Resveratrol and arctigenin production from polydatin and arctiin respectively by a thermostable β-glucosidase from Thermotoga maritima

Article abstract of DOI:10.1080/07328303.2018.1541996

For preparation of resveratrol and arctigenin from peanut hulls and arctium lappa fruits, respectively, a recombinant β-glucosidase (TmBglA) from hyperthermophile Thermotoga maritima was purified and characterized. The hydrolytic activity was the highest at 90 °C and pH 6.2 for arctiin with K_m of 1.61 mM and k_cat of 197.4 s^?1, and 90 °C and 5.8 for polydatin with K_m of 0.38 mM and k_cat of 47.6 s^?1. The enzyme produced 215.4 mg L^?1 resveratrol and 355.7 mg L^?1 arctigenin from 400 mg L^?1 polydatin and 540 mg L^?1 arctiin after 60 min of incubation at 80 °C, with capable of hydrolyzing up to 92.1 and 94.9% of polydatin and arctiin, respectively. The enzymatic hydrolysis of peanut hulls and fructus arctii displayed a conversion yield of 3.8 and 0.33 mg resveratrol and arctigenin per gram of substrate material flour, respectively. Of the reported β-glucosidase, TmBglA exhibited the highest thermostability, k_cat, k_cat/K_m, and conversion productivity for hydrolyzing polydatin and arctiin, and has great potential applications in functional food and medicine production.

Full text of DOI:10.1080/07328303.2018.1541996

Journal of Carbohydrate Chemistry
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Resveratrol and arctigenin production
from polydatin and arctiin respectively by a
thermostable β-glucosidase from Thermotoga
maritima
Yemin Xue, Zonghui Zhang, Jingjing Hou, Zhigang Cao, Lingxian Zhang, Fen
Lou & Puxu Xu
To cite this article: Yemin Xue, Zonghui Zhang, Jingjing Hou, Zhigang Cao, Lingxian Zhang, Fen
Lou & Puxu Xu (2019): Resveratrol and arctigenin production from polydatin and arctiin respectively
by a thermostable β-glucosidase from Thermotogaꢀmaritima, Journal of Carbohydrate Chemistry,
DOI: 10.1080/07328303.2018.1541996
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JOURNAL OF CARBOHYDRATE CHEMISTRY
https://doi.org/10.1080/07328303.2018.1541996
Resveratrol and arctigenin production from polydatin
and arctiin respectively by a thermostable
b-glucosidase from Thermotoga maritima
a
a
a
b
a
Yemin Xue , Zonghui Zhang , Jingjing Hou , Zhigang Cao , Lingxian Zhang ,
a
a
Fen Lou , and Puxu Xu
a
Department of Food Science and Nutrition, GinLing College, Nanjing Normal University, Nanjing,
b
PR China; Department of Urology, Nanjing First Hospital, Nanjing Medical University, Nanjing,
PR China
ABSTRACT
ARTICLE HISTORY
For preparation of resveratrol and arctigenin from peanut hulls
and arctium lappa fruits, respectively, a recombinant b-glucosi-
dase (TmBglA) from hyperthermophile Thermotoga maritima
Received 21 March 2018
Accepted 25 October 2018
KEYWORDS
was purified and characterized. The hydrolytic activity was the
Arctiin; arctigenin;
polydatin; resveratrol;
thermotoga maritima
b-glucosidase
ꢀ
highest at 90 C and pH 6.2 for arctiin with K of 1.61 mM
m
ꢁ
1
ꢀ
and kcat of 197.4 s , and 90 C and 5.8 for polydatin with K
of 0.38 mM and k
resveratrol and 355.7 mg L
00mg L polydatin and 540mg L arctiin after 60 min of
incubation at 80 C, with capable of hydrolyzing up to 92.1
and 94.9% of polydatin and arctiin, respectively. The enzym-
atic hydrolysis of peanut hulls and fructus arctii displayed a
conversion yield of 3.8 and 0.33 mg resveratrol and arctigenin
per gram of substrate material flour, respectively. Of the
reported b-glucosidase, TmBglA exhibited the highest thermo-
m
ꢁ
1
of 47.6 s . The enzyme produced
cat
ꢁ
1
ꢁ1
2
4
15.4 mg L
arctigenin from
ꢁ
1
ꢁ1
ꢀ
m
stability, kcat, kcat/K , and conversion productivity for hydrolyz-
ing polydatin and arctiin, and has great potential applications
in functional food and medicine production.
GRAPHICAL ABSTRACT
CONTACT Yemin Xue
xueyemin@njnu.edu.cn
Department of Food Science and Nutrition, GinLing
College, Nanjing Normal University, Nanjing, PR China.
Color versions of one or more of the figures in the article can be found online at www.tandfonline.com/lcar.
ß 2019 Taylor & Francis Group, LLC

2
Y. XUE ET AL.
Figure 1. Structures of polydatin and arctiin and their conversion into resveratrol and arctige-
nin by recombinant T. maritima b-glucosidase TmBglA.
Introduction
Carbohydrates are essential components of biomass and contain an array of
structural and storage polysaccharides. Polyphenolic compounds are found
abundantly in plants, showing a wide range of antioxidant activities, and are
[
1,2]
thought to exert protective effects against chronic diseases,
thereby having
received much attention. Stilbenes and lignans are polyphenolic compounds
that are important antioxidative constituents of glycosidic phytochemicals, but
[
3]
the type of polyphenols is different in different sources. Polydatin and arc-
tiin are the most well-known glycosidic phytochemicals in Polygonum cuspida-
tum and Fructus arctii, respectively, which are traditional Chinese medicine
[
4,5]
officially listed in the Chinese Pharmacopoeia.
Resveratrol, which is 3,4,5-trihydroxystilbene, and its glycosidic derivative
polydatin, which is 3,4,5-trihydroxystibene-3-monoglucoside (Figure 1), are
the two major stilbenes in Polygonum cuspidatum, which have many
pharmacological properties including anti-inflammatory activities, cardio
protective, anticancer effects, and especially protection against neuronal
[
6,7]
injury.
about 1–2%) rather than resveratrol (about 0.2%).
The main stilbenes in P. cuspidatum, however, are polydatin
[
8,9]
(
Arctigenin is a
dibenzylbutyrolactone-type lignan found most frequently in plants in free
aglycone form or its glycoside arctiin (Figure 1), which has anti-inflamma-
[
10,11]
tory, antioxidant, and antitumor activities.
Many studies have revealed
that the biological effects of stilbenes and lignans are not due to the glyco-
[
11,12]
side form, but mainly to their aglycones resveratrol and arctigenin.
Resveratrol and arctigenin are the phenolic aglycones from the glycosides
polydatin and arctiin, respectively, by removing their sugar moieties with
b-glucosidase (b-D-glucoside glucohydrolase; EC 3.2.1.21) (Figure 1).

JOURNAL OF CARBOHYDRATE CHEMISTRY
3
Industrial interest in these two molecules is now growing because of their
[
9,13]
highly bioactive properties and their unimpeded intestinal absorption.
However, their low concentration in plants compared to the form of glyco-
side limits their application. Thus, there is more interest in increasing the
amounts of the phenolic aglycones in plant materials and converting glyco-
sides to their aglycones. Due to the mild conditions, simple procedures,
lower cost, and less pollution, enzymatic hydrolysis is the best method to
prepare these aglycones and in this regard, many microbial and fungal
b-glucosidases have been explored in recent years. They include b-glucosi-
[
14,15]
[16]
[16,17]
dases from Aspergillus oryzae,
Aspergillus niger,
Lactobacillus
[
18]
[19]
[20]
kimchi,
Rhizopus,
Yeast,
Grifola frondosa mycelia
and
[
21]
[22]
almonds
and commercial b-glucosidases.
However, thermostable enzymes have not been applied to converting pol-
ydatin and arctiin into resveratrol and arctigenin. The advantages of
thermostable enzymes in industrial processes include reduced risk of con-
tamination, increased substrate solubility, higher stablility of enzymes
against denaturing agents and proteolytic enzyme, and reduced cost of
[
23]
external cooling.
Therefore, in view of an industrial biotechnological
perspective, thermostable b-glucosidases is desired for the effective hydroly-
sis of glycosides polydatin and arctiin. Thermatoga maritima grows opti-
ꢀ
mally above 80 C and is of considerable interest because of the
hyperthermostability of their enzymes. It has been shown that T. maritima
b-glucosidase (TmBglA) is an efficient catalyst to break the glycosidic bond
[
24,25]
with a high conversion rate in thermal condition.
However, there
have been no reports regarding their applications in the hydrolysis of poly-
datin and arctiin.
Peanut hulls are an important byproduct produced during peanut oil
processing. They have been shown to be a rich source of polydatin. The
fruit of Arctium lappa L. contains a large amount of lignans and has been
used as a vegetable and medicinal plant in China. To obtain resveratrol
and arctigenin form peanut hulls and arctium lappa fruits, respectively, the
thermostable TmBglA from T. maritima was targeted for hydrolysis of pol-
ydatin and arctiin. In the present study, the hydrolysis of polydatin and
arctiin by TmBglA was examined, and the optimal pH and temperature
and kinetic parameters for these reactions were investigated.
Results and discussion
Production and purification of T. maritima b-glucosidase
The recombinant plasmids pET28a-bglA and pET20b-bglA were trans-
formed into E. coli JM109 (DE3). It produced a large amount of recombin-
ant TmBglA on induction with IPTG. The highest amount of TmBglA

4
Y. XUE ET AL.
Figure 2. (A): Time-courses of TmBglA produced by E. coli JM109 (DE3) containing pET-28a-
bglA and pET-20b-bglA; (B) and (C): SDS-PAGE analysis of recombinant TmBglA. B: Lane M,
Protein marker; Lane 1, 3, 5, 7, 9 and 11, soluble fraction (Lane 1, 3, 5) and insoluble (Lane 7,
9, 11) fraction of E. coli JM109 (DE3) containing pET-28a-bglA; Lane 2, 4, 6, 8, 10, and 12, sol-
uble fraction (Lane 2, 4, 6) and insoluble (Lane 8, 10 and 12) fraction of E. coli JM109 (DE3)
containing pET-20b-bglA; C: Lane M, Protein marker; Lane 1, fraction after heat treatment of E.
coli JM109(DE3) containing the plasmid pET-28a-bglA Lane 2, crude extract of E. coli
JM109(DE3) (DE3) containing the plasmid pET-28a-bglA; Lane 3, purified TmBglA after Ni-affinity
chromatography column.
obtained by E. coli expression was 12.5 U/mL for pET20b-bglA and 38.2 U/
mL for pET28a-bglA (Figure 2A). However, the major proportion of
TmBglA in pET20b-bglA was found in inclusion bodies (Figure 2B, lanes 8,
1
0 and 12), whereas bglA gene was induced to express the TmBglA from
pET28a-bglA transformed cells, resulting in completely soluble expression
Figure 2B lanes 3 and 9). The expression yield of TmBglA obtained with
(
pET28a-bglA was three times higher than that obtained with pET20b-bglA,
which would suggest a potential application in the field of natural bioactive
compound extraction. The proteins in the cell-free extract were purified to
ꢀ
gel electrophoretic homogeneity after a heat treatment (70 C, 30 min) fol-
lowed by immobilized Ni-affinity chromatography utilizing the C-terminal
His-tag. The purity of the TmBglA was evaluated using SDS-PAGE, yield-
ing a single protein band when stained under denaturing conditions. The
molecular mass of the enzyme was estimated to be 52 kDa (Figure 2C, lane

JOURNAL OF CARBOHYDRATE CHEMISTRY
5
Figure 3. The enzyme activity (solid circle) and stability (open circle) profiles of TmBglA to
hydrolyze pNPG at various temperatures (A) and pH values (B); and TLC analysis of the products
obtained from TmBglA-catalyzed hydrolysis of polydatin (C, D) and arctiin (E, F) at different
ꢀ
temperature (C, E) and pH (D, F). A and B: pH dependence at 90 C and temperature depend-
ence at pH 6.2 when a 5-min assay was used. For pH stability, 0.075 lg purified enzyme in
ꢀ
5
0 mM PPB (pH 4.2–pH 8.2) was pre-incubated for 1 h at 37 C in the absence of substrate. Full
activity was determined at each pH value. The activity of enzyme without pre-incubation was
ꢁ
1
defined as 100% (its enzyme activity was 407U mg ). Activity was determined at their optimal
pH and temperature. For heat stability, 0.075lg purified enzyme in 50 mM PPB (pH 6.2) was
ꢀ
pre-incubated for 1 h at a temperature range of 50–95 C in the absence of substrate. C and E:
For the effect of temperature on polydatin and arctiin hydrolysis; the reaction temperature was
ꢀ
varied from 50 to 80 C at pH 6.2 for 10 min. The reactions were stopped by ice water cooling
before analysis. D and F: To determine the optimum pH of polydatin and arctiin hydrolysis;
1
.0mM polydatin and 1.0mM arctiin were incubated with purified TmBglA in 50 mM sodium
ꢀ
phosphate buffer (SPB) varying from pH 5.0 to 7.8 at 80 C for 10 min, respectively. C-F: Lanes,
M: marker.
3), which were in accordance with the theoretically calculated molecu-
lar mass.
Temperature and pH on the activity of T. maritima b-glucosidase
ꢀ
TmBglA exhibited an optimal activity at pH 6.2 and 90 C when assayed
with 2 mM pNPG (Figure 3). Thermostability experiments indicated that
ꢀ
the enzyme was stable at temperatures lower than 90 C, maintaining more
ꢀ
than 50% of maximal activity after incubation at 90 C for 1 h (Figure 3A).
TmBglA displayed stability within the pH range from pH 5.4 to 7.8
(Figure 3B). The hydrolytic activity of polydatin and arctiin by TmBglA
were investigated at different pH and temperature, their hydrolysates were
analyzed by thin-layer chromatography as shown in Figure 3C–F. The two
most intense spots, arctiin and polydatin, were completely digested with
ꢀ
ꢀ
TmBglA at pH 5.8 and 70–90 C for polydatin and at pH 6.2 and 80–90 C

6
Y. XUE ET AL.
Table 1. Hydrolytic activity and kinetic parameters of T. maritima b-glucosidase.
Specific
activity (U mg )
Relative
activity (%)
k
cat/K
m
-1
ꢁ1
ꢁ1
cat (s
ꢁ1 ꢁ1
Substrate
K
m
(mM) Vmax (U mg
)
k
)
(mM
s )
pNPGlc
Arctiin
Polydatin
pNPFuc
418.1 ± 3.8
72.9 ± 1.3
26.6 ± 1.0
302.2 ± 1.9
174.3 ± 0.63
15.2 ± 0.60
9.0 ± 0.10
3.9 ± 0.18
100
0.60 ± 0.010
1.61 ± 0.070
0.38 ± 0.040
298.6 ± 1.5
229.8 ± 17
55.4 ± 0.94
238.9 ± 3.6
197.4 ± 14
47.6 ± 0.81
398 ± 16
137 ± 16
119 ± 10
17.4 ± 1.0
6.4 ± 0.20
72.3 ± 3.6
41.7 ± 0.20
3.6 ± 0.10
2.1 ± 0.10
0.9 ± 0.01
pNPGal
Cellobiose
Gentiobiose
Lactose
Activity of TmBglA was determined at their optimal pH and temperature for the substrate concentrations of
.0 mM. The kinetic parameters were determined at their optimal pH and temperature for the substrate con-
2
centrations ranging from 0.2 to 2.0 mM for pNPG, and from 0.05 to 1.5 mM for polydatin and arctiin using the
standard assay as described in “Materials and methods”.
for arctiin. Moreover, the conversion rates to products were the highest at
ꢀ
9
0 C. These results indicated that the optimal pH and temperature of
ꢀ
TmBglA were observed at pH 5.8 and 90 C for hydrolyzing polydatin and
ꢀ
at pH 6.2 and 90 C for hydrolyzing arctiin, respectively. The optimal pH
[
21]
[14]
values of b-glucosidases from almonds,
Aspergillus oryzae sp.100,
Aspergillus niger SK34.002,
[
15]
[18]
Lactobacillus kimchi JB301
for polydatin
were 6.0, 4.5, 5.0, and 5.0, respectively. The maximal activities of b-glucosi-
[
21]
[17]
dases from Almonds,
sp.100,
Aspergillus niger SK34.002,
Aspergillus oryzae
[
15]
[18]
Lactobacillus kimchi JB301
for hydrolyzing polydatin were
ꢀ
observed at 45, 70, 65, and 30–40 C, respectively. These results suggest
that temperature for the hydrolysis of polydatin to resveratrol by TmBglA
is the highest among all reported b-glucosidases.
Substrate specificity of T. maritima b-glucosidase
The hydrolytic activity of TmBglA toward various aryl-glycosides and dis-
accharides was measured to determine substrate specificity. All of the
selected substrates could be hydrolyzed by TmBglA. The substrate specifi-
city of TmBglA were found to be in the followed order: pNPG > pNPFuc
>
pNPGal > arctiin > polydatin > cellobiose > gentiobiose > Lactose (Table 1),
compared to that from Aspergillus niger SK34.002 with the order of cellobiose
[17]
>
pNPG > polydatin > salicin > sucrose > lactose > pNPGal,
that from
Lactobacillus kimchi JB301 with the order of pNPG > polydatin
[
18]
>
gentiobiose > cellobiose,
and that from Aspergillus oryzae sp.100 with the
[15]
order of piceid > pNPG > pNPGal.
TmBglA exhibited the specific activity
for arctiin 2.7-fold higher than that for polydatin. As TmBglA exhibited
greater activity towards arctiin than polydatin, this would suggest a potential
application in proofing in the arctigenin production industry.
The observed K , V , k and k /K values of the enzyme for pNPG,
m
m
cat
cat
m
arctiin, and polydatin are summarized in Table 1. The Vm values of the
enzyme followed the order: pNPG > arctiin > polydatin, which was the

JOURNAL OF CARBOHYDRATE CHEMISTRY
7
Figure 4. Docking polydatin and arctiin into the active site of TmBglA (A, B) and time course
of enzymatic transformation of polydatin and arctiin to resveratrol and arctigenin by TmBglA
(C, D). A, B: The catalytic residues of TmBglA are colored by yellow, and polydatin and arctiin
are colored in green. C, D: Batch enzymatic transformation of 1 mL reaction volume (containing
ꢁ
1
ꢁ1
ꢀ
4
00 mg L polydatin or 540 mg L arctiin, 5% (v/v) DMSO) was performed at 80 C and
50 mM sodium phosphate buffer (pH 5.8 for polydatin and 6.2 for arctiin) with 0.2 lg purified
TmBglA. Data represent the means of three experiments and error bars represent stand-
ard deviation.
same as that observed for specific activity. The Km values of the enzyme
followed the order of arctiin > pNPG > polydatin. The K value for poly-
m
datin was 4.5-fold lower than that for arctiin. The kcat for arctiin were 4.2-
fold higher than that for polydatin, which was in accordance with our
above results of specific activity assays. Kinetic studies also confirmed the
enzyme preference for arctiin as opposed to polydatin making it suitable
for the arctigenin preparation.
Molecular docking of polydatin or arctiin as a substrate into the active
site of T. maritima b-glucosidase was performed to analyze the difference
in the specific activities of the substrates (Figures 4A and B). The binding
free energies (DG) for the substrate and enzyme docking modes were eval-
uated. The DG (ꢁ5.49kcal/mol) between the arctiin and TmBglA was
higher than that between the polydatin and TmBglA (ꢁ8.11 kcal/mol), indi-
cated that polydatin-TmBglA complex system is more stable, and its sub-
strate affinity is higher, which is consistent with the observed K . In the
m
molecular model, the distances of the catalytic residues Glu166 and Glu351
to b-glucosidic linkage of arctiin were 8.07 and 11.79 Å, respectively, which

8
Y. XUE ET AL.
Table 2. Kinetic parameters of polydatin and arctiin of b-glucosidase from different
microorganisms.
V
max
K
(s
cat
ꢁ1
ꢁ
1
ꢁ1 ꢁ1
Strain
temperature ( C)
K
m
(mM) (U mg
)
)
K
cat/K
m
(mM .s
)
Optimum
ꢀ
Optimal
pH
Ref
Hydrolysis of polydatin
Almonds
47.7
0.74
0.74
0.2
34.6
4.9
323
–
–
–
–
–
6.45
119
45
70
60
30–40
90
6.0
4.5
5.0
5.0
5.8
[21]
[17]
[14]
Aspergillus niger SK34.002
Aspergillus oryzae sp.100
Lactobacillus kimchi JB301
Thermotoga maritima
ꢂ
–
1.25
47.6
[18]
0.38
55.4
This study
Hydrolysis of arctiin
Commercial b-glucosidase
Aspergillus nigerZJUT302
Thermotoga maritima
–
–
1.61
–
–
–
–
–
–
137
40
30
90
5.0
5.0
6.2
[22]
[27]
This study
229.8
197.4
“ꢂ
ꢁ1
”, nkat mg
were closer than those of polydatin (11.04 and 15.39 Å), to enhance the
interaction between the enzyme and the substrate. Moreover, the bulkier
amino acids (tryptophan and tyrosine) at position 168 and 177 in polyda-
tin-TmBglA complexes might disrupt the binding of polydatin to the cata-
lytic residues because of their bulky size. The homology model for
substrate binding may well explain how the TmBglA has higher activity for
arctiin than for polydatin, and also further confirm the correlation between
experimental and computational results. Compared to the reported K , V
m
m
and kcat for polydatin (Table 2), the kcat and k /K of T. maritima b-glu-
cat
m
[
18]
cosidase are higher than those of Lactobacillus kimchi JB301,
which is
the highest reported thus far among enzymatic transformations, suggesting
that TmBglA is a very efficient producer of resveratrol.
Enzymatic conversion of reagent-grade polydatin and arctiin to resveratrol
and arctigenin respectively
The amount of resveratrol and arctigenin liberated from hydrolysis of com-
plex polydatin and arctiin increased with increased incubation time (Figures
4
C and D), whereas the concentration of polydatin and arctiin decreased.
ꢁ1
During the initial 5 min of incubation, 37.3 mg L of resveratrol and 86.4mg
L
amount of two aglycones increased rapidly to 157.8 mg L of resveratrol
and 307.8 mg L of arctigenin. Their percent conversion was 67.9% for pol-
ydatin and 82.2% for arctiin. After 1 h, polydatin and arctiin were almost
completely converted to their aglycones resveratrol and arctigenin, and
reached the highest value of 215.4mg L of resveratrol and 355.7 mg L of
arctigenin, with a percent conversion of 92.1 and 94.9% for polydatin and
arctiin, respectively. The previously highest reported yield was 562.5mg
resveratrol per gram of reagent-grade polydatin for a b-glucosidase from
ꢁ1
of arctigenin were obtained. In 40 min of reaction concretely, the
ꢁ1
ꢁ1
ꢁ1
ꢁ1
[
15]
Aspergillus oryzae sp.100 after incubation for 4 h,
and 33.45 mg resveratrol

JOURNAL OF CARBOHYDRATE CHEMISTRY
9
Table 3. Hydrolysis of polydatin and arctiin of b-glucosidase from different microorganisms.
Conversion of polydatin to resveratrol by b-glucosidases
Conversion
Polydatin
Resveratrol
(g/L)
Yield
(mg/g)
productivity
(mg/g/h)
Percent
conversion (%)
(g/L)
Ref
a
Rhizopus microsporus
–
40
–
0.34
22.5
–
–
–
98
100
96.7
–
[19]
[15]
[16]
[28]
Aspergillus oryzae sp. 100
Aspergillus niger and Yeast
Aspergillus niger HE01
Thermotoga maritima
562.5
140.6
b
b
33.45
33.45
6.46
b
b
–
–
16.46
0.400
0.215
537.5
537.5
92.1
This study
Conversion of arctiin to arctigenin by b-glucosidases
Arctiin
Arctigenin
(g/L)
Yield
(mg/g)
Conversion
productivity
Percent
conversion (%)
Ref
(g/L)
(
mg/g/h)
Commercial b-glucosidase
Aspergillus nigerZJUT302
Thermotoga maritima
a
–
0.06
0.540
–
–
12.59
–
695.3
0.523
–
92.3
94.9
[22]
[27]
This study
c
–
695.3
b
0.356
ꢂ
g/L, Polygonum cuspidatum fermented with the Rhizopus microspores; mg/g, P. cuspidatum roots powder; “ ” ,
ꢁ
1
c
nkat mg ; “–”, not reported. mmol/L.
per gram of P. cuspidatum roots powder for a b-glucosidase from Aspergillus
[
16]
niger and Yeast.
T. maritima b-glucosidase produced 215.4mg resveratrol
ꢁ
1
ꢁ1
ꢁ1
L
and 355.7mg arctigenin L from 400 mg polydatin L and 540 mg arc-
tiin L after 60 min of reaction in the present study, with a conversion
productivity of 537.5 mg resveratrol per gram of reagent-grade polydatin and
ꢁ1
659.3 mg arctigenin per gram of reagent-grade arctiin (Table 3). The result
which herein for conversion of polydatin to resveratrol was 3.8-fold higher
than that achieved using A. oryzae sp.100 b-glucosidase. Of the reported
b-glucosidase, T. maritima b-glucosidase converted polydatin and arctiin to
resveratrol and arctigenin, respectively, with the highest conversion.
The end products of enzymatic hydrolysis of polydatin and arctiin by
TmBglA were determined by electrospray LC-MS/MS (Figure 5). By compar-
ing both retention time and mass spectrum with a standard, two major peaks
ꢁ
were identified as resveratrol (molecular ion m/z 227.0 [Mꢁ H] ) and arcti-
ꢁ
genin (molecular ion m/z 371.1 [M ꢁ H] ) at 8.862 and 15.681 min of reten-
[9,26]
tion time, respectively, which is consistent with previous studies.
Production of resveratrol and arctigenin from peanut hulls and arctium
lappa fruits respectively
The production of resveratrol from polydatin in peanut hulls and arctige-
nin from arctiin in arctium lappa fruits were carried out under the opti-
mized conditions. Once hydrolyzed by adding TmBglA, the aglycone
contents increased with reaction time, whereas the glycosides decreased
and approximated to the complete conversion into aglycones at 1 h of reac-
tion. The aglycones became eventually the major components of peanut
hull hydrolyzates. When the reaction was carried out in arctium lappa

1
0
Y. XUE ET AL.
Figure 5. Identification of hydrolyzed products, resveratrol and arctigenin, by means of LC–MS.
fruits, the amount of arctiin decreased markedly with the reaction time
prolonging, and the content of arctigenin increased and reached the highest
at 1 h (data not shown). The conversion yields for the hydrolysis of peanut
hulls and arctium lappa fruits were 3.8 and 0.33 mg resveratrol and arctige-
nin per gram of starting materials, respectively. These results suggested the
potential of recombinant b-glucosidase from T. maritima for application in
preparing higher bioactive resveratrol and arctigenin.
Interest in the industrial usage of resveratrol and arctigenin is growing
due to their pharmacological properties such as anti-inflammatory and
[
6,7,10,11]
antitumor activities,
and health benefits. The extraction of these
phenolic aglycones from plant via the breakdown of the glycosidic bonds of
related glycosides has allowed the exploitation of these compounds in
[
19,21,27,28]
pharmaceutical and food industries.
Conclusions
Our findings demonstrated the biotechnological potential for use of
TmBglA from T. maritima in the hydrolysis of agricultural by-products

JOURNAL OF CARBOHYDRATE CHEMISTRY
11
such as peanut hulls and arctium lappa fruit residues. The enzyme exhib-
ited the highest k , k /K and optimal temperature for hydrolyzing poly-
cat cat
m
datin and arctiin with the highest conversion among all enzymes reported
to date. The high thermostablity of TmBglA allowed hot water extraction
of polydatin and arctiin from peanut hulls and arctium lappa fruits, fol-
lowed by biocatalytic conversion of these glycosyl phytochemicals to agly-
cones resveratrol and arctigenin. This method has considerable potential
applications in food and medicine production. Further work to optimize
the production of resveratrol from polydatin in peanut hulls and arctigenin
from arctiin in arctium lappa fruits using T. maritima b-glucosidase
hydrolysis to prepare high-purity resveratrol and arctigenin and to develop
a simple isolation process using the minimum solvent and time will
be performed.
Experimental section
Materials and chemicals
Escherichia coli JM109 (DE3) (Promega, Madison, WI) was used as host for
the expression of b-glucosidase A gene from T. maritima, via the expres-
sion vector pET-28a (þ) and pET-20b (þ) (Novagen, USA). Cellobiose,
gentiobiose, lactose, and p-nitrophenyl (pNP) glycoside substrates including
pNP-b-D-glucopyranoside (pNPG), pNP-b-D-galactoside (pNPGal), pNP-
b-D-fucoside (pNPFuc) were purchased from Sigma Chemical Co. (St.
Louis, MO). Oligosaccharides including cellobiose, gentiobiose, and lactose,
were purchased from Aladdin Chemical Co (China). Polydatin, arctiin, resver-
atrol and arctigenin were purchased from Extrasynthese Chemical Co.
(France). All other chemicals used were analytical grade reagents unless other-
wise stated. High-performance liquid chromatography (HPLC) grade methanol
and acetonitrile were purchased from Fisher Scientific (Hanover Park, IL).
Expression and purification
All molecular methods were performed using standard molecular biology
[
29]
[25]
techniques.
Based on plasmid pET-20b-bglA
harboring the coding
area for Thermotoga maritima b-glucosidase as a template, the fragment of
bglA (Thermotoga maritima b-glucosidase gene) was amplified by PCR
0
0
using the primer pair 5 -CCCGATATCATGAACGTGAAAAAGTTCC-3
0
0
and 5 -CCCCTCGAGCTGCAGCTGCCACAGTG-3 . This introduced NcoI
and XhoI sites, respectively (bold-faced), at each end of the gene. The PCR
product was then purified and digested with NcoI and XhoI. The digested
fragment was ligated into the expression vector pET-28a, resulting in the
recombinant plasmid pET-28a-bglA.

1
2
Y. XUE ET AL.
The recombinant protein was purified to homogeneity as follows. E. coli
JM109(DE3) cells were freshly transformed with the plasmids pET-28a-
[
25]
ꢀ
bglA and pET-20b-bglA,
and grown at 37 C in 100 mL LB media con-
ꢁ1
ꢁ1
taining kanamycin (30 lg mL ) or ampicillin (100 lg mL ). At OD of
6
00
0.6–0.8, expression was then induced by adding 0.8 mM IPTG, and growth
was continued for 16 h. The cells were harvested by centrifugation, and
washed twice with water, then resuspended in binding buffer (5 mM imid-
azole, 0.5 M NaCl, and 20 mM Tris/HCl, pH 7.9). After sonication and
ꢀ
centrifugation (30 min, 9,600 ꢃg, 4 C), the supernatant was heated for
ꢀ
3
0 min at 70 C to remove most of the host cell proteins by centrifugation.
The resulting supernatants were purified by Ni-affinity chromatography
Novagen) to homogeneity as determined by the sodium dodecyl sulfate
(
polyacrylamide gel electrophoresis (SDS-PAGE) using 12% polyacrylamide
running gels with 4% polyacrylamide stacking gels. Protein concentration
[
30]
was determined by the Bradford method using BSA as a standard.
Assays of b-glucosidase activity
Enzyme activity was quantified by p-nitrophenol (pNP) release from pNPG,
pNPGal, and pNPFuc (Sigma, USA). The reaction was initiated by adding
1
0 mL of suitably diluted enzyme to 190 lL of 2 mM pNPG, pNPGal, and
ꢀ
pNPFuc in 50 mM potassium phthalate buffer (PPB, pH 6.2) at 90 C. After
5
min, the reaction was stopped by the addition of 600 mL of 1 M Na CO ,
2 3
and the amount of pNP released was measured at 410 nm against a blank.
One unit was determined as the amount of enzyme producing 1 lmol of
pNP per minute under the above assay conditions.
For oligosaccharides, the total reaction volume was 50 mL and contained
0
1
.075 mg of the enzyme. The reaction was stopped by heating the sample to
00 C for 10 min. The amount of glucose, liberated as a product of the
ꢀ
enzymatic reaction, was measured using a glucose oxidase-peroxidase assay
Glucose Assay Kit, Nanjing Jiancheng Bioengineering Institute) in accord-
(
ance with the manufacturer’s protocol. All reactions were done in triplicate.
For determination of optimum pH and temperature of polydatin and arc-
tiin hydrolysis, the reactions were stopped by ice water cooling before analysis,
and then the hydrolyzed solutions were added to 50 lL of methanol. Aliquots
of the methanol extract were spotted on thin layer chromatography plates
using silica gel 60 F254 (Yantai Jiangyou Silica Gel Development Co., Ltd.
China). The plate was developed with chloroform/acetone/formic acid/water
(8:8:1:0.4, by vol) for the hydrolysis of polydatin, the products on the plate
were visualized by absorbance under UV light at 365 nm. For the hydrolysis
of arctiin, the plate was developed with chloroform/methanol/water (20:4:0.5,
by vol), the spots on the silica plates were stained by spraying the air-dried

JOURNAL OF CARBOHYDRATE CHEMISTRY
13
plate with a solution containing H SO /methanol (1:4) after being partitioned,
2
4
ꢀ
and heating it at 100 C for 10 minutes.
Determination of kinetic parameters
For determinations of the kinetic parameters (K , V
k /K ) of pNPG
max cat m
m
reaction was carried out at the optimum conditions using 0.2–2.0 mM
pNPG. Kinetic parameters, Km and Vmax, were determined by the
Lineweaver-Burk representation of the Michaelis-Menten model. The cata-
lytic efficiency, k /K , was calculated to determine the substrate specificity
cat
m
of each enzyme. To calculate the catalytic constant, apparent k , the
cat
amount of protein was divided by the subunit molecular mass of 52 kDa
for TmBglA. All reactions were done in triplicate, and the relative standard
deviation was <2%.
The kinetic properties of the hydrolysis of polydatin and arctiin were
determined by incubating the substrate concentrations ranging from 0.05
to 1.5 mM for polydatin and arctiin. The enzymatic hydrolysis reaction
(
final volume, 100 mL) was carried out using pure polydatin or arctiin dis-
ꢀ
solved in buffer, and allowed to react at 80 C and pH 5.8 for polydatin
and 6.2 for arctiin, with 20 pmol of TmBglA. The mixture without enzyme
was preincubated at 80 C for 5 min. After 5 min, the reactions were
ꢀ
stopped by ice water cooling, and resuspended in 50 mL methanol for
HPLC analysis. The enzyme activity using polydatin and arctiin is
expressed in Units (U). One U corresponds to the amount of enzyme
required to release 1 mmol free aglycones (resveratrol and arctigenin) per
minute under the described conditions. Each test was carried out with at
least three replicates. The relative standard deviation was recorded to <5%.
Hydrolysis of polydatin and arctiin from reagent-grade polydatin and arctiin
respectively
To evaluate the hydrolysis of TmBglA towards polydatin and arctiin, 0.2 lg
ꢁ1
of purified TmBglA was added to the 1 mL of polydatin (400 mg L ) and
ꢁ1
arctiin (540 mg L ) in 50 mM SPB (pH 5.8 for polydatin and 6.2 for arc-
ꢀ
tiin) respectively, and allowed to react at 80 C for 60 min. The reaction
mixtures were left for 2 min, 5 min, 10 min, 20 min, 40 min, and 60 min,
and then were stopped by cooled in ice bar before analysis and centrifuged
ꢀ
at 12,000 rpm and 4 C for 5 min to get the supernatant for analyzing by
HPLC to monitor the enzymatic hydrolysis process. An Agilent Zorbax SB-
C18 column (4.6 ꢃ 150 mm, 5 lm) was used for isocratic separation with a
gradient sequence using methanol (A) and 0.05% phosphoric acid solution
(
B) in the following proportions: 40% A (9 min) and 80% B (5 min) at a

1
4
Y. XUE ET AL.
ꢁ1
flow rate of 1 mL min
for enzymatic transformation of polydatin to
resveratrol, and a gradient sequence using acetonitrile (A) and water (B) in
the following proportions: 25% A (15 min), 70% A (12 min), 70% A
ꢁ1
(
10 min), and 25% A (6 min) at a flow rate of 1 mL min for enzymatic
transformation of arctiin to arctigenin. The injection volume was 10 lL and
detection was accomplished at 303 nm for polydatin, and at 280 nm
for arctiin.
LC–MS analyses
LC–MS analyses were performed using a system consisting of 1290 Infinity
LC (Agilent, USA) and 6460 QQQ MS (Agilent, USA). The separation was
carried out by using a ZORBOX-C18 reversephase column (4.6 mm
ꢃ250 mm, 5 lm, Agilent, USA) protected by a precolumn. with a gradient
sequence using methanol (A) and a mixture (B) of formic acid acid/water
(
1:100, by vol) for enzymatic transformation of polydatin to resveratrol, at
ꢁ1
a flow rate of 0.6 mL min with the following gradient: 40% A (8 min),
0% B (7 min), and 40% B (5 min). The enzymatic transformation of arctiin
8
to arctigenin was detected by a gradient sequence using acetonitrile (A)
and water (B) in the following proportions: 25% A (15 min), 70% B
ꢁ1
(
5 min), and 25% B (5min) at a flow rate of 0.6 mL min .
The mass spectral analysis was performed in a negative electrospray ion-
ization mode. The spray voltage and the splitting voltage were set at 4.0 kV
and 135V, respectively. The nebulizer gas was set at 45 psi. The capillary
ꢀ
temperature was set at 280 C. Collision induced dissociation (CID) studies
were performed using collision energy of 20%. Data acquisition is carried
out by Masshunter (Agilent).
Hydrolysis of peanut hulls and arctium lappa fruits
Peanut hulls and arctium lappa fruits were purchased from the local market
ꢀ
in Nanjing, China, and lyophilized and pulverized and stored at ꢁ20 C for
use as substrate materials (SM) for preparation of resveratrol and arctige-
nin, respectively. To confirm the hydrolysis applicability of T. maritima
TmBglA toward the glycosides polydatin and arctiin in SM. 20% (w/v) SM
was incubated with the purified TmBglA in 100 m M SPB at optimized pH
ꢀ
in a thermostatically controlled incubator at 80 C, independently. The
enzyme concentration for TmBglA was 50 U per gram SM (activity was
determined with pNPG as substrate). The reaction mixtures were left for
10 min, 20 min, 40 min, and 60 min, and then added 50 mL methanol and
analyzed by TLC. A control reaction of crude extract without enzyme was
set up in the same manner. The conversion yield of enzymatic hydrolyze

JOURNAL OF CARBOHYDRATE CHEMISTRY
15
were calculated by following equations, conversion yield ¼ G/F (mg/g). G,
the amount of certain aglycone; F, the amount of SM been processed.
Docking of polydatin or arctiin to the active site of T. maritima b-glu-
cosidase Polydatin or arctiin were docked into the active site of TmBglA
(
PDB code 1OIF) using AutoDock version 4.2 (http://autodock.scripps.
edu). The coordinate files of both protein and ligand required for docking
calculation were prepared by AutoDockTools. The substrate orientation
giving the lowest interaction energy was chosen for docking.
Funding
Huaian Key Research and Development Program grant num-
bers [HAN201621].
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