Thermally controlled biotransformation of glycyrrhizic acid via an asymmetric temperature-responsive polyurethane membrane

Article abstract of DOI:10.1039/c8ra06202a

Separating a target product from a relatively complex bioreaction system is often difficult. In this work, a "smart" bioreaction system was developed by using the special characteristic of temperature-responsive polyurethane (TRPU). By combining solvent evaporation with a wet phase inversion technique, an asymmetric membrane consisting of an integral and dense skin layer supported by a porous sublayer was prepared from a thermally responsive polyurethane that experiences a sudden free volume increase upon heating through a phase transition temperature of 56 °C. Subsequently, the asymmetric TRPU membrane served as the carrier of an immobilized enzyme, wherein β-glucuronidase was multipoint-conjugated by using biotin and streptavidin on the porous sublayer. Then, the material-asymmetric TRPU membrane served jointly as the antennae as well as the actuator, which reversibly responds to temperature to switch (on-off) the access of the reactant glycyrrhizic acid (GL). Under the optimal temperature (40 °C) and pH (7.0) conditions, the immobilized β-glucuronidase contributed to almost 33% yield of glycyrrhetinic acid 3-O-mono-β-d-glucuronide (GAMG) of the isolated counterpart for the same concentration of substrate (250 mg L^-1) reaction for 24 h, while costing 1% of that of the isolated β-glucuronidase. Kinetic results showed that V_max and K_m values were 8.89 × 10³ mg L^-1 and 2.30 × 10³ mg L^-1 h^-1, respectively. The specific functional polymer-immobilized β-glucuronidase design serves as a bioreactor of GL into GAMG, as well as a separator deliberately irritated and controlled by temperature. This "smart" support material presents a potential facilitator for the separation of complex biotransformation reactions.

Full text of DOI:10.1039/c8ra06202a

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Thermally controlled biotransformation of
glycyrrhizic acid via an asymmetric temperature-
responsive polyurethane membrane†
ad
Cite this: RSC Adv., 2018, 8, 34823
Shaoyan Wang,a Lina Zhang,a Lidong Wubd and Yi Chen‡cd
*
Xiuhong Wu,
‡
Separating a target product from a relatively complex bioreaction system is often difficult. In this work,
a “smart” bioreaction system was developed by using the special characteristic of temperature-
responsive polyurethane (TRPU). By combining solvent evaporation with
a wet phase inversion
technique, an asymmetric membrane consisting of an integral and dense skin layer supported by
a porous sublayer was prepared from a thermally responsive polyurethane that experiences a sudden
free volume increase upon heating through a phase transition temperature of 56 ꢀC. Subsequently, the
asymmetric TRPU membrane served as the carrier of an immobilized enzyme, wherein b-glucuronidase
was multipoint-conjugated by using biotin and streptavidin on the porous sublayer. Then, the material-
asymmetric TRPU membrane served jointly as the antennae as well as the actuator, which reversibly
responds to temperature to switch (on–off) the access of the reactant glycyrrhizic acid (GL). Under the
optimal temperature (40 ꢀC) and pH (7.0) conditions, the immobilized b-glucuronidase contributed to
almost 33% yield of glycyrrhetinic acid 3-O-mono-b-D-glucuronide (GAMG) of the isolated counterpart
for the same concentration of substrate (250 mg Lꢁ1) reaction for 24 h, while costing 1% of that of the
isolated b-glucuronidase. Kinetic results showed that Vmax and Km values were 8.89 ꢂ 103 mg Lꢁ1 and
Received 22nd July 2018
Accepted 17th September 2018
2.30 ꢂ 103 mg Lꢁ1
h
ꢁ1, respectively. The specific functional polymer-immobilized b-glucuronidase
design serves as a bioreactor of GL into GAMG, as well as a separator deliberately irritated and controlled
by temperature. This “smart” support material presents a potential facilitator for the separation of
complex biotransformation reactions.
DOI: 10.1039/c8ra06202a
rsc.li/rsc-advances
procedures are generally characterized by a segmented struc-
ture with a partially miscible xed phase and a reversible phase
1. Introduction
favorably imported with a typical switching temperature. When
the environmental temperature is above the phase transition
temperature, the free volume in the polymeric matrix dramati-
cally expands.12,13 More advantageously, by manipulating the
molecular structure or chemical composition, the transition of
the reversible phase can also be interlinked to a temperature of
interest for a particular application.
From a TRPU, an asymmetric TRPU (ASTRPU) membrane
can be prepared using a wet phase transfer approach, which
consists of a porous sublayer enabling its contribution to
various potential applications. In our previous studies, an
asymmetric membrane was successfully synthesized and used
in transport, medical, and environmental applications.14–16 In
continuation, we intend to investigate its applications in the
area of biotransformation by limiting the access of the substrate
to the enzyme of interest, thereby controlling the reaction
process.
Synthetic polymers that exhibit a reversible response to
temperature, pH, and light can be regarded as “smart” and hold
great potential for applications in bioprocessing and pharma-
ceuticals.1–6 These polymers that undergo changes in size,
conformation, or hydrophobicity in response to variations in
temperature or pH are gaining increased attention. In partic-
ular, there is increasing interest in their potential application
ranges, including drug delivery, bioprocessing, gas or liquid
transport, and leather industry.7–11 Temperature-responsive
polymers (TRPUs) prepared using various materials and
aSchool of Chemical and Engineering, University of Science and Technology Liaoning,
Anshan, Liaoning, 114051, PR China
bThe Key Laboratory of Control of Quality and Safety for Aquatic Products, Ministry of
Agriculture, Chinese Academy of Fishery Sciences, Beijing, 100141, PR China
cThe Key Laboratory of Leather Chemistry and Engineering, Sichuan University,
Ministry of Education, Chengdu 610065, PR China
dDepartment of Chemistry, Massachusetts of Institute of Technology, Cambridge, MA,
Biotransformation is an effective means to obtain active
components and the leading compounds of natural products in
manufacturing biotherapeutic agents.17,18 Glycyrrhizic acid (GL)
and glycyrrhetinic acid (GA) are important components
02139, USA
† Electronic supplementary information (ESI) available: HPLC assay of
biotransformation results. See DOI: 10.1039/c8ra06202a
‡ These two authors contributed equally.
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extracted from the root of Glycyrrhiza glabra and have been resin was synthesized via solution polymerization in dime-
widely used in traditional Chinese medicine. The biological thylformamide (DMF) under an N2 atmosphere via two steps.
actions of both compounds cover a wide spectrum, including During the rst step, poly(epsilon-caprolactone) (PCL) 10 000
anti-inammatory, antiviral, antioxidative, and anticancer and a two-fold molar ratio of MDI were charged into the reactor
activities.19,20 When GL is converted to GA, the intermediate and stirred at 80 ꢀC for 2 h to prepare an isocyanate-terminated
product glycyrrhetinic acid 3-O-mono-b-D-glucuronide (GAMG) prepolymer. The prepolymer was subsequently chain extended
is oen produced. GAMG exhibits even higher antitumor and at 80 ꢀC for another 2 h by successively adding a stoichiometric
anti-inammatory activities than GA. The most commonly used amount of MDI and chain extender, BDO. The nal molar ratio
approach to acquire these effective components is extraction, of MDI to PCL10000 and BDO was maintained at 1 : 1 to yield
despite its lower efficiency and higher cost. As an alternative a linear polymer. Occasionally, DMF was added into the reactor
method, chemical transformation or biotransformation has when the viscosity of the reaction mixture was too high. Finally,
become increasingly attractive, particularly biotransformation a viscous and transparent TRPU solution with 37.6 wt% solid
as it is endowed with the benet of higher selectivity and content was obtained. The above synthesis procedure and
requires less use of organic solvents. Hence, it is regarded as molecular structure of TRPU are illustrated in Scheme 1.
a sort of “green chemistry” or “green technology”.21–24 Obvi-
ously, the enzymatic activity of either the whole cell or an iso-
lated enzyme dominates the reaction rate, such as hydrolysis,
2.3 Synthesis of ASTRPU
ASTRPU was prepared as per our previous work.14 The reaction
esterication, oxidation,
reduction,
and
asymmetric
membrane was prepared using a wet phase inversion lm
formation process, i.e., casting the TRPU solution on a release
paper and then heating the membrane for 15 min at 50 ꢀC (near
its crystal temperature). Subsequently, the membrane was
immersed in water to saturate. Then, an asymmetric membrane
was obtained by washing with water to exclude the solvent and
trace metal ions. Finally, the membrane was dried at 40 ꢀC
under vacuum conditions for 10 h. The physical and chemical
parameters of ASTRPU are listed in Table 1.
synthesis.25,26 As compared to the entire cell enzyme, the iso-
lated enzymes contribute toward specic selectivity, although
they may incur a higher cost. Therefore, the use of an immo-
bilized enzyme is a considerably less expensive and convenient,
as well as recyclable, means for the corresponding bioreaction.
Among the general immobilized enzymes used, the catalytic-
functions-(CFs)-immobilized enzyme is fairly promising, but
few are obtained because of limitations of the carrier.27
In this work, a treated asymmetric polyurethane membrane
served as a switch for biotransformation and a CFs-immobilized
enzyme carrier because of its smart on–off characteristic. The
multipoint immobilization using biotin and streptavidin
referred to here has recently been regarded as the most rigid
means of immobilization.28–31
2.4 Characteristic methods
The skin and sublayer porous structure of the synthesized
ASTRPU were scanned using a scanning electron microscope
(SEM; JSM-6100 scanning microscope, JEOL, Japan).
Plasma (AutoGlow 100, Glow Research, USA) O2 treatment was
applied to activate the porous sublayer surface of the ASTRPU
membrane and was immediately available to conjugate with EDC
and biotin hydrazide. Using X-ray photoelectron spectroscopy
(XPS; PHI Versaprobe II, Japan), the elemental composition
2. Material and methods
2.1 Materials
A polycaprolactone diol with number-average molecular weight
of 10 000 g molꢁ1, 4,40-methylenebis(phenyl isocyanate) (MDI),
1,4-butanediol (BDO), and N,N0-dimethylformamide (DMF)
were purchased from Sigma Chemical Co. b-Glucuronidase
(EC.3.2.1.31, lyophilized powder from E. coli, 1 000 000–
5 000 000 units per g), GL and GA (purity > 98%), and phosphate
buffered saline (PBS) were purchased from Sigma Chemical Co.
EZ-link Amine-PEG3-Biotin and EDC were purchased from
Fisher, USA, and streptavidin was purchased from Invitrogen,
USA. Hydrazide 40-hydroxyazobenzene-2-carboxylic acid (HABA)
was purchased from Pierce, USA.
PBS: one tablet dissolved in 200 mL of deionized water yields
0.01 M phosphate buffer, 0.0027 M potassium chloride, and
0.137 M sodium chloride at a pH of 7.4 and temperature of
ꢀ
25 C.
2.2 Synthesis of TRPU
A 250 mL round-bottom, three-necked separable ask equipped
with a mechanical stirrer, nitrogen inlet thermometer, and
condenser with a drying tube was used as the reactor. The TRPU Scheme 1 Synthesis procedure and molecular structure of TRPU.
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Table 1 Physical and chemical parameters of ASTRPU
Fix phase
–NCO/–OH
(mole ratio)
contenta (wt%)
Areab (m2 gꢁ1
4.70
)
Temperatureon
56 ꢀ
Temperatureoff
<25 ꢀ
Mn (g molꢁ1
4.47 ꢂ 104
)
Mw (g molꢁ1
6.46 ꢂ 104
)
37.6
C
C
1.2
a
b
The xed phase content was calculated as the mass fraction of MDI and BDO. Measured as BET surface area.
changes in the ASTRPU membrane before and aer the plasma perforations. The SEM image involving the morphologies of the
treatment were explored. Peak analysis was performed using cross-section are shown in Fig. 2b. Fig. 2 shows that the abun-
CasaXPS version 2.1.9 soware, in which the Shirley background dant porous structure of the sublayer was available for the
was subtracted from all the spectra prior to tting.
following enzymatic conjugation.
3.2 Switching or “on–off” characteristics of ASTRPU
2.5 Analytical methods
The microstructure of the multiple nger-like pores in the
porous sublayer indicated a larger area for b-glucuronidase
loading. The transportability of TRPU was determined via the
concentration change in GL initiated by the temperature
change. By repeatedly switching the release assembly between
two shaking water baths at different temperatures, the dynamic
transport behaviors of GL through the ASTRPU membrane over
four cycles of temperature oscillations between 25 ꢀC and 45 ꢀC
were investigated, and the results are shown in Fig. 3.
As shown, the transport of GL through the ASTRPU
membrane abruptly accelerated upon heating to 45 ꢀC and then
leveled off once the temperature reached 25 ꢀC. Over four cycles
of temperature oscillations, the on–off switching transport of
GL was maintained, indicating good reproducibility and
stability as the response to temperature.
To determine the transformational temperature of the synthe-
sized ASTRPU,
a differential scanning calorimeter (DSC;
Discovery, USA) was used. The temperature range was measured
from ꢁ80 ꢀC to 270 ꢀC at an increasing temperature rate of
10 ꢀC minꢁ1. Triplicate scanning was performed; nally, an
average temperature was obtained.
To measure the surface area of the ASTRPU membrane, the
Brunauer–Emmett–Teller (BET; ASAP2020, USA) method was
applied. The sample was pretreated by drying for 12 h. Then, the
sample was measured and analyzed using the instrument
soware.
To test the molecular weight, the gel permeation chroma-
tography (GFC) method was used.
To determine the effect of biotinylating on the structure of b-
glucuronidase, circular dichroism (CD; Jasco J-1500, Japan) was
used. The spectra were measured using a 0.1 cm path length cell
and recorded at 25 ꢀC in the range of 250–195 nm at a scanning
speed of 50 nm minꢁ1, step resolution of 0.1 nm, bandwidth of
1 nm, response of 4 s, and sensitivity of 200 mdeg.
To measure the bioreaction of GL, the product GA and the
intermediate product GAMG were analyzed using a high-pressure
liquid chromatography system (HPLC; Waters, 2487 series, MA,
USA). The column was Extend C18 (150 ꢂ 4.6 mm; I.D.: 5 mm)
with a ow rate of 1.00 mL minꢁ1, detection wavelength of
254 nm, and at ambient temperature. The solvent system con-
sisted of methanol and 3.6% acetic acid at a ratio of 85 : 15 (v/v).
To verify the degree of biotinylating, an ultraviolet visible
spectrometer (UV-vis; Thermo 2010, USA) was used with HABA
assay.
3.3 Plasma treatment of ASTRPU membranes
To facilitate the conjugation of b-glucuronidase onto the
membrane surface, the membrane surface was rst treated by
O2 plasma (AutoGlow 100, Glow Research, USA) to alter the
hydrophobic surface into a hydrophilic one. The compositional
change in the porous sublayer was illustrated using XPS (PHI
5000 VersaProbe II, Japan) data obtained from the MIT Center
for Materials Science and Engineering. Peak analysis was per-
formed using the CasaXPS version 2.1.9 soware with the
Shirley background subtracted from all the spectra prior to
tting. The HR envelopes were analyzed and peak-tted aer
All the experiments were performed at the Massachusetts
Institute of Technology, USA.
3. Results and discussion
3.1 Characteristics of ASTRPU membranes
DCS analysis (Fig. 1) reveal that theꢀ prepared ASTRPU
undergoes a sharp phase transition at 56 C. The BET specic
area of the membrane was 4.70 m2 gꢁ1. Since the porous sub-
layer would make direct contact with the reactant GL and b-
glucuronidase, the morphology of the sublayer surface was
evident in Fig. 2a. Contrary to the skin layer surface (Fig. 2c), the
entire sublayer appeared to be replete with interconnected Fig. 1 DSC curve of synthesized TRPU.
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Table 2 Chemical compositions of ASTRPU membrane before and
after O2 plasma treatment
Untreated ASTRPU
Plasma treated ASTRPU
Position
BEa
Atomic (eV)
conc. %
Position
BEa
Atomic (eV)
conc. %
Peak
O 1s
C 1s
N 1s
532.9
400.4
285.0
20.44
79.24
4.31
532.0
400.0
285.0
27.72
68.29
3.99
a
BE is the binding energy.
3.4 Biotinylated ASTRPU and b-glucuronidase
Immediately, the ASTRPU membrane and the target b-glucu-
ronidase were both biotinylated using EZ-link Amine-PEG3-
Biotin and EDC following the Fisher protocol. The biotinylate b-
glucuronidase was quantied using a Pierce Biotin Quantita-
tion Kit supplied by Thermo Scientic. The kit contained the
reagent HABA, which enables a rapid estimation of the mole-to-
mole ratio of biotin to protein. Because biotin is a relatively
small molecule, it can be conjugated to many proteins without
altering the biological activity of the protein. A protein can be
conjugated with several biotin molecules, each of which can
bind one molecule of avidin, thereby considerably increasing
the sensitivity of many assays. The bond formation between
biotin and avidin is rapid; once formed, it remains unaffected
by the most extremes pH values, organic solvents, and other
denaturing agents. With regard to quantitative analysis,
Fig. 2 SEM images of the morphologies of (a) porous sublayer surface;
(b) cross-section; (c) skin layer surface of ASTRPU.
Fig. 3 Accumulative release profile of the ASTRPU membrane.
the subtraction of the Shirley background using the Gaussian–
Lorentzian peak shapes obtained from the CasaXPS soware
package. The chemical compositions of the porous surface of
ASTRPU before and aer plasma O2 treatment are listed in
Table 2. It is obvious that the most signicant change is the O
content, while the N content seldom changes. As shown in
Fig. 4, aer treatment by O2 plasma, the asymmetrical
membrane became activated with the additional introduction
of –C–O and –C]O groups (binding energy, BE: 286.5 eV and
289.2 eV, respectively). The O 1s lines also show an increase in
–C–O (BE: 533.2 eV).32
Fig. 4 XPS spectra of the C 1s and O 1s peaks of the O2-plasma-
treated asymmetric TRPU. Treatment time: 1 min.
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a solution containing biotinylated protein was added to
a mixture of HABA and avidin. Because of the higher affinity
toward avidin, biotin displaces the HABA and the absorbance at
500 nm proportionately decreases. Using this method, an
unknown amount of biotin present in a solution can be quan-
tied in a single cuvette by measuring the absorbance of the
HABA–avidin solution before and aer the addition of the
biotin-containing sample. The change in absorbance is related
to the amount of biotin in the sample via the extinction coef-
cient of the HABA–avidin complex.
3.5 Conjugate b-glucuronidase onto the ASTRPU membrane
Fig. 6 CD graphs for isolated and biotinylated b-glucuronidase.
A schematic of the immobilization process is shown in Fig. 5. The
quantitative number of both biotinylate and isolated b-glucu-
ronidase conjugated to the porous sublayer membrane was
determined using the HABA assay: 0.31 mg cmꢁ2 and 1.50 mmol
mLꢁ1, respectively. To the best of our knowledge, such an
approach of immobilized b-glucuronidase has not yet been re-
ported. The means of immobilizing b-glucuronidase presented
here was that of multipoint crosslinking (one streptavidin
molecule offers four binding sites for trapping) and it behaves
rather rigidly than that in a traditional binding approach.28 To
determine the effect of biotin on the structure of b-glucuroni-
dase, a circular dichroism (CD; Jasco J-1500, Japan) was used.
Solutions of biotinylate and original b-glucuronidase were
prepared at a concentration of 0.50 and 0.49 mg mLꢁ1 in PBS,
respectively. The structural changes in the biotinylated b-glucu-
ronidase was established by comparing the CD spectra. Typically,
three scans were accumulated and subsequently averaged.
a temperature of 35 ꢀC, the hydrolysis of GL catalyzed by b-
glucuronidase served as the probe model reaction to investigate
the integral design of the temperature-responsive ASTRPU–b-
glucuronidase bioreaction assembly. The reaction occurred in
a circular membrane surface (I.D.: 0.68 cm; area: 1.45 cm2). One
microliter of 100 mg mLꢁ1 GL solution was added into a 1.5 mL
bottle, and the porous, sublayer ASTRPU membrane served as
the cover with binding b-glucuronidase apposite to the GL
solution (i.e., on the outside of the cover) and the edges of the
membrane were thoroughly sealed to prevent leakage. This
assembly was then hung in an inverted manner in a 50 mL
conical tube containing 1.2 mL of PBS (pH 7.0) in advance.
Thereaer, the 50 mL conical tube was incubated in a shaker at
a certain temperature for 24 h. The products of the hydrolysis
reaction in the PBS were analyzed using HPLC. The HPLC
results showed that a different amount of GAMG was produced
as a function of temperature, wherein the irradiated membrane
released different concentrations of GL to access the b-glucu-
ronidase conjugated in the porous sublayer. Fig. 7 shows the
detailed results of such a tendency. The maximum GAMG
product concentration was obtained at a temperature of 50 ꢀC.
At a higher temperature, (e.g., 55 ꢀC), less GAMG was produced,
which might have been caused by a decrease in or loss of
enzymatic activity as compared to that at 50 ꢀC. Within the
model reaction, the ASTRPU membrane played the role of
a switch for GL approaching the b-glucuronidase irradiated by
the temperature alteration. To investigate the kinetic behavior
of the system, the reaction assembly was incubated in a shaker
3.6 Comparison of the before and aer structures of
biotinylated b-glucuronidase
CD was used to monitor the b-glucuronidase structure's alter-
nation, as shown in Fig. 6. Thus far, we know that the far-UV
spectra for isolated b-glucuronidase and biotinylated b-glucu-
ronidase exhibited similar spectra with strong negative ellipticity
near 220 nm, and most of the spectra of the biotinylated b-
glucuronidase overlapped with the spectra of the original type,
indicating that the secondary structure of the biotinylated b-
glucuronidase did not change during the course of biotinylating.
3.7 Reaction of GL into GAMG on biotinylated b-
glucuronidase ASTRPU membranes
With regard to isolated b-glucuronidase, under suitable reac-
tion conditions, namely, a pH of 7.0 (PBS modulated) and
Fig. 7 Temperature-dependent GL biotransformation on an immo-
Fig. 5 Schematic of ASTRPU–b-glucuronidase binding.
bilized ASTRPU membrane.
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at 40 ꢀC for 24 h. The Michaela constant, Km, and the maximum
reaction rate, Vmax, were measured using the Lineweaver–Burk
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ꢂ
103 mg Lꢁ1 and 2.3
ꢂ
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4. Conclusions
In summary, from the perspective of controlling the biocon-
version process, or alternatively, to obtain intermediate prod-
ucts of interest (e.g., GAMG) in this work, the characteristic of
TRPU was used. A catalytic bioreaction assembly coupled with
the smart “on–off” switch was designed. The b-glucuronidase
multipoint was rigidly immobilized onto ASTRPU via biotin and
streptavidin. The ASTRPU–b-glucuronidase reactor succeeded
in controlling the access of the reactant GA toward b-glucu-
ronidase by deliberately alternating the temperature, thereby
controlling the bioconversion.
The developed reactor exhibited the potential advantage of
obtaining important intermediate products or some of the steps
during the reaction. Meanwhile, the reactant, product, and
catalytic enzyme used were independent of each other,
beneting from the separation. Moreover, in addition to the
immobilized enzyme that could be repeatedly used in most of
the reaction system, the reactant could be repeatedly used in
this system, which seldom occurs in most cases.
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Conflicts of interest
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There are no conicts to declare.
Acknowledgements
The authors thank Professor Alexander M. Klibanov for help
applying the experimental condition for all the experiments at
the Massachusetts Institute of Technology. They gratefully
acknowledge the National Nature Science Foundation of China
[Grant Number 21576127] for support. They also thank
Professor Chun Li for applying the extract GAMG to the chem-
ical standard and Sappi North America for applying for the
release paper.
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