Synthesis of isorhamnetin-3-O-rhamnoside by a three-enzyme (Rhamnosyltransferase, Glycine Max Sucrose Synthase, UDP-Rhamnose Synthase) cascade using a UDP-rhamnose regeneration system

Article abstract of DOI:10.3390/molecules24173042

Isorhamnetin-3-O-rhamnoside was synthesized by a highly efficient three-enzyme (rhamnosyltransferase, glycine max sucrose synthase and uridine diphosphate (UDP)-rhamnose synthase) cascade using a UDP-rhamnose regeneration system. The rhamnosyltransferase

Full text of DOI:10.3390/molecules24173042

molecules
Article
Synthesis of Isorhamnetin-3-O-Rhamnoside by a
Three-Enzyme (Rhamnosyltransferase, Glycine Max
Sucrose Synthase, UDP-Rhamnose Synthase) Cascade
Using a UDP-Rhamnose Regeneration System
Anna Chen 1,2, Na Gu 1,2, Jianjun Pei 2,3, Erzheng Su 1 , Xuguo Duan 1, Fuliang Cao 1 and
Linguo Zhao 1,2,3,
*
1
Co-Innovation Center for Sustainable Forestry in Southern China, Nanjing Forestry University,
Nanjing 210037, China
College of Chemical Engineering, Nanjing Forestry University, Nanjing 210037, China
Jiangsu Key Lab of Biomass Based Green Fuels and Chemicals, Nanjing 210037, China
Correspondence: lg.zhao@163.com; Tel.: +86-025-85427962
2
3
*
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 4 August 2019; Accepted: 19 August 2019; Published: 22 August 2019
Abstract: Isorhamnetin-3-O-rhamnoside was synthesized by a highly efficient three-enzyme
(rhamnosyltransferase, glycine max sucrose synthase and uridine diphosphate (UDP)-rhamnose
synthase) cascade using a UDP-rhamnose regeneration system. The rhamnosyltransferase gene (78D1)
from Arabidopsis thaliana was cloned, expressed, and characterized in Escherichia coli. The optimal
◦
activity was at pH 7.0 and 45 C. The enzyme was stable over the pH range of 6.5 to 8.5 and
◦
had a 1.5-h half-life at 45 C. The Vmax and Km for isorhamnetin were 0.646 U/mg and 181
µM,
respectively. The optimal pH and temperature for synergistic catalysis were 7.5 and 25 ◦C,
and the optimal concentration of substrates were assayed, respectively. The highest titer of
isorhamnetin-3-O-rhamnoside production reached 231 mg/L with a corresponding molar conversion
of 100%. Isorhamnetin-3-O-rhamnoside was purified and the cytotoxicity against HepG2, MCF-7,
and A549 cells were evaluated. Therefore, an efficient method for isorhamnetin-3-O-rhamnoside
production described herein could be widely used for the rhamnosylation of flavonoids.
Keywords: rhamnosyltransferase; one-pot synthesis; isorhamnetin-3-O-rhamnoside; catalysis
synergistic; UDP-rhamnose
1. Introduction
In nature, isorhamnetin glycosides, as the main derivatives of isorhamnetin, not only improve
the solubility and stability of isorhamnetin but also exhibit anti-inflammatory [1], antinociceptive [2],
and antioxidant activities [3]. Furthermore, isorhamnetin harboring different sugar residue results in
diverse biological activities. Isorhamnetin-3-O-galactoside has been reported to have antithrombotic
and profibrinolytic activities [
antigenotoxic activity in human chronic myelogenous leukemia cell line K562 [
4
], while isorhamnetin-3-O-robinobioside can enhance antioxidant and
]. On the other
5
hand, there was an interesting finding that the rhamnose moiety of steroidal alkaloids could affect
the binding specificity to steroid receptors and triggering death of human hepatoma cell (Hep3B)
by apoptosis [6]. In addition, some pharmacological studies also showed that many rhamnoside
compounds possess significant pharmacological activities. For example, the myricetin-3-O-rhamnoside,
isolated from Myrtus communis leaves, participates in the cellular defense system by regulating the
oxidative stress response of cells and DNA damage repair [7]. Quercetin-7-O-rhamnoside has a
strong inhibitory effect on the replication of the porcine diarrhea virus [
8] and its structural analogue,
Molecules 2019, 24, 3042; doi:10.3390/molecules24173042
www.mdpi.com/journal/molecules

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quercetin-3-O-rhamnoside also shows a good inhibitory effect on the human influenza A/WS/33
virus [ ]. As one kind of isorhamnetin glycosides, the isorhamnetin-3-O-rhamnoside only has been
found in the Cruciferae [10], Caesalpinia gilliesii [11], Formosan A. Purpurea [12], and Asturian cider
8,9
apple [13]. The isorhamnetin-3-O-rhamnoside was reported to exhibit moderate antioxidant activity [14
]
and the other biological properties remain to be studied.
The methods of extraction for the isorhamnetin-3-O-rhamnoside have been reported [10–14].
These methods are mainly based on the chemical reagents and plants resources and could be unfriendly
to the environment. What’s worse, they are limited by the low concentration of product and complex
secondary metabolites in the extracts. In comparison with chemical methods, enzymatic methods have
many potentials for the isorhamnetin-3-O-rhamnoside production because of their high efficiency and
environmental compatibility. The research about biological synthesis of isorhamnetin 3-O-glucoside
using engineered glucosyltransferase has been reported [15]. The glycosyltransferases, which can
transfer sugar molecules in their activated forms to acceptors, were considered to catalyze the most
of glycosylation reactions in nature [16–20]. However, glycosylation reactions were limited by sugar
donors. Sugar nucleotides are most commonly used as sugar donors and the uridine diphosphate
(UDP) sugars are the largest group of nucleotide sugars. Nevertheless, most UDP sugars, especially
UDP-rhamnose, which can be attached to small molecules such as secondary metabolites to improve
their solubility, stability, and pharmacological properties [21–24], are expensive and hard to acquire.
In our previous work, a cofactor self-sufficient UDP-rhamnose regeneration system has been established.
This system can synthesize UDP-rhamnose from sucrose, Nicotinamide adenine dinucleotide (NAD+),
and UDP by the catalysis of UDP-rhamnose synthase (VvRHM-NRS, cloned from Vitis vinifera) coupling
with Glycine max sucrose synthase (GmSUS) and can be applied to the glycosylation of flavonoids by
using the A. thaliana glycosyltransferase (78D1) [25].
In this paper, 78D1 was cloned, expressed, and characterized. Moreover, we have developed a
process for the biosynthesis of isorhamnetin-3-O-rhamnoside by a three-enzyme cascade on the basis of
the self-sufficient UDP-rhamnose regeneration system (Figure 1). Then, isorhamnetin-3-O-rhamnoside
was prepared and used to evaluate the cytotoxicity against HepG2, MCF-7, and A549 cells.
Figure 1. Schematic diagram of synergistic catalysis for isorhamnetin-3-O-rhamnoside by the
combination of uridine diphosphate (UDP)-rhamnose regeneration and glycosylation of isorhamnetin.
2. Results and Discussion
2.1. Characterization and Purification of 78D1
Rhamnosyltransferase, which is widely found in nature, is involved in the production of secondary
metabolites and plays an important role in the structural composition and various physiological
functions of organisms. Some rhamnosyltransferases, which can catalyze the rhamnosylation of

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phenolic hydroxyl groups in flavonoids at different positions, were cloned and identified from different
plants. For example, a flavonol 3-O-glucoside (1 6) rhamnosyltransferase GmF3G6”Rt was cloned
→
from soybean [26], which can convert kaempferol 3-O-glucoside to kaempferol 3-O-rutinoside and utilize
3-O-glucosylated/galactosylated flavonols and UDP-rhamnose as substrates. Casas et al. [27] cloned
the glycosyltransferase UGT91L1 from the maize, and the results of enzyme function identification
showed that UGT91L1 could use isoorientin and UDP-rhamnose as substrates and convert them to
rhamnosylisoorientin. However, most of these rhamnosyltransferases have not been characterized.
Jones et al. [28] cloned 78D1 from Arabidopsis thaliana and confirmed that 78D1 catalyzes the transfer of
rhamnose from UDP-rhamnose to the 3-OH position of quercetin and kaempferol, which means that
78D1 can be widely applied to the rhamnosylation of flavonoids. Thus, it is necessary to characterize
the rhamnosyltransferase 78D1.
In order to improve the expression level of 78D1 in E. coli, we designed and optimized codons
of 78D1 for the E. coli expression system. The recombinant 78D1 was expressed by adding 0.1 mM
Isopropyl β
-d-Thiogalactoside (IPTG) at 20 ◦C for approximately 12 h. Recombinant 78D1 was purified
by Nickel affinity (Ni-NTA) column. The purification gave a single band on a 12% polyacrylamide gel
electrophoresis (SDS-PAGE) gel and the molecular mass of the enzyme was less than 55 kDa without
undesired bands (Figure 2), which was as similar as the theoretical molecular mass of the monomer
(50019 Da).
Figure 2. Polyacrylamide gel electrophoresis (SDS-PAGE) analysis of recombinant enzymes from E. coli.
Lane 1: protein marker, lane 2: the crude extract of E. coli BL21, lane 3: the crude extract of 78D1, and
lane 4: purified 78D1.
The biochemical properties of the 78D1 were characterized by using the purified recombinant
78D1. The optimal temperature of 78D1 is 45 ◦C (Figure 3a), and the enzyme activity was higher than
80% of the maximum activity when the temperature ranges from 35 to 55 ◦C. The thermostability of

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78D1 was assayed and indicated that the residual activity of the enzyme was more than 95% after
being incubated at 40 ◦C for 90 min, and more than 50% after being incubated at 45 ◦C for 90 min
(Figure 3b). The optimal pH of 78D1 was 7.0 (Figure 3c), while its activity was higher than 80% of the
maximum activity when the pH ranges from 6.5 to 8.5. The 78D1 showed good stability after being
treated in 50 mM phosphate buffers of different pH levels at 4 ◦C for 24 h, and the residual activity
was more than 95% (Figure 3d). Apparent Km and Vmax of 78D1 for isorhamnetin were 181
µM and
0.646 U/mg respectively, and the Vmax/Km value of 78D1 for isorhamnetin was 3.56 U/(mg·mM).
Figure 3. The effects of pH and temperature on the activity and stability of 78D1. (
a) Effects of
temperature on 78D1 activity. ( ) The thermostability of 78D1. ( ) Effects of pH on 78D1 activity.
b
c
(d) Effects of pH on 78D1 stability. The initial activity was defined as 100%.
It is important for 78D1 to adapt to the reaction conditions of synergistic catalysis. We investigated
the effects of reaction substrates on the activity of 78D1. The effects of sucrose and fructose on the
activity of 78D1 were not significant (Figure 4a,b), which suggested that 78D1 can adapt to the reaction
conditions of GmSUS. The effect of NAD+ on 78D1 activity was not significant either (Figure 4c), which
indicated that the enzyme can adapt to the reaction conditions of VvRHM-NRS. The enzyme activity
was slightly increased when the concentration of Dimethyl sulfoxide (DMSO) was increased from 1%
to 5% (Figure 4d). The enzyme activity was inhibited when the concentration of UDP was gradually
increased (Figure 4e), which indicated that it is necessary to avoid the accumulation of UDP in the
reaction system. These researchers suggest that recombinant 78D1 represents a potent candidate for the
production of isorhamnetin-3-O-rhamnoside on the basis of the UDP-rhamnose regeneration system.
A certain amount of flavonoid product was synthesized by 78D1, GmSUS, and VvRHM-NRS
(Figure 5c), while no flavonoid product was synthesized when the reaction mixture only had 78D1 or
GmSUS and 78D1 (Figure 5a,b), which means that there is a synergistic reaction between three enzymes.
A comparison of the m/z values of the molecular ion (M – H)− of the product (461.1104) showed that
the differences corresponded to a rhamnose residue in isorhamnetin (315.0518) (Figure 5d,e).

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Figure 4. Effects of sucrose (
purified 78D1. The initial activity was defined as 100%.
a), fructose (b (c), DMSO (d), and UDP (e) on the activity of
), NAD+
2.2. Optimizing the Ratio among 78D1, GmSUS, and VvRHM-NRS in a One-Pot Synthesis of
Isorhamnetin-3-O-Rhamnoside
The effects of the ratio among 78D1, GmSUS, and VvRHM-NRS on isorhamnetin-3-O-rhamnoside
production were determined (Table 1). Isorhamnetin-3-O-rhamnoside production increased 457%
when the amount of VvRHM-NRS was increased from 125 to 500
183% and 280% when the amounts of GmSUS and 78D1 were raised from 8 to 88
95 g/mL, respectively. This result indicates that the one-pot synthesis of isorhamnetin-3-O-rhamnoside
µg/mL, whereas it only increased
µg/mL and from 9.5 to
µ
catalyzed by VvRHM-NRS is the rate-limiting step in the present system. Isorhamnetin-3-O-rhamnoside
production increased slightly when 78D1, GmSUS, or VvRHM-NRS continued to increase without
changing the amounts of the other two enzymes. Thus, the optimal ratio among 78D1, GmSUS, and
VvRHM-NRS was determined to be 57:50:375 (µg/mL).

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Figure 5. HPLC and LC/MS analysis of products synthesized from isorhamnetin. (
the reaction system with 78D1 only. ( ) HPLC analysis of the reaction system with 78D1 and GmSUS.
) HPLC analysis of the reaction system with 78D1, GmSUS, and VvRHM. ( ) LC/MS analysis of the
product of the synergistic catalysis.
a) HPLC analysis of
b
(c
d,e
Table 1. Effect of Enzyme Concentration on Product Yields of Isorhamnetin-3-O-rhamnoside a.
Entry
78D1 (µg/mL)
GmSUS (µg/mL)
VvRHM-NRS (µg/mL)
Isorhamnetin-3-O-rhamnoside (mM)
1
2
3
4
5
6
7
8
9
95
95
95
95
9.5
19
38
95
57
57
57
57
57
8
250
250
250
250
250
250
250
250
125
188
250
375
500
0.024
0.034
0.039
0.044
0.015
0.025
0.033
0.042
0.014
0.017
0.041
0.059
0.064
16
32
88
50
50
50
50
50
50
50
50
50
10
11
12
13
a
The reaction mixture was incubated in 25 ◦C for 2 h. Values shown are the mean of duplicate experiments, and the
variation about the mean was below 5%.

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2.3. Optimizing the Conditions of Synergistic Catalysis
Temperature and pH are important factors in the one-pot synthesis of isorhamnetin-3-O-rhamnoside
because of the effect on enzyme-specific activities and stabilities. The result showed that the optimal
pH and temperature for synergistic catalysis were pH 7.5 and 25 ◦C (Figure 6a,b), respectively.
◦
Isorhamnetin-3-O-rhamnoside production was 16% of the maximum production at 35 C because
of the poor thermostability of VvRHM-NRS, and the lower temperature is more beneficial to sustaining the
thermostabilities of the enzymes. Therefore, the temperature 25 ◦C was used for the following experiments.
Figure 6. Optimization of bioconversion conditions for isorhamnetin-3-O-rhamnoside production by
synergistic catalysis. Effects of temperature (a), pH (b), DMSO (c), and isorhamnetin (d) concentration
on production. Values shown are the mean of duplicate experiments, and the variation about the mean
was below 5%.
The influence of DMSO concentration on the synergistic catalysis was studied (Figure 6c).
The isorhamnetin solubility can be improved by DMSO to solve the problem that the poor solubility of
isorhamnetin in the reaction mixture inhibits the activity of enzyme 78D1. The production was slightly
increased when the concentration of DMSO was increased from 1% to 2%. When the concentration of
DMSO exceeded 15%, isorhamnetin-3-O-rhamnoside production rapidly decreased. The production of
isorhamnetin-3-O-rhamnoside increased as the concentration of isorhamnetin increased from 0.1 mM
to 0.5 mM and the maximal production reached 0.25 mM in 15 h (Figure 6d).
2.4. Effects of Uridine Diphosphate (UDP), NAD+, and Sucrose on the One-Pot Synthesis of
Isorhamnetin-3-O-rhamnoside
The isorhamnetin-3-O-rhamnoside production rate was measured when the initial concentrations
of sucrose (60–525 mM; 0.5 mM UDP; 1 mM NAD+), UDP (0.02–2 mM; 300 mM sucrose; 1 mM NAD+),
and NAD+ (0.1–8 mM; 0.5 mM UDP; 300 mM sucrose) were varied, respectively. The results are
shown in Figure 7. The isorhamnetin-3-O-rhamnoside production rate was significantly decreased as
the concentration of sucrose was increased from 420 mM to 525 mM (Figure 7a). UDP is a complex
factor of synergistic catalysis, which is not only essential for GmSUS but is also a potent inhibitor of
78D1. The isorhamnetin-3-O-rhamnoside production rate was increased approximately three-fold
when the concentration of UDP was increased from 20 to 500 µM and was decreased by half when the

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concentration of UDP exceeded 0.5 mM (Figure 7b). The isorhamnetin-3-O-rhamnoside production
rate was also increased approximately six-fold when the concentration of NAD+ was increased from
0.1mM to 2 mM (Figure 7c). The production rate decreased by half when the concentration exceeded
2 mM because of the fact that NAD+ was also a potential inhibitor of the activity of VvRHM-NRS and
high concentration of NAD+ could inhibit nicotinamide adenine dinucleotide (NADH) to serve as the
cofactor of VvRHM-NRS. Based on the conversion efficiency, 400 mM sucrose, 0.5 mM UDP, and 2 mM
NAD+ were used for the next experiments.
Figure 7. Isorhamnetin-3-O-rhamnoside production rate (rp) by synergistic catalysis as a function of
(a) sucrose, (b) UDP, and (c
) NAD+ concentrations. Values shown are the mean of duplicate experiments,
and the variation about the mean was below 5%.
2.5. The Time Courses of Isorhamnetin-3-O-rhamnoside Production and Preparation
On the basis of the consequence presented above, the optimal conversion conditions were
determined and used for the production of isorhamnetin-3-O-rhamnoside. The specific productivity
was 4.5 mg/L/h during the first hour after the initiation of synergistic catalysis (Figure 8). The specific
productivity gradually increased as the reaction proceeded. The specific productivities were 9.86 mg/L/h
over a reaction time of 1–3 h, 7.98 mg/L/h over a reaction time of 3–12 h, 5.36 mg/L/h over a reaction
time of 12–24 h, 2.10 mg/L/h over a reaction time of 24 48 h, and 0.65 mg/L/h over a reaction time of
−
48–72 h. The main cause of the reduction in the specific productivity was the effect of product inhibition.
After 72 h, 231 mg/L isorhamnetin-3-O-rhamnoside was produced with a corresponding molar
conversion of 100%. After reparative liquid chromatography, rotary evaporation, and freeze-drying,
isorhamnetin-3-O-rhamnoside was prepared from isorhamnetin in 89.8% overall yield and the final
production of product was 50.1 mg. The structure of the product from synergistic catalysis was
determined. The 1H and 13C-NMR spectra were recorded on a Bruker AVANCE IIII 400 spectrometer
at 400 MHz (1H-NMR) and 100 MHz (13C-NMR) in DMSO-d6, with Me4Si as the internal standard.
1H-NMR (400 MHz, DMSO-d6)
δ
7.43 (d, J = 2.0 Hz, 1H, 60-H), 7.37 (dd, J = 8.3, 2.1 Hz, 1H, 50-H),

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6.93 (d, J = 8.3 Hz, 1H, 20-H), 6.40 (s, 1H, 8-H), 6.18 (s, 1H, 6-H), 5.28 (d, J = 1.7 Hz, 1H, 1”-H), 4.95
(d, J = 4.4 Hz, 1H, 5”-H), 4.75 (d, J = 4.0 Hz, 1H, 2”-H), 4.63 (s, 1H, 3”-H), 3.96 (s, 1H, 4”-H), 3.84
(s, 3H, 30-OCH3), 3.50 (d, J = 10.8 Hz, 2H, 3”, 4”-OH), 0.78 (d, J = 5.7 Hz, 3H, 2”-CH3). 13C-NMR
(100 MHz, DMSO-d6)
δ 177.32 (4-C), 172.96 (7-C), 156.61 (5-C), 149.78 (9-C), 147.26 (2-C), 133.98
(3-C), 122.48 (30-C), 120.60 (40-C), 115.46 (10-C), 112.69 (60-C), 104.04 (50-C), 103.28 (20-C), 101.68
(10-C), 99.26 (8-C), 94.07 (6-C), 72.51 (1”-C), 71.12 (5”-C), 70.03 (2”-C), 69.77 (3”-C), 63.06 (4”-C),
55.71 (30-OCH3), 17.48 (2”-CH3) (Figures S1 and S2).The H-NMR spectrum was analyzed and
1
compared to reference compounds10. The result confirmed that the catalysis synergistic product
was isorhamnetin-3-O-rhamnoside. Obtained Isorhamnetin-3-O-rhamnoside thus meets commercial
standards of quality and was used for the next research.
Figure 8. Time courses of isorhamnetin-3-O-rhamnoside production and isorhamnetin reduction
in a synergistic reaction system. The circle represents isorhamnetin-3-O-rhamnoside. The triangle
represents isorhamnetin.
2.6. Evaluation of Cytotoxicity against HepG2, MCF-7, and A549 Cells
In this paper, we evaluated antiproliferative activities of isorhamnetin-3-O-rhamnoside and
cisplatin (DDP) against HepG2, MCF-7, and A549 cells using the Thiazolyl Blue Tetrazolium Bromide
(MTT) assay. The cell proliferation inhibition rates are shown in Figure 9. There is no inhibiting effect
on the HepG2, MCF-7, and A549 cells when the isorhamnetin-3-O-rhamnoside and DPP do not exist.
Moreover, the isorhamnetin-3-O-rhamnoside and DPP had lower inhibitory effects on human hepatoma
cell line HepG2 and human lung cancer cell line A549. 160 µM isorhamnetin-3-O-rhamnoside showed
strong inhibitory effects on human breast adenocarcinoma cell line MCF-7 proliferation (inhibition rate
= 51%), though the same concentration DPP (positive control) showed the strongest inhibitory effects
on human breast adenocarcinoma cell line MCF-7 proliferation (inhibition rate = 97%).

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Figure 9. Isorhamnetin-3-O-rhamnoside evaluation of cytotoxicity against HepG2, MCF-7, and
A549 cells. Various concentrations of Isorhamnetin-3-O-rhamnoside and DDP (0, 40, 80, or 160 M)
were added.
µ

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3. Materials and Methods
3.1. Strains, Plasmids, Media, and Chemicals
The plasmid propagated by Escherichia coli Top10 and recombinant enzymes were produced by
Escherichia coli BL21 (DE3). PET-28a was purchased from Novagen (Darmstadt, Germany). The strains
were cultured at 37 ◦C in Luria-Bertani (LB) medium supplemented with appropriate antibiotics. UDP
and NAD+ were purchased from Sigma Chemical Co (St. Louis, MO, USA). UDP-rhamnose was
purchased from Xiyuan Biotechnology Co (Shanghai, China). Isorhamnetin was purchased from MUST
Bio-Technology (Chengdu, China).
3.2. Plasmid Construction
We designed and optimized codons of 78D1 for E. coli expression system. The BamHI and EcoRI
sites were added to the 50 and 30 ends of the gene, respectively. The synthesized 78D1 was digested
with BamHI and EcoRI and subcloned into the expression plasmid PET-28a at the Bam HI and EcoRI
sites to create PET-28a-78D1.
3.3. Purification of Recombinant Enzymes
Recombinant enzymes GmSUS and VvRHM-NRS were prepared in our previous work [25].
The PET-28a-78D1 plasmid was introduced into E.coli BL21 (DE3) and induced to express by adding
0.1 mM isopropyl-
β
-d-thiogalactopyranoside (IPTG) at an OD600 of approximately 0.7. Then, the strain
◦
was incubated at 20 C for approximately 24 h. Recombinant cells (1000 mL) were harvested by
centrifugation at 5000
procedures were performed according to the previous publication [29]. The recombinant 78D1 was
purified by Ni2+-NTA affinity chromatography column and was examined by SDS-PAGE. The protein
concentration was determined by the Bradford method using Albumin from bovine serum (BSA) as
a standard.
◦
×
g for 10 min at 4 C and washed twice with distilled water. The detailed
3.4. Glycosyltransferase Activity
To determine the activity of 78D1, the reaction mixture was incubated at 35 ◦C for 30 min and
contained 50 mM phosphate buffer (pH 7.5), excess UDP-rhamnose, 0.5 mM isorhamnetin, a certain
amount of 78D1 in 100
µL. The reaction was stopped by adding 200 µL of methanol and was then
assayed via high-performance liquid chromatography (HPLC). One unit of enzyme activity was
defined as the amount of enzyme necessary to synthesize 1
min under the assay conditions.
µmol isorhamnetin-3-O-rhamnoside per
The effects of temperature on the enzyme activity were determined over a temperature range of
35–60 ◦C in 50 mM phosphate buffer, pH 7.5. To determine the effect of temperature on the stability of
78D1, the enzyme in the 50 mM phosphate buffer (pH 7.5) was pre-incubated for various times at 40 ◦C
and 45 ◦C in the absence of the substrate. The effects of pH on the enzyme activity were assayed over a
pH range of 6.0–8.5 at 35 ◦C for 30 min using 50 mM phosphate buffer. To determine the effects of pH
on the stability of 78D1, the enzyme in the 50 mM phosphate buffer (pH 6.5–8.5) was pre-incubated for
24 h at 4 ◦C in the absence of the substrate.
The effects of sucrose (0, 10, 50, 100, 200, and 500 mM), fructose (0, 10, 50, 100, and 200 mM), NAD+
(0, 0.1, 0.5, 1, 2, and 4 mM), dimethyl sulfoxide (DMSO) (1%, 5%, 10%, 15%, and 20% v/v), and UDP
(0, 0.01, 0.05, 0.1, 0.5, 1, and 5 mM) on the 78D1 activity were assayed, respectively. The enzyme was
incubated with different concentrations of substrates for 5 min at 35 ◦C before adding UDP-rhamnose
to start the enzymatic reaction. The activity of 78D1 was determined as described above and the
activity without pre-incubation was defined as 100%. The kinetic constant of 78D1 was determined by
measuring the initial rates at different isorhamnetin concentrations under standard reaction condition.

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3.5. Synergistic Catalysis
Reaction mixture containing 0.5 mM isorhamnetin, 300 mM sucrose, 1 mM NAD+, 0.5 mM UDP,
50 mM phosphate buffer (pH 7.5), 10% DMSO (v/v), rhamnosyltransferase 78D1, sucrose synthase
GmSUS, and VvRHM-NRS in 100 µ
L was incubated for 15 h at 30 ◦C. 1 mM DTT was added to keep
the stability and activity of VvRHM-NRS. The reaction was stopped by adding 200
assayed via HPLC.
µL of methanol and
The effects of pH on the synergistic catalysis were assayed over a pH range of 7.0–8.5 at 30 ◦C for
15 h in 50 mM phosphate buffer. The effects of temperature on synergistic catalysis were determined
over a temperature range of 25–35 ◦C in 50 mM phosphate buffer, pH 7.5. To optimize the conversion
conditions, the concentrations of substrates and DMSO were varied separately: UDP (0–2 mM), sucrose
(0–525 mM), NAD (0–8 mM), isorhamnetin (0–1.5 mM), and DMSO (1%–20%, v/v). To determine the
optimal ratio among GmSUS, VvRHM-NRS, and 78D1, different ratios of enzymes were added into
standard reaction mixtures.
3.6. Synthesis and Purification of Isorhamnetin-3-O-Rhamnoside
The substrate concentrations in the conversion were improved. The reaction mixture contained
0.5 mM isorhamnetin, 400 mM sucrose, 0.5 mM UDP, 2 mM NAD+, 50 mM phosphate buffer (pH 7.5), 2%
DMSO (v/v), 57
VvRHM-NRS in 100
thermomixer. The reaction was ended by adding methanol and harvested by centrifugation at 20,000
for 10 min. The supernatant was applied to preparative liquid chromatography (SHIMADZU, Japan)
and a C18 column with methanol (A) and distilled water (B) at the A/B ratio of 55:45. The product
was collected and evaporated to remove the methanol, then the product was frozen to dryness and
analyzed by HPLC and NMR.
µ
g/mL rhamnosyltransferase 78D1, 50
µg/mL sucrose synthase GmSUS, and 375 µg/mL
µ
L. The reaction volume was 250 mL and was incubated at 25 ◦C and 150 rpm in a
×
g
3.7. HPLC, LC/MS Analysis and Structural Identification
HPLC analysis of isorhamnetin and isorhamnetin-3-O-rhamnoside (5,7-dihydroxy-2-
(4-hydroxy-3-methoxyphenyl)-3-(((2R,3S,4S,5R)-3,4,5-trihydroxy-6-methyltetrahydro-2H-pyran-2-yl)oxy)-
4H-chromen-4 -one) was performed by using an HPLC 1200 system (Agilent, USA) and a C18
(250 × 4.6 mm; 5 µm) column with methanol (A) and distilled water (B) at A/B ratios of 55:45 for 17 min.
The flow rate was 0.8 mL/min, and detection was performed by monitoring the absorbance at 368 nm.
Liquid Chromatograph Mass Spectrometer (LC/MS) for isorhamnetin and isorhamnetin-3-O-rhamnoside
were analyzed in an LTQ Orbitrap XL LC/MS in negative mode with an anion trap analyzer. The ion
spray was operated at 25 Arb N2/min, 3.5 kV, and 300 ◦C. The 1H and 13C-NMR spectra were recorded on
a Bruker AVANCE IIII 400 spectrometer at 400 MHz (1H-NMR) and 100 MHz (13C-NMR) in DMSO-d6,
with Me4Si as the internal standard.
3.8. Cell Culture and Cytotoxicity Assay
Human hepatoma cell line HepG2, human breast cancer cell line MCF-7, and human lung cancer
cell line A549 were purchased from the Cell Bank of the Chinese Academy of Sciences. HepG2, MCF-7,
and A549 cells were grown in dulbecco’s modified eagle medium (DMEM) medium supplemented
with 10% fetal bovine serum. All cells were kept in a humidified incubator with 5% CO2 and 95% air at
37 ◦C. Cells were routinely collected by centrifugation at 800× g for five minutes.
The effect of isorhamnetin-3-O-rhamnoside on the proliferation of tumor cells was tested by MTT
assay. After trypan blue staining, 3
×
103 tumor cells were inoculated into each well of a 96-well plate.
After 6 h, different concentrations of isorhamnetin-3-O-rhamnoside and cisplatin (DPP) were added to
each well, and then the plate was cultured at 37 ◦C in a 5% CO2 atmosphere for 72 h. MTT solution
(20
µ
L, 4 mg/mL) was added to the culture medium. After 4 h, the plate was centrifuged at 1000
×
g for
5 min, and the supernatant was removed. DMSO (200
µL) was added to the wells and mixed until

Molecules 2019, 24, 3042
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the precipitate completely dissolved. The absorbance at 540 nm was measured and recorded. All
experiments were performed in at least four parallels and repeated three times. The percentage of cell
proliferation inhibition was calculated using the following formula: Cell proliferation inhibition = (1
ODsample/ODcontrol) × 100%.
−
4. Conclusions
An enzymatic method for isorhamnetin-3-O-rhamnoside production has not been reported,
and the product was seldom reported in other literatures. In conclusion, the rhamnosyltransferase
gene (78D1) from Arabidopsis thaliana was cloned, expressed, and characterized in Escherichia coli.
Furthermore, this paper described a novel and simple multienzyme method for the synthesis of
isorhamnetin-3-O-rhamnoside with high efficiency. We have reported that the quercetin can be the
substrate of the three-enzyme synergistic system [25]. This work confirmed that isorhamnetin can
also be the substrate of the three-enzyme synergistic system. The result confirmed that the regenerate
UDP-rhamnose system, which was reported in our previous work, can be applied to the rhamnosylation
of flavonoids.
Supplementary Materials: The following are available online, Figure S1: 1H-NMR(A) and 13C-NMR(B)
spectra of isorhamnetin production by synergistic catalysis.
of isorhamnetin-3-O-rhamnoside.
Figure S2: The structural formula
Author Contributions: Data curation, A.C.; Funding acquisition, L.Z.; Investigation, N.G.; Methodology, A.C., J.P.
and L.Z.; Supervision, E.S., X.D., F.C. and L.Z.; Writing – original draft, A.C.; Writing – review and editing, A.C.
Funding: This work was supported by the National Natural Science Foundation of China (31570565), the Science
and Technology project of Jiangsu Province (SZ-XZ2017023), and the Forestry Achievements of Science and
Technology to Promote Projects ((2017) 10).
Acknowledgments: This work was support by Advanced Analysis and Testing Center, Nanjing Forestry University.
Conflicts of Interest: The authors declare no conflict of interest.
References
1.
2.
3.
Antunes-Ricardo, M.; Gutierrez-Uribe, J.A.; Lopez-Pacheco, F.; Alvarez, M.M.; Serna-Saldivar, S.O. In vivo
anti-inflammatory effects of isorhamnetin glycosides isolated from Opuntia ficus-indica (L.) Mill cladodes.
Ind. Crop. Prod. 2015, 76, 803–808. [CrossRef]
Kucukboyaci, N.; Akkol, E.K.; Suntar, I.; Calis, I. In vivo Anti-Inflammatory and Antinociceptive Activities
of the Extracts and Chemical Constituents of an Endemic Turkish Plant, Salsola grandis. Rec. Nat. Prod.
2016, 10, 369–379.
Park, J.Y.; Kim, S.I.; Lee, H.J.; Kim, S.S.; Kwon, Y.S.; Chun, W. Isorhamnetin-3-O-Glucuronide Suppresses
JNK and p38 Activation and Increases Heme-Oxygenase-1 in Lipopolysaccharide-Challenged RAW264.7
Cells. Drug Dev. Res. 2016, 77, 143–151. [CrossRef]
4.
5.
Ku, S.K.; Kim, T.H.; Lee, S.; Kim, S.M.; Bae, J.S. Antithrombotic and profibrinolytic activities of
isorhamnetin-3-O-galactoside and hyperoside. Food Chem. Toxicol. 2013, 53, 197–204. [CrossRef]
Boubaker, J. Isorhamnetin 3-O-robinobioside from Nitraria retusa leaves enhance antioxidant and antigenotoxic
activity in human chronic myelogenous leukemia cell line K562. BMC Complement. Altern. Med. 2012, 12, 135.
[CrossRef]
6.
7.
Chang, L.C.; Tsai, T.R.; Wang, J.J.; Lin, C.N.; Kuo, K.W. The rhamnose moiety of solamargine plays a crucial
role in triggering cell death by apoptosis. Biochem. Biophys. Res. Commun. 1998, 242, 21–25. [CrossRef]
Hayder, N.; Bouhlel, I.; Skandrani, I.; Kadri, M.; Steiman, R.; Guiraud, P.; Mariotte, A.;
Ghedira, K.; Dijoux-Franca, M.; Chekir-Ghedira, L. In vitro antioxidant and antigenotoxic potentials of
myricetin-3-O-galactoside and myricetin-3-O-rhamnoside from Myrtus communis: Modulation of expression
of genes involved in cell defence system using cDNA microarray. Toxicol. Vitr. 2008, 22, 567–581. [CrossRef]
Choi, H.J.; Kim, J.H.; Lee, C.H.; Ahn, Y.J.; Song, J.H.; Baek, S.H.; Kwon, D.H. Antiviral activity of quercetin
7-rhamnoside against porcine epidemic diarrhea virus. Antivir. Res. 2009, 81, 77–81. [CrossRef] [PubMed]
8.

Molecules 2019, 24, 3042
14 of 15
9.
Choi, H.J.; Song, J.H.; Park, K.S.; Kwon, D.H. Inhibitory effects of quercetin 3-rhamnoside on influenza A
virus replication. Eur. J. Pharm. Sci. 2009, 37, 329–333. [CrossRef] [PubMed]
10. Atta, E.M.; Hashem, A.I.; Ahmed, A.M.; Elqosyd, S.M.; Jasparse, M.; El-Sharkawc, E.M. Phytochemical
studies on Diplotaxis harra growing in Sinai. Eur. J. Chem. 2011, 2, 535–538. [CrossRef]
11. Osman, S.M.; El-Haddad, A.E.; El-Raey, M.A.; Abd El-Khalik, S.M.; Koheil, M.A.; Wink, M. A New
Octadecenoic Acid Derivative from Caesalpinia gilliesii Flowers with Potent Hepatoprotective Activity.
Pharmacogn. Mag. 2016, 12, S332–S336. [CrossRef] [PubMed]
12. Chang, F.R.; Wei, J.L.; Teng, C.M.; Wu, Y.C. Antiplatelet aggregation constituents from Annona purpurea.
J. Nat. Prod. 1998, 61, 1457–1461. [CrossRef] [PubMed]
13. Ramirez-Ambrosi, M.; Lopez-Marquez, D.M.; Abad-Garcia, B.; Dapena, E.; Berrueta, L.A.; Gallo, B.
Comparative study of phenolic profile of fruit and juice samples of a progeny of ‘Meana’ x ‘Florina’
from an Asturian cider apple breeding program. Eur. Food Res. Technol. 2015, 241, 769–784. [CrossRef]
14. Osman, S.M.; Abd El-Khalik, S.M.; Abd El-Khalik, A.E.; Wink, M. A new steroidal compound
(β-sitosterol-3-O-butyl) isolated from Caesalpinia gilliesii flowers. Int. J. Appl. Res. Nat. Prod. 2015
,
8, 14–19.
15. Kim, B.G.; Su, H.S.; Na, R.J.; Chong, Y.; Ahn, J.H. Biological synthesis of isorhamnetin 3-O-glucoside using
engineered glucosyltransferase. J. Mol. Catal. B Enzym. 2010, 63, 194–199. [CrossRef]
16. Kim, B.G.; Kim, H.J.; Ahn, J.H. Production of bioactive flavonol rhamnosides by expression of plant genes in
Escherichia coli. J. Agric. Food Chem. 2012, 60, 11143–11148. [CrossRef]
17. Lim, E.K.; Ashford, D.A.; Hou, B.K.; Jackson, R.G.; Bowles, D.J. Arabidopsis glycosyltransferases as
biocatalysts in fermentation for regioselective synthesis of diverse quercetin glucosides. Biotechnol. Bioeng.
2010, 87, 623–631. [CrossRef]
18. Kim, H.J.; Kim, B.G.; Ahn, J.H. Regioselective synthesis of flavonoid bisglycosides using Escherichia coli
harboring two glycosyltransferases. Appl. Microbiol. Biotechnol. 2013, 97, 5275–5282. [CrossRef]
19. Lairson, L.L.; Henrissat, B.; Davies, G.J.; Withers, S.G. Glycosyltransferases: Structures, functions, and
mechanisms. Annu. Rev. Biochem. 2008, 77, 521. [CrossRef]
20. Pei, J.; Dong, P.; Wu, T.; Zhao, L.; Fang, X.; Cao, F.; Tang, F.; Yue, Y. Metabolic Engineering of Escherichia coli
for Astragalin Biosynthesis. J. Agric. Food. Chem. 2016, 64, 7966–7972. [CrossRef]
21. Diantini, A.; Subarnas, A.; Lestari, K.; Halimah, E.; Susilawati, Y.; Supriyatna; Julaeha, E.; Achmad, T.H.;
Suradji, E.W.; Yamazaki, C.; et al. Kaempferol-3-O-rhamnoside isolated from the leaves of Schima wallichii
Korth. inhibits MCF-7 breast cancer cell proliferation through activation of the caspase cascade pathway.
Oncol. Lett. 2012, 3, 1069–1072. [CrossRef] [PubMed]
22. Rho, H.S.; Ghimeray, A.K.; Yoo, D.S.; Ahn, S.M.; Kwon, S.S.; Lee, K.H.; Cho, D.H.; Cho, J.Y. Kaempferol and
kaempferol rhamnosides with depigmenting and anti-inflammatory properties. Molecules 2011, 16, 3338.
[CrossRef] [PubMed]
23. Lee, S.H.; Ko, C.I.; Ahn, G.; You, S.; Kim, J.S.; Heu, M.S.; Kim, J.; Jee, Y.; Jeon, Y.J. Molecular characteristics and
anti-inflammatory activity of the fucoidan extracted from Ecklonia Cava. Carbohydr. Polym. 2012, 89, 599–606.
[CrossRef] [PubMed]
24. Tatsimo, S.J.N.; Tamokou, J.D.D.; Havyarimana, L.; Csupor, D.; Forgo, P.; Hohmann, J.; Kuiate, J.R.; Tane, P.
Antimicrobial and antioxidant activity of kaempferol rhamnoside derivatives from Bryophyllum pinnatum.
BMC Res. Notes 2012, 5, 1–6. [CrossRef] [PubMed]
25. Pei, J.J.; Chen, A.; Sun, Q.; Zhao, L.G.; Cao, F.L.; Tang, F. Construction of a novel UDP-rhamnose regeneration
system by a two-enzyme reaction system and application in glycosylation of flavonoid. Biochem. Eng. J.
2018, 139, 33–42. [CrossRef]
26. Rodas, F.R.; Rodriguez, T.O.; Murai, Y.; Iwashina, T.; Sugawara, S.; Suzuki, M.; Nakabayashi, R.;
Yonekura-Sakakibara, K.; Saito, K.; Kitajima, J. Linkage mapping, molecular cloning and functional analysis
of soybean gene Fg2 encoding flavonol 3-O-glucoside (1
84, 287–300. [CrossRef] [PubMed]
→
6) rhamnosyltransferase. Plant Mol. Biol. 2014,
27. Casas, M.I.; Falcone-Ferryra, M.L.; Jiang, N.; Mejia Guerra, M.K.; Rodriguez, E.J.; Wilson, T.; Engelmeier, J.;
Casati, P.; Grotewold, E. Identification and characterization of maize salmon silks genes involved in
insecticidal maysin biosynthesis. Plant Cell 2016, 28, 1297. [CrossRef] [PubMed]

Molecules 2019, 24, 3042
15 of 15
28. Jones, P.; Messner, B.; Nakajima, J.; Schäffner, A.R.; Saito, K. UGT73C6 and UGT78D1, glycosyltransferases
involved in flavonol glycoside biosynthesis in Arabidopsis thaliana. J. Biol. Chem. 2003, 278, 43910–43918.
[CrossRef]
29. Pei, J.; Chen, A.; Zhao, L.; Cao, F.; Ding, G.; Xiao, W. One-pot synthesis of hyperoside by a three-enzyme
cascade using a UDP-galactose regeneration system. J. Agric. Food. Chem. 2017, 65, 6042–6048. [CrossRef]
Sample Availability: Samples of the compounds are available from the Dr. Jianjun Pei.
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2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
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