Biomimetic-functionalized, tannic acid-templated mesoporous silica as a new support for immobilization of nhase

Article abstract of DOI:10.3390/molecules22101597

Tannic acid-templated mesoporous silica (TAMS) was synthesized using a simple nonsurfactant template method and dopamine-functionalized TAMS (Dop-TAMS), which was prepared via a biomimetic coating, was developed as a new support for immobilization of NHase (NHase@Dop-TAMS). The Dop-TAMS was thoroughly characterized by the transmission electron microscopy (TEM), scanning electron microscopy (SEM), Brunauer–Emmett–Teller (BET), and Fourier transform infrared (FT-IR) and the results showed that the Dop-TAMS possessed sufficiently large pore size and volume for the accommodation of NHase. Studying the thermal stability, storage, shaking stability, and pH stability of the free and immobilized NHase indicated that the catalytic properties of NHase@Dop-TAMS were significantly enhanced. Moreover, the NHase@Dop-TAMS exhibited good reusability. All the results demonstrated that Dop-TAMS could be used as an excellent matrix for the immobilization of NHase.

Full text of DOI:10.3390/molecules22101597

molecules
Article
Biomimetic-Functionalized, Tannic Acid-Templated
Mesoporous Silica as a New Support for
Immobilization of NHase
Jun-kai Gao 1, Zi-jun Zhang 1, Yan-jun Jiang 2, Yan Chen 1,* and Shu-feng Gao 3
1
School of Port and Transportation Engineering, Zhejiang Ocean University, Zhoushan 316022, China;
gaojk@zjou.edu.cn (J.-k.G.); 18857088135@163.com (Z.-j.Z.)
School of Chemical Engineering and Technology, Hebei University of Technology, Tianjin 300130, China;
2
yanjunjiang@hebut.edu.cn
YinzhouKefeng New Material of Polymer Co. Ltd., Ningbo 315100, China; gsf324@163.com
3
*
Correspondence: chenyan@zjou.edu.cn; Tel./Fax: +86-580-2554-491
Received: 23 August 2017; Accepted: 19 September 2017; Published: 25 September 2017
Abstract: Tannic acid-templated mesoporous silica (TAMS) was synthesized using a simple
nonsurfactant template method and dopamine-functionalized TAMS (Dop-TAMS), which was
prepared via a biomimetic coating, was developed as a new support for immobilization of NHase
(NHase@Dop-TAMS). The Dop-TAMS was thoroughly characterized by the transmission electron
microscopy (TEM), scanning electron microscopy (SEM), Brunauer–Emmett–Teller (BET), and Fourier
transform infrared (FT-IR) and the results showed that the Dop-TAMS possessed sufficiently large
pore size and volume for the accommodation of NHase. Studying the thermal stability, storage,
shaking stability, and pH stability of the free and immobilized NHase indicated that the catalytic
properties of NHase@Dop-TAMS were significantly enhanced. Moreover, the NHase@Dop-TAMS
exhibited good reusability. All the results demonstrated that Dop-TAMS could be used as an excellent
matrix for the immobilization of NHase.
Keywords: dopamine; mesoporous silica; tannic acid; immobilization; nitrile hydratase
1. Introduction
Enzymes have a wide range of applications, including in disease diagnosis, clinical treatment,
and in the food industry, due to several advantages, such as mild reaction conditions, high efficiency,
and specificity [
adaptability, poor operational stability, and poor recyclability [
these drawbacks and the immobilization of enzymes on solid carriers has been demonstrated to
improve their adaptability to changes in surroundings and raise their recycling rate [ ]. The methods
1
,2
]. However, the application of free enzymes is still limited by low environmental
3
]. It is important, therefore, to overcome
4,5
proposed for enzyme immobilization are distinguished as either physical or chemical. Physical
methods involve physisorption and entrapment and chemical methods are divided into ion binding,
covalent binding, and cross-linking [6,7].
In the preparation of immobilized enzymes, it is essential to select an appropriate carrier to
improve the operational stability of the biocatalyst [8,9]. Many carriers of enzymes have been studied,
including polymer supports, metallic and glass materials, organic and inorganic nanometer compound
materials, carbon nanotubes, and colloidal supports [10]. Among the numerous carriers, mesoporous
silica has attracted significant research attention due to its large specific surface area, adjustable pore
structure with ordered arrangement, good mechanical stability, and biocompatibility [11,12]. For
example, Hung et al. utilized 3-aminopropyltrimethoxysilane and glutaraldehyde-functionalized
mesoporous silica nanoparticles (IBN-4) as the carrier of Horseradish peroxidase (HRP) and the
Molecules 2017, 22, 1597; doi:10.3390/molecules22101597
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results exhibited that IBN-4 greatly increased stability and activity of HRP enzymes [13]. Ali et al.
covalently immobilized the Candida rugosa lipase on fibrous silica nanoparticles (KCC-1), and the
resulting immobilized lipase exhibited wider reaction temperatures and pH regionsin comparison
to the free lipase [14]. Gao et al. synthesized monodisperse core-shell magnetic organosilicanano
flowers as the support for Candida antarctica lipase B (CALB) immobilization and the study results
showed that the the resulting immobilized enzyme (CALB/nanoflowers) exhibited improved pH
stability, improved thermal stability, and satisfactory storage stability [15]. Li et al. Developed
hierarchically macro/mesoporous silica sphere with structural macropores and skeleton mesopores
for immobilization of catalase. The study results indicated that the immobilized catalase showed
comparable catalytic efficiency to the free catalase and possessed appreciable reusability [16]. However,
the traditional synthetic method for mesoporous silica involves the application of surfactant, such as
triblock copolymers and cetyltrimethylammonium bromide [17–19] and these surfactant templating
agents are toxic, expensive, and difficult to remove [20 21]. Therefore, a templating agent that is
,
cheap, non-toxic, and easy to remove is needed. Recent research has found that tannic acid may be
a suitable template agent. Tannic acid is a class of polyphenol compound with complex structure and
is found in numerous plants [22,23]. It is not only cheap and environmental friendly, but can also be
dissolved in water or alcohol. Therefore, when tannic acid is used as template agent, it can be easily
removed by water or alcohol. Additionally, mesoporous silica synthesized with tannic acid as template
agent had large interconnected mesopores, which benefited the accommodation of enzymes [18].
Considering these advantages, tannic acid-templated mesoporous silica (TAMS) has great potential for
the immobilization of enzymes in practical applications.
Typical methods for immobilizing enzymes in mesoporous silicas rely on grafting suitable
functional groups on their surfaces, which can react with the enzymes by covalent conjugation [24,25].
However, such immobilization strategies are usually accompanied by enzyme deactivation [25]. The
utilization of dopamine as a surface functionalization reagent has attracted much attention because
it is facile, simple, and cost-effective and the dopamine molecules contain both primary amine and
catechol, which have the ability to immobilize biomolecules [26,27]. Furthermore, it avoids calcination
of the TAMS during synthesis and therefore allows more silanol groups on the surface of TAMS to be
retained. This is favorable for the grafting of dopamine, which subsequently enhances the ability of the
dopamine-functionalized TAMS to immobilize biomolecules. However, to the best of our knowledge,
dopamine-functionalized tannic acid-templated mesoporous silica (Dop-TAMS) has never been used
as an enzyme immobilization support.
Nitrile hydratase (NHase; EC 4.2.1.84) is a kind of microbial enzyme, which catalyzes the
hydration of nitriles to high value amides, with extensive applications in industrial production
processes [28]. The use of free NHase in industrial applications is not conducive to improving
efficiency due to low stability, poor adaptability to environment, and poor recovery rate [6].
Therefore, immobilization of NHase on a suitable support is very important. Thus, in this work, the
Dop-TAMS was synthesized, and NHase was immobilized in Dop-TAMS by physisorphtion (named
NHase@Dop-TAMS). The reaction scheme for immobilization of NHase in Dop-TAMS is shown
in Figure 1. Additionally, the activity, operational stability, and reusability of NHase@Dop-TAMS
were investigated detailedly. The results showed that the Dop-TAMS had great potential for the
immobilization of NHase in practical applications.

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Dopamine·HCl
TAMS
Dop-TAMS
NHase
Dop-TAMS
NHase@Dop-TAMS
Polydopamine
NHase
Figure 1. The reaction scheme for immobilization of NHase in dopamine-functionalized tannic
acid-templated mesoporous silica (Dop-TAMS).
2. Results and Discussion
2.1. Characterization of the Dop-TAMS and NHase@Dop-TAMS
The SEM and TEM images of Dop-TAMS are shown in Figure 2. Spherical and monodisperse
particles can be viewed from the typical SEM photograph of Dop-TAMS (Figure 2a) and the average
diameter of the monodisperse particles was about 120–180 nm. The TEM image clearly exhibits that the
Dop-TAMS had porous structure with irregular pore arrangement (Figure 2b), and after immobilization
of NHase, the NHase@Dop-TAMS exhibited a small amount of aggregation (Figure 2c). Figure 3 shows
the N2 adsorption/desorption isotherms and pore size distribution of the Dop-TAMS. According to
the BET surface area measurements, the specific surface area of Dop-TAMS was 389 m2/g and the
calculated BJH pore volume and pore size were 0.71 cm3/g and 6.1 nm, respectively. The large pore
size and pore volume were beneficial for the enzyme entering the internal structure of the Dop-TAMS
and ensuring the accommodation of NHase.
Figure 2. (a) SEM and (b) TEM images of Dop-TAMS and (c) TEM image of NHase@Dop-TAMS.
Fig−u1re 4 shows the FT-IR spectra of TAMS, Dop-TAMS, and NHase@Dop-TAMS. The peaks at
−1
1646 cm and 3419 cm were attributed to the stretching vibration of water molecules and hydroxyl,
respectively [29]. The typical peaks at 1097 cm−1 and 802 cm−1 corresponded to the asymmetric
stretching and symmetric stretching of Si-O-Si bonds, respectively [30]. The adsorption band at
977 cm−1 was attributed to the symmetric stretching of Si-OH groups [31]. The adsorption peak at
1504 cm−1 of Dop-TAMS was attributed to benzene ring stretching from dopamine [32
,33], which
demonstrated its successful graft. For the NHase@Dop-TAMS, the peaks at 1541 cm−1 and 1454 cm−1
were due to the deformation vibration of the amino groups and the bending vibration of the -C-H
groups [3], which proved that the NHase was successfully immobilized in the Dop-TAMS.

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540
450
360
270
180
90
0.12
0.08
0.04
0.00
0
15
30
45
60
Pore Diameter (nm)
0.0
0.2
0.4
0.6
0.8
1.0
Relative Pressure (P/P0)
Figure 3. N2 adsorption/desorption isotherms and pore size distribution of the Dop-TAMS.
1454
NHase@Dop-TAMS
1541
802
Dop-TAMS
1504
TAMS
1646
977
3419
1097
4000 3500 3000 2500 2000 1500 1000 500
Wavenumber (cm-1)
Figure 4. FT-IR spectra of the TAMS, Dop-TAMS, and NHase@Dop-TAMS.
The enzyme loading capacities and activities of NHase@TAMS and NHase@Dop-TAMS are shown
in Table 1. According to the results, when the initial concentration of NHase in the phosphate buffer
solution was 1.04 mg/mL and the dosage of TAMS was 10 mg/mL, the amount of NHase immobilized
in NHase@Dop-TAMS was 63.1 mg/g and the activity recovery was 40.9%. In the control experiment,
the enzyme loading amount of NHase@TAMS was 47.7 mg/g and the activity recovery was 35.2%.
The improved NHase loading capacity and specific activity of NHase@Dop-TAMS could be attributed
to the catechol and primary amines in dopamine molecules reacting with the NHase molecules to form
strong conjugation of covalent bonds.
Table 1. The enzyme loading capacities and activities of NHase@TAMS and NHase@Dop-TAMS.
NHase Loading Capacity
(mg/g)
Specific Activity
(U/mg)
Activity Recovery
(%)
Samples
NHase@Dop-TAMS
NHase@TAMS
63.1
47.7
2.14
1.85
40.9
35.2
2.2. Thermal Stability of Free and Immobilized NHase
Figure 5 shows the thermal stabilities of free NHase and immobilized NHase inphosphate buffer
◦
solution (50 mM, pH 7.0) over different times at 50 C. The rate of decrease in NHase@Dop-TAMS was
lower than in free NHase and NHase@TAMS. After 7 h of incubation at 50 ◦C, the relative activity of

Molecules 2017, 22, 1597
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free NHase and NHase@TAMSretained 13.2% and 42.6%, respectively, whereas the relative activity of
NHase@Dop-TAMS maintained 50.7%. These results indicate that the NHase@Dop-TAMS had better
thermal stability than the free NHase and NHase@TAMS, and this can be mainly attributed to the fact
that Dop-TAMS can enhance the rigidity of NHase and protect it from unfolding, thus preventing
denaturation of NHase molecules at high temperatures [34–36].
100
NHase@Dop-TAMS
NHase@TAMS
80
free NHase
60
40
20
0
0
1
2
3
4
5
6
7
8
Time (h)
Figure 5. The thermal stability of free and immobilized NHase.
2.3. pH Stability of Free and Immobilized NHase
The pH stabilities of free NHase and immobilized NHase were mensurated by incubating them
in phosphate buffer solutions at pH 3.0 and pH 9.0, respectively, and the results are shown in Figure 6.
After 0.5 h of incubation at pH 3.0 or pH 9.0, the relative activity of the free NHase declined quite
sharply; in constrast, the decreases in relative activity for the immobilized NHase varieties were
considerably smaller. After 6 h of incubation at pH 3.0, the free NHase and NHase@TAMS retained
4.5% and 62.8% of their original activity, respectively, and the remaining activity of NHase@Dop-TAMS
was 71.7%. After 6 h of incubation at pH 9.0, the relative activity of free NHase and NHase@TAMS
retained 11.2% and 66.5%, respectively, and that of NHase@Dop-TAMS retained 79.3%. Therefore,
under both acidic or alkaline conditions, NHase@Dop-TAMS was more stable than free NHase
or NHase@TAMS. This might be attributed to the strong conjugation of covalent bonds between
NHase and dopamine-functionalized TAMS, which can restrain the conformational change of the
NHasemolecules caused by H+ or OH− [6,37,38].
100
a
80
60
NHase@Dop-TAMS
40
NHase@TAMS
freee NHase
20
0
0
1
2
3
4
5
6
7
Time (h)
Figure 6. Cont.

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100
80
60
40
20
0
b
NHase@Dop-TAMS
NHase@TAMS
free NHase
0
1
2
3
4
5
6
7
Time (h)
Figure 6. pH stability of free NHase and immobilized NHase at (a) pH 3.0; and (b) pH 9.0.
2.4. Stability of Free and Immobilized NHase in Shaking Conditions
Figure 7 shows the stability of free NHase andimmobilized NHase in shaking conditions (200 rpm).
The relative activity of free NHase varied tremendously and decreased to zero after shaking for
3 d and the adsorption of NHase in TAMS or Dop-TAMS improved its stability. Under the same
conditions, the relative activity of NHase@TAMS and NHase@Dop-TAMS could maintain 33.4% and
46.1%, respectively, after 8 d. The decrease of activity in free and immobilized NHase was because
vigorous shaking led to the autolysis and denaturation of the enzyme molecules [6,39]. Compared to
NHase@TAMS, the improved storage stability of NHase@Dop-TAMS might be attributed to strong
conjugation of covalent bonds between the NHase and dopamine-functionalized TAMS, which would
not only prevent leaching of NHase from NHase@Dop-TAMS, but also increase conformational stability
of the NHase molecules [39–41].
NHase@Dop-TAMS
NHase@TAMS
free NHase
100
80
60
40
20
0
-1
0
1
2
3
4
5
6
7
8
9
Time (day)
Figure 7. The stability of free and immobilized NHasein shaking conditions.
2.5. Storage Stability of Free and Immobilized NHase
The storage stability of enzymes is also a crucial factor in practical applications [42]. Figure 8
shows the storage stability of the free NHase and immobilized NHase. It was observed that the activity
of the free NHase decreased faster than that of the immobilized NHase. After being stored for 30 d,
the free NHase and NHase@TAMS maintained 11.4% and 31.8% of their initial activities, respectively,
whereas the residual activity of the NHase@Dop-TAMS maintained 56.3%. Thus, the investigation
proved that the immobilization of NHase in Dop-TAMS could prolong its storage life. The improved

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storage stability of NHase@Dop-TAMS might be related to protecting the active sites of the enzyme
from deactivating more effectively, compared with free NHase under the same conditions [36,38].
NHase@Dop-TAMS
100
80
60
40
20
0
NHase@TAMS
free NHase
-5
0
5
10
15
20
25
30
35
Time (day)
Figure 8. The storage stability of free and immobilized NHase.
2.6. Kinetics of Free and Immobilized NHase
The kinetic parameters of freeNHase and immobilized NHase are shown in Table 2. The Km values
of the free NHase, NHase@TAMS and NHase@Dop-TAMS were 1.71 mM, 2.48 mM, and 2.35 mM,
respectively. Distinctly, the immobilized NHase exhibited a higher Km value than the free NHase,
demonstrating that the substrate’s affinity capacity for the NHase@TAMS or NHase@Dop-TAMS was
lower than for the free NHase. This might be ascribed to the conformational change of the NHase
after its immobilization, which could lower the substrate’s accessibility toward the active sites of
the immobilized NHase [43,44]. The Km value of the NHase@Dop-TAMS was lower than that of the
NHase@TAMS, suggesting that the NHase@Dop-TAMS could enhance the NHase’s affinity capacity
for the substrate. The Vmax. values for the NHase@TAMS and NHase@Dop-TAMS were lower than for
the free NHase, indicating that while the substrate diffused and accessed the enzyme molecules, the
mass transfer limitation of the immobilized NHase was larger than for the free NHase [42]. Moreover,
the Vmax. value of NHase@Dop-TAMS was higher than for NHase@TAMS, demonstrating that the
polydopamine on the surface of Dop-TAMS enhanced the catalytic ability of NHase@Dop-TAMS [38].
Table 2. Kinetic parameters of free and immobilised NHase.
Samples
Free NHase
NHase@TAMS
NHase@Dop-TAMS
Km (mM)
1.71
4.46
2.61
2.48
3.59
1.45
2.35
3.96
1.69
Vmax. (mM/min)
−1
V
max./Km (s
)
2.7. Reusability of Immobilised NHase
Studying the reusability of the immobilized NHase is very meaningful for large-scale industrial
applications. Figure 9 shows the results for reusability of NHase@TAMS and NHase@Dop-TAMS.
After recycling for 8 batches, the relative activity ofNHase@TAMS and NHase@Dop-TAMS was
found to be 52.4% and 62.2%, respectively. The activity loss for the enzyme can be primarily
attributed to leakage of NHase from the carrier and denaturation of the NHase molecules [19,23].
Compared with the NHase@TAMS, the activity of NHase@Dop-TAMS decreased slowly, and the
improved operational stability of NHase@Dop-TAMS can be ascribed to the strong conjugation of
covalent bonds between the NHase molecules and the polydopamine on the surface of Dop-TAMS,

Molecules 2017, 22, 1597
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which could have prevented the leakage of NHase from the NHase@Dop-TAMS [36]. The favorable
reusability of NHase@Dop-TAMScould reduce the cost of biocatalysts and improve economic benefits
in practical applications.
100
A
B
80
60
40
20
0
0
1
2
3
4
5
6
7
8
9
Cycle number
Figure 9. The reusability of (A) NHase@Dop-TAMS; and (B) NHase@TAMS.
3. Materials and Methods
3.1. Materials
Dopamine hydrochloride and tannic acid were purchased from Sigma-Aldrich (Shanghai, China).
Tetraethoxysilane (TEOS) and ammonium hydroxide were purchased from Meryer (Shanghai, China).
NHase was purchased from Hangzhou Biosci Biotech Co. (Hangzhou, China). Ethanol was purchased
from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Acrylonitrile was purchased from
Alfa Aesar (Shanghai, China). All other chemical reagents were of AR grade and used as received.
3.2. Preparation of Dopamine-Functionalized TAMS (Dop-TAMS)
TAMS was synthesized using tannic acid as the template, according to the reported method [18].
Dopamine-functionalized TAMS was synthesized using the post-grafting method. Typically, 1.0 g of
TAMS was added into 200 mL of 1 g/L dopamine solution, which was freshly prepared in phosphate
buffer (pH 8.5), and the suspension was stirred for 3 h. Then the slurry was centrifuged and washed
with phosphate buffer (pH 8.0) three times. The material prepared in this manner was denoted
as Dop-TAMS.
3.3. Immobilization of NHase in Dop-TAMS
To immobilize NHase in Dop-TAMS, 50 mg of Dop-TAMS was mixed with 5 mL of NHase
phosphate buffer solution (1.04 mg/mL, 0.1 M PBS, pH 7.0). The resulting solutionwas kept under
constant stirring (200 rpm) at ambient temperature for several hours. After this step, the Dop-TAMS
loading NHase was separated from the solution by centrifugation and washed with PBS (0.1 M,
pH 7.0) several times. The resulting product was denoted as NHase@Dop-TAMS, which was stored
◦
in PBS at 4 C until use. The quantity of NHase in the solution was measured by Bradford assay
and the loading amounts of NHase in the matrix were calculated [23]. NHase was also immobilized
in TAMS by physical adsorption (named NHase@TAMS), in order to make comparisons with the
NHase@Dop-TAMS.

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3.4. Enzymatic Activity Assay
Enzymatic activities offree NHase or immobilized NHase were assayed according to the reported
procedure [ 45]. Specifically, an appropriate amount of native NHase or immobilized NHase was
6,7,
added to 125 mM of acrylonitrile solution prepared in phosphate buffer (50 mM, pH 7.0) and the
◦
suspension was stirred for 5 min at 30 C. Finally, 0.2 mL of 0.6 M HCl was added to the mixture
to stop the reaction. High-performance liquid chromatography (HPLC, 600E-2487, Waters, Milford,
MA, United States) was utilized to measure the formed acrylamide concentration. One unit of NHase
activity was defined as the amount of NHase which released 1
above conditions.
µM of acrylamide per minute under the
3.5. Thermal and pH Stability
For ther◦mal stability studies, free NHase or immobilized NHase was incubated in PBS (50 mM,
pH 7.0) at 50 C for several hours and the residual activities were measured periodically by the method
described in Section 2.4. The relative activity was calculated from the ratio of the residual activity to
the initial activity.
pH stabilities were assayed by incubating the free NHase or immobilized NHase in pH 3.0 and
pH 9.0 PBS at 25 ◦C for 6 h and, after each incubation period, the residual activities were measured.
The pH of the PBS was adjusted by 0.1 M NaOH or 0.1 M HCl and the relative activities werecalculated
as described above.
3.6. Stability of Free and Immobilized NHase in Shaking and Storage Conditions
The stabilities in shaking conditions were determined by shaking the free NHase or immobilized
NHase (200 rpm) in pH 7.0 phosphate buffer solutions at room temperature for several days and the
residual activities were measured periodically. The relative activities were therefore calculated.
In order to determine storage stability, the free NHase or immobilized NHase were immersed
in pH 7.0 phosphate buffer solutions at 4 ◦C for several days. The residual activities were measured
periodically and the relative activities were calculated.
3.7. Determination of Kinetic Parameters
The kinetic parameters of freeNHase and immobilized NHase were calculated by using the
Michaelis–Menten equation (1/V versus 1/[S]). The reactions were carried out using acrylonitrile as
substrate in the initial concentration ranges of 2 to 50 mM. At the end of the reaction, the acrylamide
yields were measured by the HPLC method.
3.8. Reusability
The reusability of NHase@TAMS and NHase@Dop-TAMS were determined according to the
enzyme activity assay method described in Section 2.4. At the end of each batch reaction, the
immobilized NHase was centrifuged, washed with PBS (50 mM, pH 7.0), and then reused in the
next reaction.
3.9. Characterizations
The scanning electron microscopy (SEM) images of Dop-TAMS were gained by a Hitachi S-4800
SEM (Tokyo, Japan). The transmission electron microscopy (TEM) images were gained by a JEOL
JEM-2100F TEM (Tokyo, Japan) at 200 kV. The Brunauer-Emmett-Teller (BET) surface area was
calculated according to the adsorption data, which was measured by a BEL BET BELSORP-max,
and the pore size distributions were obtained according to the Barrett-Joyner-Halenda (BJH) method.
The Fourier transform infrared (FT-IR) spectra of TAMS, Dop-TAMS and immobilized NHase were
recorded using a Bruker VECTOR22 Fourier transform infrared spectrometer (Karlsruhe, Germany) by
KBr pellet.

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4. Conclusions
In summary, tannic acid-templated mesoporous silica (TAMS) was synthesized using a simple,
cost-effective, and environmentally-friendly nonsurfactant template method and dopamine-functionalized
TAMS (Dop-TAMS) was developed as a carrier to immobilize NHase (NHase@Dop-TAMS). The study
results suggest that the thermal stability, storage stability, shaking stability, and pH stability of the
NHase@Dop-TAMS were significantly enhanced. Furthermore, the NHase@Dop-TAMS showed good
reusability. Based on these advantages, the efficient immobilization of NHase in Dop-TAMS has great
potential in practical applications.
Acknowledgments: The authors are grateful for the financial support of the Zhejiang Provincial Natural Science
Foundation of China (No. LY18E060007), the Scientific Research Foundation of Zhejiang Ocean University
(No. Q1608), the Scientific Research Foundation of Zhejiang Ocean University (No. Q1609), and the National
Natural Science Foundation of China (No. 51606168).
Author Contributions: J.-k.G., Y.-j.J., and Y.C. conceived and designed the experiments; J.-k.G., Z.-j.Z., and Y.-j.J.
carried out the experiments and wrote the manuscript text; J.-k.G., Z.-j.Z., and Y.C. analyzed the data; J.-k.G.,
Y.-j.J., Y.C., and S.-f.G. discussed the results and reviewed the manuscript.
Conflicts of Interest: The authors declare no conflicts of interest.
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Sample Availability: Samples of the compounds are not available from the authors.
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