Preparation of isoquercitrin by biotransformation of rutin using α-L-rhamnosidase from Aspergillus niger JMU-TS528 and HSCCC purification

Article abstract of DOI:10.1080/10826068.2019.1655763

Isoquercitrin is a flavonoid with important applications in the pharmaceutical and nutraceutical industries. However, a low yield and high production cost hinders the industrial preparation of isoquercitrin. In the present study, isoquercitrin was prepared by biotransformation of rutin using α-L-rhamnosidase from Aspergillus niger JMU-TS528 combined with high-speed countercurrent chromatography (HSCCC) purification. As a result, the optimum transformation pH, temperature, and time were pH 4.0, 60 °C, and 60 min, respectively. The K_m and V_max were 0.36 mM and 0.460 mmol/min, respectively. For isoquercitrin production, the optimal rutin concentration and transformation time were approximately 1000 mg/L and 60 min. The rutin transformation rate reached 96.44%. The isoquercitrin was purified to a purity of 97.83% using one-step purification with HSCCC. The isoquercitrin was identified using UPLC-Q-TOF-MS. The comprehensive results indicated that the biotransformation procedure using the α-L-rhamnosidase from A. niger JMU-TS528 combined with HSCCC was a simple and effective process to prepare isoquercitrin, which might facilitate the production of isoquercitrin for industrial use.

Full text of DOI:10.1080/10826068.2019.1655763

Preparative Biochemistry and Biotechnology
ISSN: 1082-6068 (Print) 1532-2297 (Online) Journal homepage: https://www.tandfonline.com/loi/lpbb20
Preparation of isoquercitrin by biotransformation
of rutin using α-L-rhamnosidase from Aspergillus
niger JMU-TS528 and HSCCC purification
Li Jun Li, Xiao Qing Liu, Xi Ping Du, Ling Wu, Ze Dong Jiang, Hui Ni, Qing Biao
Li & Feng Chen
To cite this article: Li Jun Li, Xiao Qing Liu, Xi Ping Du, Ling Wu, Ze Dong Jiang, Hui Ni, Qing
Biao Li & Feng Chen (2019): Preparation of isoquercitrin by biotransformation of rutin using
α-L-rhamnosidase from Aspergillusꢀniger JMU-TS528 and HSCCC purification, Preparative
Biochemistry and Biotechnology, DOI: 10.1080/10826068.2019.1655763
To link to this article: https://doi.org/10.1080/10826068.2019.1655763
Published online: 23 Aug 2019.
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PREPARATIVE BIOCHEMISTRY AND BIOTECHNOLOGY
https://doi.org/10.1080/10826068.2019.1655763
Preparation of isoquercitrin by biotransformation of rutin using
a-L-rhamnosidase from Aspergillus niger JMU-TS528 and HSCCC purification
Li Jun Lia,b,c, Xiao Qing Liua, Xi Ping Dua,b,c, Ling Wua,b,c, Ze Dong Jianga,b,c, Hui Nia,b,c, Qing Biao Lia,b,c, and
Feng Chena,d
b
aCollege of Food and Biological Engineering, Jimei University, Xiamen, China; Fujian Provincial Key Laboratory of Food Microbiology and
c
d
Enzyme Engineering, Xiamen, China; Research Center of Food Biotechnology of Xiamen City, Xiamen, China; Department of Food,
Nutrition and Packaging Sciences, Clemson University, Clemson, SC, USA
KEYWORDS
ABSTRACT
a-L-rhamnosidase; biotrans-
formation; high-speed
countercurrent chromatog-
raphy; isoquercitrin; rutin
Isoquercitrin is a flavonoid with important applications in the pharmaceutical and nutraceutical
industries. However, a low yield and high production cost hinders the industrial preparation of iso-
quercitrin. In the present study, isoquercitrin was prepared by biotransformation of rutin using
a-L-rhamnosidase from Aspergillus niger JMU-TS528 combined with high-speed countercurrent
chromatography (HSCCC) purification. As a result, the optimum transformation pH, temperature,
and time were pH 4.0, 60 ꢀC, and 60 min, respectively. The Km and Vmax were 0.36 mM and
0.460mmol/min, respectively. For isoquercitrin production, the optimal rutin concentration and
transformation time were approximately 1000 mg/L and 60 min. The rutin transformation rate
reached 96.44%. The isoquercitrin was purified to a purity of 97.83% using one-step purification
with HSCCC. The isoquercitrin was identified using UPLC-Q-TOF-MS. The comprehensive results
indicated that the biotransformation procedure using the a-L-rhamnosidase from A. niger JMU-
TS528 combined with HSCCC was a simple and effective process to prepare isoquercitrin, which
might facilitate the production of isoquercitrin for industrial use.
Introduction
and the glucoside bond, which simultaneously yields quer-
cetin and isoquercitrin, decreasing the isoquercitrin product-
ivity and increasing the purification cost.[16] Enzymes such
as naringinase, hesperidinase, and a-L-rhamnosidase (Fig. 1)
have been proposed for transforming rutin to isoquerci-
trin.[9,14,20–23] Naringinase and hesperidinase also yield quer-
cetin and isoquercitrin simultaneously,[24,25] as they have the
activities of both a-L-rhamnosidase and b-D-glucosidase.[19]
In contrast, the enzyme a-L-rhamnosidase specifically
hydrolyzes the terminal L-rhamnose from natural glycosides,
which avoid the generation of quercitrin and thus transform
rutin solely to isoquercitrin. Some a-L-rhamnosidases were
reported to transform rutin to isoquercitrin;[26] however, the
low enzyme activity or low affinity of the enzyme to rutin
resulted in a low transformation rate, causing difficulties in
purification. Therefore, various purification methods are
involved in the preparation of isoquercitrin, leading to long
purification times and high production costs.[21] For
instance, Li et al. applied macroporous adsorptive resin col-
umn chromatography in combination with Sephadex LH-20
column chromatography to purify isoquercitrin.[27] Wen
et al. developed a combined procedure consisting of high-
speed countercurrent chromatography (HSCCC) and pre-
parative high-performance liquid chromatography to purify
Currently, the world’s population is steadily facing aging
and noncommunicable, chronic, and progressive dis-
eases.[1–3] Reactive oxygen, such as hydrogen peroxide,
superoxide anion, and hydroxyl radicals, can cause oxidative
damage to cellular components, which plays an important
role in the aging process.[4] Hyperglycemic emergencies are
important causes of morbidity and mortality among patients
with diabetes.[5] Neurodegeneration is damage to the brain
that results in the deterioration of central nervous system
function and memory loss.[6] Natural flavonoids that widely
exist in herbs, fruits, vegetables and other plant-derived
[7,8]
foods
could effectively reduce or alleviate these diseases
and symptoms.[7–12] Isoquercitrin is one such flavonoid that
has attracted increasing interest from scientists and food
manufacturers because this chemical has shown strong bio-
logical functions for anti-aging and the prevention of
chronic and progressive diseases,[13–15] as well as desirable
solubility and bioavailability in the digestive tract.[11,12]
In industry, isoquercitrin is prepared by hydrolyzing the
terminal rhamnose from rutin by means of acid hydroly-
sis,[16] heating,[14] microbial transformation,[17,18] or enzym-
atic transformation.[16,19] Among these, acidic and thermal
hydrolyzes nonspecifically hydrolyze the rhamnoside bond isoquercitrin.[28]
CONTACT Qing Biao Li
kelqb@xmu.edu.cn
College of Food and Biology Engineering, Jimei University Xiamen, Fujian 3616021, China.
Color versions of one or more of the figures in the article can be found online at www.tandfonline.com/lpbb.
ß 2019 Taylor & Francis Group, LLC

2
L. J. LI ET AL.
Figure 1. Transformation products of rutin using acid hydrolysis, heating, naringinase, hesperidinase, and a-L-rhamnosidase. Note: The red and blue moieties are
rhamnose and glucose, respectively.
Based on the above studies, it is important to identify a characterize the kinetics for isoquercitrin production; and
a-L-rhamnosidase that can transform rutin with high effi-
ciency and thus improve the purification of isoquercitrin. In
our previous studies, a-L-rhamnosidase from A. niger JMU-
TS528 was shown to act on rutin,[29] and HSCCC was
developed to purify a number of bioactive compounds
effectively.[30] In this context, the present study aimed to
improve the effectiveness of isoquercitrin preparation with
the aid of rutin transformation using the a-L-rhamnosidase
from A. niger JMU-TS528 and HSCCC purification. The
specific goals included the following: (1) optimize the trans-
(3) develop the HSCCC purification of isoquercitrin.
Materials and methods
Materials
Rutin and isoquercitrin standards were purchased from
Shanghai Aladdin Bio-Chem Technology Co., Ltd.
(Shanghai, China). HPLC-grade methanol and acetonitrile
were purchased from Merck & Co., Inc. (Darmstadt,
formation condition for isoquercitrin production; (2) Germany). Analytical grade ethyl acetate and formic acid

PREPARATIVE BIOCHEMISTRY AND BIOTECHNOLOGY
3
were purchased from Sinopharm Chemical Reagent Co., Ltd. conduct the enzymatic transformation. After the reaction,
the concentrations of rutin and isoquercitrin were detected
to calculate the rutin transformation rate and isoquercitrin
generation yield.
(Shanghai, China). All reagents used for culturing microor-
ganisms were purchased from Guangdong Huankai
Microbial Sci. & Tech. Co., Ltd. (Guangdong, China).
The rutin transformation rate and isoquercitrin gener-
ation yield were calculated using the following equations.
Preparation of a-L-rhamnosidase
ð Þ
Rutin transformation rate %
The gene for a-L-rhamnosidase r-rha 1 from A. niger JMU-
TS528 was integrated into plasmid pPIC9K with the AOX1
promoter, which was expressed in P. pastoris GS115. P. pas-
toris GS115 containing the gene encoding the a-L-rhamnosi-
dase r-rha 1 was preserved at ꢁ80 ꢀC in 80% (v/v) glycerol.
The deposited strain was inoculated into yeast extract pep-
tone dextrose medium (YPD) consisting of 1% (w/v) yeast
extract, 2% (w/v) peptone, and 2% (w/v) glucose by the plate
scribing method. After cultivation at 30 ꢀC for 2 days, colo-
nies had grown on the YPD plate. Then, the strain was ino-
culated in 100 mL of the buffered glycerol-complex medium
(BMGY) consisting of 1% (w/v) yeast extract, 2% (w/v) pep-
tone, 100 mM potassium phosphate pH 6.0, 1.34% (w/v)
yeast nitrogen base (YNB), 4 ꢂ 10ꢁ5% (w/v) biotin, and 1%
(v/v) glycerol, followed by cultivation on a rotary shaker at
30 ꢀC and 200 rpm. When the OD600 reached the range of
3.0ꢁ6.0, the culture was harvested by plain sedimentation
for 12 hours and then resuspended in 100 mL of buffered
methanol-complex medium (BMMY) consisting of 1% (w/v)
yeast extract, 2% (w/v) peptone, 100 mM potassium phos-
phate pH 6.0, 1.34% (w/v) yeast nitrogen base (YNB),
4 ꢂ 10ꢁ5% (w/v) biotin, and 0.5% (v/v) methanol. For the
induction of a-L-rhamnosidase, the strain was cultured at
30 ꢀC and 200 rpm for 6 days, during which methanol was
added to the medium daily at a concentration of 0.5% (v/v).
Finally, the supernatant was harvested by centrifugation at
8,000 rpm and 4 ꢀC for 5 min.[29]
ð
Þ
consumptive molar amount of rutin mol
¼
ꢂ100
ð
Þ
initial molar amount of rutin mol
ð Þ
Isoquercitrin generation yield %
ð
Þ
molar amount of isoquercitrin mol
¼
ꢂ100
ð
Þ
initial molar amount of rutin mol
Kinetics study of rutin transformation
For the kinetics study, the reaction mixture was composed
of 1980 mL of rutin solution at different concentrations and
20 mL of enzyme (preinactivated enzyme for the blank) and
immediately incubated at 60 ꢀC for 15 min, followed by heat-
ing at 100 ꢀC for 10 min to inactivate the enzyme. The initial
concentrations of rutin solutions were 0.04, 0.08, 0.16, 0.33,
0.49, 0.66, and 0.82 mM. After denaturation of the enzyme,
the reaction solutions were filtered through 0.22 mm mem-
branes and then submitted to HPLC analysis. The chemical
scheme of a-L-rhamnosidase is shown in Fig. 2. The meas-
ured rutin transformation was applied to calculate enzymatic
activity. One unit of enzyme activity was defined as the
amount of enzyme required to hydrolyze 1 mg rutin per
minute. A Lineweaver–Burk chart was plotted between 1/S
and 1/V, where S (mmol/mL) was the rutin concentration,
ꢃ
and V (mmol/(min mg)) was the reaction rate at a given
rutin concentration. Then, the kinetic constants, i.e.,
Michaelis constant (Km) and maximum velocity (Vmax
were calculated.
)
Optimization of pH and temperature for rutin
transformation
To determine the optimal pH to yield isoquercitrin, the
reaction system was set at a series pH values, i.e., 3.0, 4.0,
5.0, 6.0, 7.0, and 8.0, to operate the transformation reaction
using 1 mL of 300 mg/mL rutin solution in citric acid-diso-
dium hydrogen phosphate buffer and 20 mL of enzyme at
60 ꢀC for 15 min. To optimize the reaction temperature, the
Optimization of rutin concentration and reaction time
for isoquercitrin production
To determine the desirable rutin concentration for isoquer-
citrin production, the solubility of rutin in the reaction solu-
tion (50 mM citric acid/disodium monohydrogen phosphate
temperature was set at 30, 40, 50, 60, 70, and 80 ꢀC to buffer) was investigated. The rutin concentration was set at
Figure 2. The reaction scheme for isoquercitrin production using a-L-rhamnosidase.

4
L. J. LI ET AL.
0, 200, 400, 600, 800, 1000, 1200, and 1400 mg/mL in the The total run time was 12 min at a flow rate of 0.3 mL/min.
solution. After incubation at 60 ꢀC for 60 min, 1 mm of the
rutin solutions were clarified by filtration through a 0.22-mm
filter to remove the undissolved rutin. The resultant filtrates
were maintained at 60 ꢀC until HPLC analysis.
To investigate the reaction time for isoquercitrin yield,
the reaction system was composed of 1980 mL of the rutin
solution (1 mg/mL, pH 4.0) and 20 mL of a-L-rhamnosi-
dase, followed by incubation at 60 ꢀC for 10, 20, 30, 40, 50,
and 60 min. Then, the reaction solutions were fetched to
analyze rutin from the reaction mixture and isoquercitrin
concentrations, which were further used to calculate the
rutin transformation rate and isoquercitrin gener-
ation yield.
The column and sample temperatures were 40 ꢀC and 4 ꢀC,
respectively. The injection volume was 1.0 mL, and the
detection wavelength was 200–700 nm. A quadrupole time-
of-flight (Q-TOF) mass spectrometry (MS) instrument
(Waters, Massachusetts, USA) was used for the identifica-
tion of isoquercitrin. Negative-ion mode was used at the
optimized conditions as follows: the capillary and sampling
cone voltages were set at 3.0 kV and 20 V, respectively; the
source temperature was 100 ꢀC; the desolvation temperature
was 400 ꢀC; the desolvation gas (nitrogen) flow rate was
700 l/h; and the collision energy was 6 V. Mass detection
was performed in full scan mode for m/z in the
range 20–1000.
Purification of isoquercitrin
HPLC analysis of rutin and isoquercitrin
The isoquercitrin was purified using a high-speed counter-
current chromatography (HSCCC) method according to a
previous study.[31] The HSCCC separation was performed
on a TBE-300C high-speed countercurrent chromatography
instrument (Shanghai Tauto Biotech Co., Ltd., Shanghai,
China). HSCCC was carried out using ethyl acetate-water
solution (1:1, v/v) as the two-phase solvent system,[31] in
which the upper and lower phase is the solution in which
ethyl acetate and water are saturated with each other,
respectively. The coils were filled with the upper phase.
After the column was entirely filled, the apparatus was
rotated at 800 rpm, while the lower phase (mobile phase)
was pumped into the column at a flow rate of 2.0 ml/min.
After hydrodynamic equilibrium was reached, as indicated
by the emergence of the mobile phase front, 10 mL of trans-
formation solution was injected into the column through
the injection valve. The effluent from the tail end of the col-
umn was continuously monitored with a UV detector at
254 nm. The temperature of the apparatus was set to 25 ꢀC.
The peak fractions were collected manually according to the
All solutions were filtered through 0.22 mm microfilters prior
to HPLC analysis. HPLC analysis was performed in a
Shimadzu HPLC equipped with a UV detector and an
InertSustain C18 column (4.6 ꢂ 250 mm, 5 mm). The flow
rate and temperature were set to 0.6 ml/min and 35 ꢀC,
respectively. Sample injection was 20 mL, the detection wave-
length was 360 nm and the running time was 20 min. The
mobile phase was composed of ultrapure water (solvent A),
methanol (solvent B), and acetonitrile (solvent C). The gra-
dient elution was as follows: 62% solvent A, 12% solvent B,
and 26% solvent C within 0–9 min; 15% solvent A, 35%
solvent B, 50% solvent C within 9–17 min; and then 62%
solvent A, 12% solvent B, and 26% solvent
C
within 17–20 min.
For plotting the calibration curves, a series of rutin (50,
100, 200, 300, 400, 500, and 600 mg/mL) and isoquercitrin
(50, 100, 200, 300, 400, 500, and 600 mg/mL) solutions were
prepared with reaction solution. The calibration curves were
drawn by regressively matching peak areas against
UV signal in the detector, and further evaporated under concentrations. The resultant calibration curves with regres-
sive equations y ¼ 45076x ꢁ 889655 (R2 ¼ 0.995) and
reduced pressure. The condensed eluents were dissolved in
methanol for purity analysis using both HPLC and thin-
y ¼ 53034x ꢁ 2844195 (R2 ¼ 0.997) were used to calculate
layer chromatography (TLC) analysis that was conducted on
silica gel GF254 precoated plates (Puke Separation Materials
Co., Ltd., Qingdao, China) using a mobile phase consisting
of ethyl acetate, formic acid, and water (8:1:1, v/v/v). The
developed spots on TLC were visualized using the iodine
stain method.
the concentrations of rutin and isoquercitrin, respectively.
The purity of isoquercitrin was the mass percentage of
isoquercitrin against the separated product using the follow-
ing equation:
ð Þ
Isoquercitrin purity %
mass of isoquercitrin mg
ð
Þ
ð
¼
ꢂ 100:
Identification of the purified isoquercitrin
total mass of HSCCC fraction mg
Þ
The isoquercitrin obtained from HSCCC was identified
using TLC, HPLC, and UPLC-Q-TOF. The ultraperform-
ance liquid chromatography (UPLC) system (Waters,
Massachusetts, USA) equipped with a BEH-HILIC silica
column (1.7 mm, 2.1 mm ꢂ 100 mm) was used to separate
isoquercitrin. The mobile phase A was acetonitrile, and the
mobile phase B was 0.1% aqueous formic acid. The gradi-
ent elution was as follows (B%): 0–5 min, 85–75%;
Statistical analysis
All of the experiments were performed in 3–5 replicates.
Experimental results were expressed as the means SD from
the values of the replications of each sample. Statistical ana-
lysis of experimental results was carried out using Microsoft
6.0–8.0 min, 75–62%; 8–10 min, 62–0%; 10–12 min, 0%. Excel 2013 and Origin Pro 8.0.

PREPARATIVE BIOCHEMISTRY AND BIOTECHNOLOGY
5
Figure 3. HPLC analysis of the rutin standard (A), isoquercitrin standard (B), transformation product (C), and HSCCC eluent (D). Note: in structural formula of rutin
and isoquercitrin, the red moiety is rhamnose, and the blue moiety is glucose.
solubility of substrates.[33] To investigate the optimal pH
value and temperature for rutin transformation, rutin and
isoquercitrin in the reaction solutions were analyzed by
HPLC. Rutin and isoquercitrin were determined to have
retention times of 6.272 min and 7.139 min (Fig. 3A,B),
respectively. The rutin seemed to entirely transform to
Results and discussion
Optimization of reaction pH and temperature for rutin
transformation
pH and temperature play critical roles in biotransformation
reactions because they affect enzyme activity[32] and the

6
L. J. LI ET AL.
isoquercitrin when the pH was controlled within the range between 1/S and 1/V (Fig. 5B), which was modulated to have
the regressive equation of y ¼ 0.0008x þ 0.0022 (R2 ¼ 0.9928).
of 3.0–5.0 (Fig. 4A). In contrast, both rutin transformation
and isoquercitrin generation dramatically decreased when
the pH was in excess of 5.0 (Fig. 4A). This result might be
attributed to the fact that a-L-rhamnosidase is an acidic
enzyme that has been previously shown to be stable in
acidic solutions.[29] In addition, when the reaction tempera-
ture was within 30–60 ꢀC, it was observed that more rutin
was transformed as the temperature increased, (Fig. 4B).
Temperatures over 60 ꢀC led to a decrease in both rutin
transformation and isoquercitrin generation (Fig. 4B). This
result was consistent with the fact that the enzyme had the
optimal activity at 60 ꢀC and became unstable over 60 ꢀC, as
illustrated in our previous study.[29]
From this regressive equation, the Km and Vmax were
estimated to be 0.36mM and 0.46mmol/min. Compared to
other studies (Table 1), the a-L-rhamnosidase from A. niger
JMU-TS528 had a Km towards rutin that was similar to that
of the a-L-rhamnosidase from L. acidophilus[34] but less than
that of a-L-rhamnosidases and hesperidinase from other
sources,[22,34–41] indicating that the a-L-rhamnosidase had a
stronger affinity towards rutin than most of the other
a-L-rhamnosidases. In addition, the a-L-rhamnosidase from
A. niger JMU-TS528 had a catalytic efficiency (Kcat/Km)
higher than those of a-L-rhamnosidases from Aspergillus
terreus[34] and Bifidobacterium breve.[37] This result was
explained by the fact that the present enzyme could lead to
high rutin transformation and isoquercitrin generation as
shown in Table 1.[34,35,37,38]
Kinetic characterization of rutin transformation
Based on the enzyme activities at different substrate concen-
trations (Fig. 5A), the Lineweaver-Burk chart was plotted
350
300
250
200
150
100
50
(A)
(A)
100
Rutin transformation rate
Isoquercitrin generation yield
100
80
60
40
20
0
80
60
40
20
0
0
0.00
0.20
0.40
0.60
0.80
Rutin concentration (mM)
(B)
3
4
5
6
7
8
pH
0.02500
0.02000
0.01500
0.01000
0.00500
0.00000
(B)
Rutin transformation rate
Isoquercitrin generation yield
100
y = 0.0008x + 0.002
R² = 0.9928
100
80
60
40
20
0
80
60
40
20
0
1/V = Km/Vmax × 1([S]) + 1/Vmax
Km ≈ 0.36 mM
Vmax ≈ 4.60× 10-1 mM*min
0.00
5.00
10.00
15.00
20.00
25.00
30.00
1/[S] (ml/µmol)
30
40
50
60
70
80
Temperature (℃)
Figure 5. The detected a-L-rhamnosidase activities at various rutin concentra-
Figure 4. Rutin transformation rates and isoquercitrin generation yields at dif- tions (A) and the Lineweaver–Burk plot analysis (B). Note: rutin concentrations
ferent pH values (A) and temperatures (B).
are within 0.04–0.82 mM. Reaction pH and temperature are pH 4.0 and 60 ꢀC.
Table 1. Kinetic parameters of the present a-L-rhamnosidase and other related enzymes towards rutin.
Specific activity
Km (mM) Vmax (mM min) Vm/Km (minꢁ1
)
Kcat/Km (sꢁ1 Mꢁ1
)
ꢃ
Enzymes
Source
Substrate
(U/mg)
a-L-rhamnosidase[35]
a-L-rhamnosidase[36]
Hesperidinase[38]
A. terreus
Commercial
ND
B. breve
ND
L. plantarum
L. acidophilus
Chloroflexus aurantiacus
Bacteroides thetaintaomicron
A. terreus
pNPR
Rutin
Rutin
Rutin
Rutin
Rutin
Rutin
Rutin
pNPR
pNPR
Rutin
451.5
96
ND
ND
ND
ND
ND
154.4
0.57
454
ND
0.481
ND
ND
ND
ND
ND
0.446
ND
0.114
ND
ND
8.5 ꢂ 103
ND
2.240
2.010
1.400
0.500
0.310
ND
9.99 ꢂ 10ꢁ1
5.72 ꢂ 10ꢁ2
1.60 ꢂ 10ꢁ1
ND
ND
a-L-rhamnosidase[37]
Hesperidinase[22]
1.2 ꢂ 103
ND
a-L-rhamnosidase[34]
a-L-rhamnosidase[34]
a-L-rhamnosidase[39]
a-L-rhamnosidase[40]
a-L-rhamnosidase[41]
ND
ND
ND
ND
ND
ND
2.87
ND
ND
6.07 ꢂ 10ꢁ1
8.67 ꢂ 105
6.7 ꢂ 104
0.476
0.360
ND
ND
1.263
a-L-rhamnosidase (this work) A. niger JMU-TS528
4.60 ꢂ 10ꢁ1
Note: the numbers in square brackets indicate the numerical order of the cited literature.

PREPARATIVE BIOCHEMISTRY AND BIOTECHNOLOGY
7
Theoretical rutin concentration
Actual rutin concentration
Optimization of rutin concentration and reaction time
for isoquercitrin production
(A)
1400
1200
1000
800
600
400
200
0
It has been reported that rutin has a low solubility in aqueous
solutions.[42] In this study, rutin was detected to have a solu-
bility of approximately 1000 mg/mL in the transformation
solution (Fig. 6A), indicating that a rutin concentration of
1000mg/mL is suitable for isoquercitrin production. At the
optimal rutin concentration (1000mg/mL), the transformation
reaction reached equilibrium after the reaction proceeded for
60min (Fig. 6B). Under this reaction condition, the rutin
transformation rate and isoquercitrin generation rate were
measured to be 96.44% and 95.12%, respectively, using the
HPLC method (Fig. 4C). Previous studies indicated that
acidic hydrolysis led to an isoquercitrin yield of 9.6%;[16] nar-
inginase transformation resulted in an isoquercitrin yield of
61%.[24] In addition, another a-L-rhamnosidase obtained an
isoquercitrin yield of 60%.[35] Notably, the present study
showed a much higher isoquercitrin yield (95.12%) than those
reported in other studies (Table 2).
0
200
400
600
800
1000
1200
1400
Rutin concentration added in the solution (μg/mL)
Rutin transformation rate (%)
Isoquercitrin generation yield (%)
(B)
100
80
60
40
20
0
100
80
60
40
20
0
0
10
20
30
40
50
60
Purification and identification of isoquercitrin by HSCCC
Reaction time (min)
Figure 6. The solubility of rutin in reaction solution (A) and rutin transform-
ation rates and isoquercitrin generation yields at various reaction times (B).
HSCCC is an effective separation technique based on a
liquid–liquid partition mechanism, which is widely used for
Table 2 Isoquercitrin yields in the present study and other studies.
Method
Rutin concentration (g/L)
Reaction time (h)
Isoquercitrin generation yield (%)
[16]
2.5% H3PO4
1% HCl[16]
0.5% H2SO4
1
1
1
1
1
20
20
20
36
36
10
6
2
4
1
9.60
0.69
1.25
43.2
3.07
60.0
61.0
76.1
82.9
95.1
[16]
Hesperidinase[16]
Snailase[16]
a-L-rhamnosidase[35]
Naringinase[24]
4.88
0.46
18.3
9.77
1
a-L-rhamnosidase[39]
a-L-rhamnosidase[41]
a-L-rhamnosidase (this work)
Figure 7. HSCCC of the transformation product (A) and TLC analysis of the main eluent of HSCCC (B). Note: HSCCC was running with the solvent systems composed
of ethyl acetate-water in ratio of 1:1 (v/v); flow rate is 2 mLꢄminꢁ1; revolution speed is 800 rpm; detection wavelength is 254 nm. The Lane 1 in B is isoquercitrin
standard; the Lane 2 is the eluent from HSCCC.

8
L. J. LI ET AL.
Disclosure statement
We declare that there are no known conflicts of interest associated with
this publication, and there is no significant financial support for this work
that could have influenced its outcome. We confirm that the manuscript
has been read and approved by all named authors, and no other persons
satisfy the criteria for authorship but not listed. We further confirm that
the order of authors listed in the manuscript has been approved.
Funding
This work was supported by the National Natural Science Foundation
of China [No. U1805235] and Xiamen Municipal Bureau of Science
and Technology [No. 3502Z20183029].
Figure 8. Mass spectra of the HSCCC eluent (A) and isoquercitrin standard (B).
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rutin and specifically transforms almost all of the rutin to
isoquercitrin.
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