids: an
overview. Sci World J 2013:162750
Mohamad NR, Marzuki NH, Buang NA, Huyop F, Wahab RA (2015) An overview
of technologies for immobilization of enzymes and surface analysis
techniques for immobilized enzymes. Biotechnol Biotechnol Equip
Xiudong X, Ying W, Xiaoli L, Ying L, Jianzhong Z (2016) Soymilk residue (okara)
as a natural immobilization carrier for Lactobacillus plantarum cells
enhances soymilk fermentation, glucosidic isoflavone bioconversion,
and cell survival under simulated gastric and intestinal conditions. PeerJ
4:e2701
2
9:205–220
Yim JS, Kim YS, Moon SK, Cho KH, Bae HS, Kim JJ, Park EK, Kim DH (2004) Meta‑
bolic activities of ginsenoside Rb1, baicalin, glycyrrhizin and geniposide
to their bioactive compounds by human intestinal microflora. Biol Pharm
Bull 27:1580–1583
Nemeth K, Plumb GW, Berrin JG, Juge N, Jacob R, Naim HY, Williamson G, Swal‑
low DM, Kroon PA (2003) Deglycosylation by small intestinal epithelial cell
beta‑glucosidases is a critical step in the absorption and metabolism of
dietary flavonoid glycosides in humans. Eur J Nutr 42:29–42
Pham TQ, Cormier F, Farnworth E, Tong VH, Van Calsteren MR (2000) Antioxi‑
dant properties of crocin from Gardenia jasminoides Ellis and study of the