Engineering peptide-based biomimetic enzymes for enhanced catalysis

Article abstract of DOI:10.1039/c6ra05778k

Herein, we design and synthesize a novel hydrolase model by integrating the supramolecular self-assembly of an amphiphilic short peptide (Fmoc-FFH) and electrostatic complexation (with PEI) at an aqueous liquid-liquid interface to synthesize stable peptid

Full text of DOI:10.1039/c6ra05778k

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Engineering peptide-based biomimetic enzymes
for enhanced catalysis†
Cite this: RSC Adv., 2016, 6, 40828
a
b
acd
a
acd
Guohua Zhang, Renliang Huang,* Wei Qi,*
and Zhimin He
Yuefei Wang, Rongxin Su
a
Herein, we design and synthesize a novel hydrolase model by integrating the supramolecular self-assembly
of an amphiphilic short peptide (Fmoc-FFH) and electrostatic complexation (with PEI) at an aqueous liquid–
liquid interface to synthesize stable peptide–polymer Fmoc-FFH/PEI hybrid capsules (FPCs). After
treatment with glutaraldehyde as a crosslinking agent, we can obtain novel Fmoc-FFH/PEI/GA hybrid
capsules (FPGCs). The FPGCs with imidazolyl groups as the catalytic centers exhibit high catalytic activity
for the hydrolysis of p-nitrophenyl acetate (PNPA). The resulting hydrolase model (FPCs or FPGCs) shows
kinetics behavior typical of natural enzymes, and the catalytic activity is higher than that of a Fmoc-FFH
hydrogel. The enhanced catalytic activity may be attributed to the high density of catalytic sites on the
inner surface of the hybrid capsule. Additionally, the FPGCs retained 93% of their productivity after
fifteen cycles, suggesting high stability and excellent recyclability. This novel hybrid capsule is expected
to be applied as a substitute for natural hydrolases in industrial production applications.
Received 4th March 2016
Accepted 16th April 2016
DOI: 10.1039/c6ra05778k
www.rsc.org/advances
It is well known that various weak supramolecular interac-
tions play important roles in the recognition of substrates,
Introduction
Enzymes catalyze a considerable variety of reactions with stabilization of transition states, and enhancement of catalytic
1
5,16
extremely high catalytic efficiency and specicity under mild activities.
conditions.
Based on the unique advantages of supramolec-
However, problems such as low operational ular chemistry, researchers can construct highly ordered
stability, high costs associated with preparation and purica- nanostructures to mimic the characteristics of natural
1,2
9,17–22
tion, and particularly difficulties in recovery and recycling enzymes.
In this respect, Liu and co-workers developed
3,4
5,10,23,24
greatly limit the applications of enzymes. To overcome the many articial enzymes via self-assembly.
For instance,
23
aforementioned limitations, mimetic enzymes have rapidly Zou et al. reported a supramolecular vesicle with controllable
5–11
emerged as an active eld of research.
Over the past several glutathione peroxidase (GPx) activity via the self-assembly of
decades, the development of enzyme mimics has been the focus supra-amphiphiles formed by host–guest recognition between
of considerable research efforts. However, the high complexities cyclodextrin and adamantane derivatives. Additionally, Sober-
25
of enzymes have prevented researchers from replicating the ats et al. prepared an articial enzyme by combining the
enzymatic features. Therefore, it is completely unrealistic to catalyst 2-aminobenzimidazole with resorcinarene cavitands to
develop synthetic chemical equivalents to natural enzymes in mimic the enzymatic hydrolysis of p-nitrophenylcholine
26
terms of structure, catalytic efficiency, specicity, selectivity, carbonate. Zaramella et al. synthesized an articial enzyme
and so forth. To address this problem, a new strategy of through the self-assembly of histidine-containing peptides H1–
supramolecular chemistry for enzymatic mimicking has H3 on the surface of AuMPC 1, which triggers their esterolytic
5,6,12–14
recently been developed.
activity. Recently, in our group, the catalytic triad (Ser/His/Asp)
of natural hydrolase was introduced into the peptide segment of
9
-uorenylmethoxycarbonyl diphenylalanine (Fmoc-FF). Based
a
State Key Laboratory of Chemical Engineering, School of Chemical Engineering and on the self-assembly and co-assembly of Fmoc-peptides, a series
Technology, Tianjin University, Tianjin 300072, P. R. China. E-mail: qiwei@tju.edu.cn
of supramolecular nanobers were obtained as articial
hydrolases. These highly ordered nanostructures tend to have
similar advantages: (i) dynamic supramolecular structures, (ii)
amphiphilic supramolecular architectures, (iii) supramolecular
interactions as driving forces for substrate recognition, and (iv)
b
Tianjin Engineering Center of Biomass-derived Gas/Oil Technology, School of
Environmental Science and Engineering, Tianjin University, Tianjin 300072, P. R.
China. E-mail: tjuhrl@tju.edu.cn
27
c
Collaborative Innovation Center of Chemical Science and Engineering (Tianjin),
Tianjin 300072, P. R. China
d
Tianjin Key Laboratory of Membrane Science and Desalination Technology, Tianjin specic and/or hydrophobic microenvironments for catalysis,
University, Tianjin 300072, P. R. China
Electronic supplementary information (ESI) available: Supplementary gures.
See DOI: 10.1039/c6ra05778k
which provides new ideas and methods for the development of
articial enzymes.
†
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Generally, the use of native enzymes in their free form always peptide was dissolved in an alkaline aqueous solution (pH
ꢀ1
faces difficulties in recovery and reuse. Hence, the need to 9.0) at a nal concentration of 4 mg mL by adding 0.5 M
preserve the catalytic properties of enzymes becomes increas- NaOH. Then, 1.5 mL of Fmoc-FFH solution was injected drop-
ingly important. Immobilization may result in improved wise (5 mL) into 5.0 mL of a 1.0 wt% PEI solution (pH 8.0)
stability of the enzymes toward harsh reaction conditions and through an Eppendorf pipette. Aer incubating for 30 min at
moreover enable their easier separation from the reaction room temperature, the excess PEI solution was then poured off,
28–35
media and their reuse.
Analogous to native enzymes, and the formed Fmoc-FFH/PEI capsules were washed twice with
supramolecular enzyme mimics also face recycling and reuse potassium phosphate buffer (10 mM, pH 8.0).
problems in many cases due to the small size of the supramo-
lecular assemblies. In this study, we attempt to address this
Synthesis of Fmoc-FFH/PEI/GA hybrid capsules
problem via the immobilization of biomimetic enzymes.
In a typical experiment, the formed Fmoc-FFH/PEI capsules
Herein, we used the imidazolyl-containing amphiphilic tri-
were crosslinked with glutaraldehyde (10 mL, 1.0 wt%) for 2 h to
peptide Fmoc–Phe–Phe–His (Fmoc-FFH), which was obtained
obtain new capsules (Fmoc-FFH/PEI/GA capsules, FPGCs). The
by incorporating imidazolyl groups into the supramolecular
FPGCs were then washed twice with potassium phosphate
framework, to simulate the catalytic function of native hydro-
buffer (10 mM, pH 8.0).
lase. Fmoc-FFH is an aromatic tripeptide that possesses an
Fmoc-FF segment, which can self-assemble into nanobers in
36,37
an anti-parallel b-sheet arrangement.
We previously re- Characterization of hybrid capsules
ported the hierarchical self-assembly of ordered materials at an
Zeta potential measurements of the Fmoc-FFH solution and PEI
solutions were performed using a Zetasizer Nano-ZS (Malvern
aqueous liquid–liquid interface via the combination of an
38–40
Fmoc-FF peptide solution with a cationic polymer solution.
ꢁ
Instruments, UK) at 25 C. The pH value of the Fmoc-FFH (4 mg
Using this strategy, hybrid peptide/polymer capsules with high
stability and recyclability were obtained. Similar to Fmoc-FF,
Fmoc-FFH molecules also possess strong negative charges and
can be dispersed in water in their monomeric state. Therefore,
in this work, we used an aqueous solution of polyethyleneimine
ꢀ1
mL ) solution was maintained at 9.0. For the PEI solutions (1.0
wt%), different pH values from 3.0 to 12.0 were obtained by
adding concentrated HCl or NaOH. A pH meter (Microbench pH
6
00, Singapore) was used to measure the pH values of all the
solutions.
For scanning electron microscopy (SEM) analysis, the hybrid
capsules were rst rinsed using ddH O for 5 min. The water was
(PEI) to induce the self-assembly of Fmoc-FFH at a liquid–liquid
interface, aiming to form hybrid Fmoc-FFH/PEI capsules with
Fmoc-FFH nanobers on the inner surface. Furthermore, the
capsules were treated with glutaraldehyde to improve their
stability. The catalytic performance (e.g., activity, stability, and
recyclability) of the immobilized Fmoc-FFH nanobers serving
as biomimetic enzymes was evaluated.
2
then removed by quickly freezing in liquid nitrogen and
subsequent freeze drying under vacuum. The samples were
then coated with gold using an Emitech K550 sputter coater
(Hitachi High-Technologies Co., Japan) and then imaged using
an S-4800 eld-emission scanning electron microscope (SEM,
Hitachi High-Technologies Co., Japan) at an accelerating
voltage of 3 kV.
Experimental
Chemicals and materials
Enzyme activity assay
Fmoc–diphenylalanine–histidine peptide (Fmoc-FFH) was
purchased from the Shanghai Gil Biochemical Co., Ltd.;
cationic polyethyleneimine (PEI), glutaraldehyde (GA), sodium
hydroxide and hydrochloric acid were purchased from the
Tianjin Guangfu Fine Chemical Research Institute. All other
chemicals, such as p-nitrophenyl acetate (p-NPA), p-nitrophenol
The enzyme activity was determined using p-nitrophenyl acetate
(
PNPA) as the substrates. Considering the UV spectrum of the
hydrolytic product, which exhibits a strong absorption peak at
00 nm, the reaction rates were determined by monitoring the
4
absorption increase at 400 nm with a Shimadzu 2450 UV-VIS-
NIR spectrophotometer. Then, we can calculate the corre-
sponding product concentration.
(p-NP), anhydrous ethanol and anhydrous acetonitrile, were of
analytical grade and were obtained from commercial sources.
All chemicals were used as received without any further puri-
cation. An Fmoc-FFH solution was prepared by dispersing the
In a typical experiment, approximately y hybrid capsules
or 250 mL of FHs (Fmoc-FFH hydrogels, both including 1 mg of
lyophilized Fmoc-FFH powder) was added to 4750 mL of phos-
phate buffer solution (10 mM, pH 8.0). Aer incubating for 2
lyophilized peptide powder in ddH O by sonication, followed by
2
adding an appropriate amount of a 0.5 M NaOH solution to the
suspension with subsequent stirring for 30 min.
ꢁ
min at 37 C, the reaction was initiated by adding 250 mL of
a PNPA acetonitrile solution (100 mM); eventually, the substrate
concentration became 5 mM. The absorption increase at l ¼
Synthesis of Fmoc-FFH/PEI capsules
4
00 nm due to the release of the product 4-nitrophenol (PNP)
A 1.0 wt% polyethyleneimine (PEI) solution was prepared by was recorded.
diluting a 50 wt% PEI solution in deionized water. The pH of the
The self-hydrolysis of PNPA and its hydrolysis in the pres-
PEI solution was then adjusted to 8.0 by adding an appropriate ence of Fmoc-FF/PEI capsules or Fmoc-FF/PEI/GA capsules were
amount of concentrated HCl. The lyophilized Fmoc-FFH tested as the control experiment. The preparation of Fmoc-FF/
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PEI capsules and Fmoc-FF/PEI/GA capsules and the activity in 5 mL of fresh phosphate buffer (10 mM, pH 8.0) for the next
assay is the same as the procedure described above.
reaction cycle.
Effects of pH and temperature on the activity of the hybrid
capsules and FHs
Storage stability and thermostability of hybrid capsules and
FHs
The optimal pH for the hybrid capsules and FHs was deter-
mined using PNPA as a substrate in the pH range of 6.0 to 10.0
at 37 C. The pH buffer was a 10 mM phosphate-buffered
Hybrid capsules and FHs were stored in phosphate buffer (10
mM, pH 8.0) at 4 C for 30 days (or pH 7.0, pH 10 at room
ꢁ
ꢁ
temperature for one day). The storage stability was compared in
terms of relative activity, dened as the ratio of the activity of
hybrid capsules or FHs aer storage to their initial activity.
The thermostability of the enzyme was measured by
analyzing the residual activity aer incubating the hybrid
solution (pH 8.0). The optimal temperature for the hybrid
capsules and FHs was determined by measuring the initial
ꢁ
reaction rate in the temperature range of 30 to 70 C in phos-
phate buffer (10 mM, pH 8.0). All of these reactions were per-
formed according to the method described in the Enzyme
activity assay section.
ꢁ
capsules and FHs at 70 C in 10 mM phosphate buffer (pH 8.0)
for 1 h. All these reactions were performed according to the
method described in the Enzyme activity assay section.
Determination of kinetic parameters of Fmoc-FFH/PEI hybrid
capsules
Results and discussion
Characterization of hybrid capsules
The kinetic parameters (Kcat and K ) of the Fmoc-FFH/PEI
m
hybrid capsules were determined by assaying the enzymatic
ꢁ
activity in 10 mM phosphate buffer (pH 8.0) at 37 C with seven
Fmoc-FFH can be dissolved in a basic solution (pH S 9.0). As
the pH decreases, the magnitude of the charge of Fmoc-FFH
molecules decreases, allowing them to self-assemble into
different concentrations (10, 20, 25, 33, 50, and 100 mM) of
PNPA, 4-nitrophenyl butyrate (PNPB) or 4-nitrophenyl phos-
phate (PNPP) as a substrate. The Michaelis–Menten equation
was used to t all the kinetic data to a Lineweaver–Burk plot,
and SigmaPlot soware (Systat Soware, Chicago, IL, USA) was
used to calculate the kinetic parameters. The initial reaction
rate (V) at different substrate concentrations ([S]) was plotted
according to the Lineweaver–Burk double-reciprocal model
10
nanobers through p–p interactions (Fig. S1†). A positively
charged polymer, PEI, was used to induce and direct the self-
assembly of Fmoc-FFH at an aqueous interface, leading to the
rapid synthesis of stable Fmoc-FFH/PEI hybrid capsules (FPCs)
and Fmoc-FFH/PEI/GA hybrid capsules (FPGCs, Fig. S3†).
To demonstrate the concept, an aqueous solution (pH 9.0) of
ꢀ1
(eqn (1)).
Fmoc-FFH (4 mg mL ) was added dropwise (5 mL) to an
aqueous solution (pH 8.0) of PEI (1.0 wt%) through a pipette.
When the transparent liquid droplets came into contact with
the PEI solution, white-colored capsules were formed within
seconds. Fig. 1a and b present photographs of the synthetic
FPCs and FPGCs, respectively, which are approximately 2 mm in
diameter and exhibit good uniformity. To investigate the exte-
rior and interior of the capsules, we fractured the capsules prior
to SEM imaging. As shown in Fig. 1c, the exteriors of the FPCs
have a smooth and compact surface structure, whereas their
interiors exhibit a nanobrous morphology (Fig. 1d). The
compact membrane structure of the capsule exterior (Fig. 1c) is
probably generated by the PEI-induced self-assembly of Fmoc-
FFH and deposition of PEI. The porous and brous structure
of the capsule interior is similar to that of FHs, suggesting that
the internal layer may be formed by the self-assembly of Fmoc-
FFH alone without PEI. As shown in Fig. 1e, the surface struc-
1
K
m
1
1
¼ V
ꢂ
¼
þ V
(1)
(2)
V
max
½Sꢃ
max
V
max
K
cat
½
Eꢃ
where V is the apparent initial catalytic rate, Vmax is the
maximum apparent initial reaction rate, [S] is the substrate
concentration, Km is the apparent Michaelis–Menten constant,
Kcat measures the number of substrate molecules turned over
per enzyme molecule per second, and [E] is the enzyme
concentration. The kinetic parameters, including K
and Kcat/K can be calculated from eqn (1) and (2).
m
, Vmax, Kcat,
m
Recycling assay of Fmoc-FFH/PEI and Fmoc-FFH/PEI/GA
hybrid capsules
Recycling studies of Fmoc-FFH/PEI and Fmoc-FFH/PEI/GA ture of the exterior of the FPGCs appears to be more compact,
hybrid capsules were conducted on a 5 mL scale for the whereas the interior still exhibits a nanobrous morphology
hydrolysis of PNPA using the same procedure described in the that is slightly more compact (Fig. 1f). The reason for this result
Enzyme activity assay section. The capsules were added to is that GA crosslinking primarily acts on polyethyleneimine,
phosphate buffer (10 mM, pH 8.0), yielding a nal enzyme which mainly leads to changes of the external structure. In
ꢀ1
concentration of 0.2 mg mL . Then, 250 mL of a PNPA aceto- contrast, small amounts of PEI deposition occur in the capsule
nitrile solution (100 mM) was added to a nal concentration of 5 interior during the synthesis of stable FPGCs, which has a slight
ꢁ
mM and shaken in a shaking bath (150 rpm) at 37 C for 20 min. inuence on the self-assembly of Fmoc-FFH.
The capsules were collected by ltration aer each reaction
batch, washed three times with phosphate buffer (10 mM, pH FPCs was investigated. The experiment indicated that stable
.0) to remove residual product or substrate, and then dispersed capsules were formed at pH # 11.0, while no capsule was
The effect of the pH of the PEI solution on the synthesis of
8
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Fig. 3 Effect of glutaraldehyde concentration on the recovery of
activity of the Fmoc-FFH/PEI hybrid capsules. Data represent the
means of three experiments, and error bars represent the standard
deviation.
electrostatic repulsion was eliminated by the neutralization of
charges. Self-assembly of Fmoc-FFH therefore occurred at the
Fig. 1 Morphology characterization of synthetic FPCs and FPGCs. (a) interface and formed capsules (Fig. S2†). A similar self-assembly
Photograph of hybrid capsules formed in a 1.0 wt% PEI solution (pH mechanism was also found in other molecules, such as peptide
ꢀ
1
8
.0) by adding 4 mg mL Fmoc-FFH solution. (b) Photograph of
41
amphiphiles.
hybrid capsules formed in 1.0 wt% PEI solution (pH 8.0) by adding 4 mg
mL Fmoc-FFH solution and then crosslinking with glutaraldehyde.
c) SEM images of the exterior of FPCs. (d) SEM images of the interior of
Additionally, the effect of glutaraldehyde on the activity
recovery was investigated by adding glutaraldehyde at different
ꢀ1
(
FPCs and partial enlarged details. (e) SEM images of the exterior of nal concentrations (10–125 mM). The results showed that the
FPGCs and a cross-section of the capsule membrane with a thickness hybrid capsules retained 76.8 ꢄ 2.5% of their activity aer cross-
of 6.67 mm. (f) SEM images of the interior of the synthetic FPGCs and
partial enlarged details.
ꢁ
linking with 25 mM glutaraldehyde at 25 C for 2 h (Fig. 3). We
hypothesized that the addition of glutaraldehyde led to a more
compact structure, making enzyme activity weaker but the
structure more stable.
formed at a pH of 12.0. The zeta potential of the PEI solution at
pH values from 3 to 12 was measured to explain this phenom-
enon. As shown in Fig. 2, the PEI solution shows a nearly zero
potential at pH 11.0, and the static charge gradually decreases
over the pH range from 3 to 12. In this experiment, the Fmoc-
FFH solution at a pH of 9.0 showed a negative zeta potential
Enzyme activity assay
The specic activity was measured according to the increase in
the absorption of the hydrolytic product p-nitrophenol at 400
nm. To investigate the effect of PEI, glutaraldehyde and
microstructure of capsules on the self-hydrolysis of PNPA, we
prepared the Fmoc-FF/PEI capsules and Fmoc-FF/PEI/GA
capsules, which had a similar structure as FPCs and FPGCs,
respectively. As shown in Fig. S6,† the results indicate these
factors have no inuence on the self-hydrolysis. Therefore, the
self-hydrolysis of PNPA was used as the background in the
catalytic reaction.
(ꢀ60.9 mV, Fig. 2); thus, it is difficult to self-assemble in this
solution due to the electrostatic repulsion. When Fmoc-FFH
molecules came into contact with positive PEI chains, the
As shown in Fig. 4, the specic activity of FHs for the rst 20
ꢀ
1
ꢀ1
min reached 25.31 U g , far below that of FPCs (54.79 U g
)
ꢀ1
and FPGCs (41.34 U g ), which is possibly due to the limited
mass transfer of PNPA in the Fmoc-FFH hydrogel. In contrast,
FPCs and FPGCs have porous structures, in which the substrate
and products are able to pass through the capsule membrane.
In comparison with FPCs, FPGCs have a more compact
membrane, which limit the mass transfer of substrates.
Effects of pH and temperature on the activity of the hybrid
capsules
The Fmoc-FFH hybrid capsules and FHs both showed an
optimal pH of 8.0 (Fig. 5). As expected, over the pH range from 6
Fig. 2 Zeta potentials of PEI solution (1.0 wt%) at different pH values
and of Fmoc-FFH (4 mg mL ) solutions at a pH of 9.0.
ꢀ1
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Fig. 4 The specific activities of FHs, FPCs and FPGCs in a shaking bath
ꢁ
(
150 rpm, 37 C). Data represent the means of three experiments, and
error bars represent the standard deviation.
Fig. 6 Lineweaver–Burk plots for the hydrolysis of PNPA (a) and PNPB
(b) catalyzed by FPCs and FPGCs.
the research focus and compared the hydrolysis rates under
different concentrations of PNPA or PNPB solution. As shown in
Fig. 6, the results show that FPCs and FPGCs have catalytic
characteristics typical of natural enzymes, namely, they simu-
late the substrate recognition and the catalytic process of
natural enzymes to some extent.
Based on the Lineweaver–Burk double-reciprocal model

tting, we can calculate the Michaelis constants for articial
hydrolytic enzymes. As shown in Table 1, the values of Kcat/Km
indicate that the FPCs have a higher catalytic efficiency
compared to FPGCs, which is in accordance with the result as
described before. In comparison with PNPA, PNPB has a higher
Fig. 5 Effects of pH (a) and temperature (b) on the specific activity of
the FPCs, FPGCs and FHs.
affinity (smaller K
m
values) to Fmoc-FFH nanobers (Table 2). It
value, indicative of a higher
also has a slightly larger Kcat/K
m
catalytic efficiency. However, we nd that the capsule can not
catalyze the hydrolysis reaction of PNPP under the same
conditions.
to 10, the Fmoc-FFH hybrid capsules exhibited higher catalytic
activity than the FHs. The optimal temperature of the hybrid
ꢁ
capsules and FHs was 37 C. Additionally, the result indicates
ꢁ
that the hybrid capsules still have high enzyme activity at 60 C,
Reusability assay of hybrid capsules
indicative of a high temperature resistance.
The reusability of the hybrid capsules was evaluated through
PNPA hydrolysis for successive batches. As shown in Fig. 7, the
Determination of kinetic parameters of hybrid capsules
93% productivity of FPGCs retained aer een cycles (total
Michaelis–Menten kinetics is an important characteristic that time: 300 min) is considerably higher than the 44% productivity
distinguishes biological enzyme catalysts from general chem- of FPCs retained aer ve cycles (total time: 100 min), sug-
istry catalysts. We selected FPCs and FPGCs with high activity as gesting the high stability and excellent recyclability of FPGCs.
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Table 1 Kinetic parameters of capsules using PNPA as substrate
ꢀ
ꢀ1
4
K
cat (mM
m
Kcat/K (10
min mg
ꢀ
1
ꢀ1
ꢀ1
Catalyst
FPCs
K
m
(mM)
min mg
)
)
11.09
7.39
0.031
27.95
FPGCs
0.017
23.01
Table 2 Kinetic parameters of capsules using PNPB as substrate
cat (mM (10
ꢀ
ꢀ1
4
K
Kcat/K
m
ꢀ1
ꢀ1
ꢀ1
Catalyst
K
m
(mM)
min mg
)
min mg
)
FPCs
3.09
1.66
0.011
35.59
FPGCs
0.005
27.38
Fig. 8 The storage stability (a) and thermal stability (b) of FPCs and
FPGCs. The specific activities of the initial enzymes were normalized to
1
00%.
stability than that (71.83% residual activity) of the FPCs. As
shown in Fig. 8, the hybrid capsules also have good thermal
ꢁ
stability. Aer incubating the hybrid capsules for 1 h at 70 C
in 10 mM phosphate buffer (pH 8.0), the residual activities of
the FPCs and FPGCs were 87.49% and 94.00%, respectively.
Additionally, the capsules were stored in an phosphate buffer
(
pH 7.0 or pH 10) for 24 h. As shown in Fig. S7,† there is no
obvious change in the catalytic activity of FPCs and FPGCs,
indicating the high basic tolerability.
Conclusions
In conclusion, we designed and synthesized a novel hydrolase
model: hybrid capsules (FPGCs). Because of the hydrophobic
microenvironment provided by the Fmoc-FFH aromatic ring
segments and the high density of catalytic histidines, this
Fig. 7 Recycling and reuse of FPCs (a) and FPGCs (b) for the hydrolysis model exhibits higher catalytic activity for the hydrolysis of
of PNPA (20 min per cycle).
PNPA compared to Fmoc-FFH hydrogels. In addition, the
resulting hydrolase model exhibits kinetics behaviour that is
typical of natural enzymes as well as high stability and excel-
However, as expected, it is difficult to recover the Fmoc-FF lent recyclability, and it is expected to be applied as a substi-
hydrogels via the simple centrifugation due to the small size tute for natural hydrolases in industrial production
of Fmoc-FFH nanobers.
applications.
Storage stability and thermal stability of hybrid capsules
Acknowledgements
The storage stability of the hybrid capsules was also deter-
ꢁ
mined. Aer storage at 4 C for 30 days, the residual activity This work was supported by the Natural Science Foundation of
of the FPGCs was 85.45%, demonstrating a better storage China (no. 21476165 and 21306134).
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2
2 M. Resmini, K. Flavin and D. Carboni, Top. Curr. Chem.,
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