Multiscale immobilized lipase for rapid separation and continuous catalysis

Article abstract of DOI:10.1039/c8nj01950a

Enzyme-inorganic hybrid nanoflowers have drawn extensive research interest for enzyme immobilization owing to their enhanced enzymatic activity, high surface area, and excellent chemical stability. However, their low mechanical stability and challenging s

Full text of DOI:10.1039/c8nj01950a

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Multiscale immobilized lipase for rapid
separation and continuous catalysis
1
, 2
1
2
1
2
2
2
Weifan Ren , Xu Fei* , Jing Tian* , Yao Li , Muzi Jing , Huan Fang , Longquan Xu , Yi
2
Wang .
1
2
. Instrumental Analysis Center, Dalian Polytechnic University, Dalian 116034, China
. School of Biological Engineering, Dalian Polytechnic University, Dalian 116034, China
#
*
Correspondence to: Xu Fei, Instrumental Analysis Center, Dalian Polytechnic University, 1
Qinggongyuan Road, Dalian 116034, P. R. China
Eꢀmail: feixudlpu@163.com
Tel: +86ꢀ411ꢀ86323691ꢀ201
Fax: +86ꢀ411ꢀ86322038
*
Correspondence to: Jing Tian, School of Biological Engineering, Dalian Polytechnic
#
University, 1 Qinggongyuan Road, Dalian 116034, P. R. China
Eꢀmail: tianjing@dlpu.edu.cn
Tel: +86ꢀ411ꢀ86324485
1

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Abstract
The enzymeꢀinorganic hybrid nanoflowers have drawn extensive research interest for enzyme
immobilization owing to their enhanced enzymatic activity, high surface area, and excellent
chemical stability. However, its low mechanical stability and challenge in separation hinder its
application. Herein, we report
a
facile and effective strategy to construct
nanoflowersꢀPVAꢀchitosan composite hydrogels (NPC) that have excellent mechanical strength,
high catalytic activity, and are easy to be separated. The lipaseꢀCu (PO ) •3H O nanoflowers
3
4 2
2
were uniformly immobilized in the hydrogel networks, and the substrate and product can travel
freely. The excellent mechanical properties of the hydrogels effectively protect the fragile
nanoflowers from the external stress, ensuring the enzyme activity. Compared with free lipase
o
(
97.28 U/g), NPC3 exhibited higher enzymatic activity (146.12 U/g) at 37 C and pH 7.4. In
further, hydrogels as bulk materials can be processed into various physical forms, such as thin
films and spheres. The product can easily flow out without a separation process. It is expected
that this immobilization method would have great potential in industrial catalysis.
Keywords: Lipase, Hybrid nanoflower, Composite hydrogel, Continuous catalysis
2

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1
. Introduction
Enzymes are environmentally friendly biocatalysts with outstanding prospects in industrial
processes, which include high catalytic efficiency and mild reaction conditions, high specificity
1
ꢀ5
and high selectivity. However, the application of enzymes is limited due to their short
lifetimes, unstable structure and extensive separation costs. Most free enzymes are unstable due
6
,7
to a harsh storage and catalytic environment. Additionally, most enzymes are water soluble,
8
which make recovery of this expensive catalyst complex almost impossible. In order to
overcome these difficulties, the methods of enzyme immobilization have been developed in the
past decades, which commonly conclude: embedding, adsorption, crosslinking and covalent
9
ꢀ12
binding.
Enzyme immobilization is an efficient strategy to improve the catalytic
performance of enzymes in nonnatural environments, providing many important advantages
over the use of natural enzymes; these advantages include enzyme activity, stability, reusability,
1
3ꢀ16
continuous operation, and process economics.
Recently, Zare's group has reported the preparation of enzymeꢀinorganic hybrid
nanoflowers through the coꢀprecipitation of Cu (PO ) and enzymes in an aqueous buffer
3
4 2
17
solution. Unlike conventional immobilization methods, this synthetic method does not require
any toxic agents and is very simple to fabricate immobilized enzymes by adding enzyme to
18,19
metal ion solution.
Due to the high specific surface area and confinement of enzymes in the
nanoflowers, the enzymeꢀinorganic hybrid nanoflowers exhibited enhanced enzymatic activity,
2
0ꢀ23
durability and stability compared with the free enzyme.
Hence, enzymeꢀinorganic hybrid
2
4ꢀ26
nanoflower as a new nanostructured immobilized enzyme has got more attention.
In recent
3

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2
7
28,29
36
30
31
years, αꢀacetolactate decarboxylase. glucose oxidase,
papain, soybean peroxidase,
3
2
33
34
35
37
lipase, urease, lactoperoxidase, trypsin, αꢀamylase, and chymotrypsin, have been
chosen as organic component and their enzymatic activities were increased generally. Among
these enzymes, lipase as a versatile biocatalyst can catalyze various types of reactions, such as
3
8
hydrolysis, esterification, and aminolysis. Therefore, lipase has been widely used in food,
pharmaceuticals, biodiesel preparation, detergents and cosmetics. Many studies have been
carried out to improve the catalytic properties of lipases due to their poor stability, narrow pH
range adaptability, and sensitivity to harsh environments. Using this selfꢀassembly
immobilization method, a surfactantꢀactivated lipaseꢀinorganic hybrid nanoflower was
successfully prepared and the enzymatic activity and stability of lipase was significantly
3
2
improved. However, there are still many issues that need to be addressed. For example, it is
difficult separate the nanoflowers from the catalytic system due to their nano or micron scale
18,38
size,
Furthermore, the nanoflowers have low mechanical strength, which always lead to
3
9
damage during the stirring and separation process, These drawbacks hinder their applications
in industry. Therefore, it is necessary to find an effective way to overcome the disadvantages of
nanoflowers.
Hydrogels are threeꢀdimensional polymeric networks that can absorb up to large amounts of
water without dissolution. They have been the center of attraction for biomaterials scientists
due to their good biocompatibility, functionality and mechanical properties. They are promising
entrapment matrices for enzyme immobilization. PVA and chitosan composite hydrogel with
high porosity can be physically crosslinked by repeated freezing and thawing, not
4

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4
0
compromising biological activity of enzyme. .At the same time, both PVA and chitosan were
41ꢀ43
successfully used for enzyme immobilization
is a good carrier for hybrid nanoflowers.
Therefore, PVAꢀchitosan composite hydrogel
In this work, the Pseudomonas cepacia lipase, which is an efficient lipase in the
transesterification of prochiral or racemic alcoholswith acetates, was activated by cetyltrimethyl
ammonium bromide (CTAB) and selfꢀassembled to form the lipaseꢀCu (PO ) •3H O
3
4 2
2
nanoflowers. And the nanoflowers were immobilized into a hydrogel network, which
effectively protect the fragile nanoflowers from the external stress and simplify separation
process. The enzymatic activity of lipase was improved by selfꢀassembly, and the
lipaseꢀCu (PO ) •3H O nanoflowers were uniformly immobilized in polyvinyl alcohol (PVA)
3
4 2
2
and chitosan composite hydrogel networks by repeated freezing and thawing. The substrates
could travel freely through the hydrogel networks and contact the lipaseꢀCu (PO ) •3H O
3
4 2
2
nanoflowers. It is an effective immobilization method to keep high catalytic activity and
stability of the enzyme, further simplify the separation process.
2
2
. Experimental
.1. Materials
The lipase from Pseudomonas cepacia L05 was obtained from Shenzhen Lvwei Kang
bioengineering co., Ltd. PꢀNitrophenyl palmitate (pꢀNPP) was purchased from Aladdin Reagent
Corporation. Cetyltrimethyl ammonium bromide (CTAB) was purchased from Tianjin Damao.
Triton Xꢀ100 (TXꢀ100) and pꢀNP (pꢀNitrophenol) were supplied by Tianjin Guangfu Fine
Chemical Corporation. Arabic gum, Chitosan and polyvinyl alcohol were purchased from
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Sinopharm Chemical Reagent Corporation. CuCl , NaCl, KCl, Na HPO , KH PO and Na CO
3
2
2
4
2
4
2
were purchased from Tianjin Damao Chemical Reagent Corporation. Aspergillus niger was the
strain deposited in our laboratory. All of these chemicals were analytical reagent grade quality
and used without further purification. Deionized water was used throughout all experiments.
2
.2. Measurements
The UVꢀVis absorption spectra were taken by a PerkinꢀElmer LAMBDA35 (USA). Scanning
electron microscopy (SEM) was performed on a JEOL JSM 7800 electron microscope with
primary electron energy of 5.0 kV and energy dispersive Xꢀray spectroscopy (EDS) was
recorded with Oxford INCA. The FTꢀIR spectra were taken on a PerkinꢀElmer Spectrum TWO
infrared spectrophotometer. The Xꢀray diffraction (XRD) was measured on a Shimadzu
XRDꢀ7000 diffractometer with Cu Kα radiation (50 kV, 200 mA, λ= 0.154 nm) and a scanning
o
step of 0.02 . The mechanical properties of the NPC were characterized by tension test at a
speed of 10 mm/min and compression test at a speed of 1 mm/min. The size of the hydrogel
specimens for tension and compression tests were length: 40 mm, width: 15 mm, thickness: 5
mm and thickness: 12 mm, diameter: 20 mm, respectively.
2
.3. Preparation of enzyme-inorganic hybrid nanoflowers
The lipaseꢀCu (PO ) •3H O nanoflowers were synthesized by selfꢀassembly method. Firstly,
3
4 2
2
lipase (225 mg, 97.28 U/g), CTAB (82 mg), and phosphate buffer solution (900 mL, pH=7.4,
.01 M) were mixed and stirred for 10 min. The CTAB was used as an activating agent to open
active form of lipase, because that most lipases were generally “inactive” closed form due to a
0
3
2,44
hydrophobic amino acids chain covering their active site
The mixture solution was
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incubated for 10 h to achieve the activated lipase solution. Secondly, CuCl aqueous solution (6
2
mL, 120 mM) was added to the activated lipase solution (900 mL), and then the mixture was
incubated at 25 °C for 3 days. The lipases with an active form were anchored in hierarchical
nanoflowers via selfꢀassembly. Finally, the blue precipitates were collected by centrifugation
(3500 rpm for 5 min), and then were washed for three times by deionized water. The blue
precipitates were dried into the powder by using the technology of the vacuum freezeꢀdrying.
2
.4. Preparation of the NPC
LipaseꢀCu (PO ) •3H O nanoflowers (10 mg, 245.46 U/g), 10 wt% PVA aqueous solution (0.8
3
4 2
2
g), and 5 wt% chitosan aqueous solution (0.2 g) were mixed. And then, the mixture was frozen
at ꢀ20 °C for 10 h and thawed at room temperature for five cycles to form the
nanoflowersꢀPVAꢀchitosan composite hydrogels (NPC1). The NPC2, NPC3, NPC4 and NPC5
were prepared as the same procedure. And the weight percentage of nanoflowers in NPC2,
NPC3, NPC4 and NPC5 were 2 wt%, 3 wt%, 4 wt% and 5 wt% respectively and their feed
ratios were provided in supporting information.
2
.5. Weight percentage of enzyme in the lipase-Cu (PO ) •3H O
3
4 2
2
nanoflowers
To determine the enzyme content, the dried lipaseꢀCu (PO ) •3H O nanoflowers were calcined
3
4 2
2
at 700 °C for 2 h by muffle furnace. After removing the crystallization water and the organic
enzyme in the lipaseꢀCu (PO ) •3H O nanoflowers, Cu (PO ) as the inorganic metal salt was
3
4 2
2
3
4 2
obtained (Figure S2, S3). The weight percentage of enzyme in lipaseꢀCu (PO ) •3H O
3
4 2
2
nanoflowers was calculated as follow equation:
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G −G0
N
W =
×100%
GN
where W is the weight percentage enzyme (%), G is the weight of nanoflowers, and G is the
N
0
weight of Cu (PO ) •3H O.
3
4 2
2
2
.6. Determination of enzymatic activity
The enzymatic activity of lipase, lipaseꢀCu (PO ) •3H O nanoflowers, and NPC were
3
4 2
2
determined by using pꢀnitrophenylpalmitate (pꢀNPP) as substrate. Lipase can catalyze pꢀNPP
hydrolyzed into pꢀnitrophenol (pꢀNP) which can be detected at 402 nm (the standard curve of
pꢀNP was shown in Fig. S1). 1 vol of a 16.5 mM solution of pꢀNPP in 2ꢀpropanol was mixed
with 9 vol of PBS (0.01 M, pH 7.4) containing 0.4% (w/v) Triton Xꢀl00 (emulsifier) and 0.1%
(
w/v) Arabic gum (emulsifier) as the substrate solution. After preheating for 5 min (150 rpm,
7 °C), 4 mL of this substrate solution and 1 mL PBS (0.01 M, pH 7.4) containing 1 mg lipase
were mixed. Then the mixture was incubated for 5 min (water bath shaking at 180 rpm and
7 °C) and terminated by adding 5 mL of 0.25 M Na CO (5 min, 150 rpm, 37 °C). After that,
3
3
2
3
the mixture was rapidly centrifuged (5000 rpm, 5 min). Then the supernatant was measured at
4
5,46
4
02 nm by UV–Vis absorption spectroscopy.
As a comparison, the activity of lipaseꢀ
Cu (PO ) •3H O nanoflowers and NPC (the amount of lipase embedded in nanoflowers and
3
4 2
2
NPC were equivalent to 1 mg lipase) determined as the same procedures for the free lipase.
One enzyme unit was defined as the amount of lipase releasing 1ꢁmol pꢀNP from pꢀNPP per
minute under experimental conditions. The calculation of enzymatic activity was used in the
following equation:
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VT •
[
C −C
]
S
B
U =
G •T
E
where U is the enzymatic activity (U/g), C is the concentration of pꢀNP in the sample
S
(
ꢁmol/mL), and C is the concentration of pꢀNP in the blank (ꢁmol/mL), G is the amount of
B
E
enzyme in NPC (g), V is the total volume of reaction solution (mL), T is the reaction time
T
(min).
2
.7. Stabilities and kinetic parameters of free lipase and
immobilized lipase
The storage stabilities of free lipase, lipaseꢀCu (PO ) •3H O nanoflowers and NPC3 were
3
4 2
2
o
studied by incubation in PBS buffer at 25 C for 15 day. The residual activities of the enzymes
were determined every three days. The initial enzyme activity was set to 100%, and the residual
enzyme activity relative to the initial enzyme activity ratio was studied. The kinetic parameters
(
K and V ) of free lipase, lipaseꢀCu (PO ) •3H O nanoflowers and NPC3 were calculated by
m max 3 4 2 2
the LineweaverꢀBurk doubleꢀreciprocal plot method. The concentration of pꢀNPP (as substrate)
was set from 0.1 mM to 1.0 mM and the enzyme concentration was 1 mg/mL. The enzymatic
o
reaction was carried at 37 C and pH 7.4 in PBS.
2
.8. Mechanical properties of the NPC
The mechanical properties of NFC1, NPC2, NPC3, NPC4 and NPC5 hydrogels were measured
by tension and compression tests. The composite hydrogels for compression test were prepared
for the same size column (diameter 40 mm, thickness 12 mm), and the rate of compression test
was 1 mm/min. The samples of tension test were moulded into cuboid shape (length 60 mm,
width 20 mm, height 5 mm) and were measured at the speed of 10 mm/min. The mechanical
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properties were tested at room temperature.
2
.9. Antibacterial experiment of NPC3
The antimicrobial activity of NPC3 was detected by plate coating method. 10 g of the NPC3
solution was poured into a petri dish and was physically crosslinked by the five
freezingꢀthawing cycles. Then the Aspergillus niger spore suspension was added dropwise to
the hydrogel surface and was spread evenly with a glass spreading rod. After being dried for 30
min, NPC3 was placed in a thermostatic incubator at 30 °C to observe the growth of colonies. A
composite hydrogel without chitosan was used as a control.
3
3
. Results and discussion
.1. Synthesis and characterization of NPC
For this study, the formation mechanism was illustrated in Fig. 1. The required
lipaseꢀCu (PO ) •3H O nanoflowers were synthesized and immobilized into PVAꢀchitosan
3
4 2
2
hydrogels. FTIR, XRD and EDS were used to define the structure and composition of
lipaseꢀCu (PO ) ·3H O nanoflowers and NPC. As shown in Fig. 2A(a), the strong characteristic
3
4 2
2
ꢀ
1
ꢀ1
absorptions at 1049 cm (asymmetric stretching), 990 cm (symmetric stretching), and 630
ꢀ1
cm (bending) were attributed to PꢀO vibrations and indicated the existence of phosphate
ꢀ1
ꢀ1
groups. In Fig. 2A(b), typical lipase absorption peaks occurred at 1653 cm and 1541cm for
ꢀ1
ꢀ1
ꢀ1
ꢀ
CONH, 3000 cm to 2800 cm for ꢀCH and ꢀCH , and around 3300 cm for ꢀOH. Fig. 2A(c)
3 2
indicated that the characteristic absorption peaks of lipase and Cu (PO ) ·3H O were
3
4 2
2
maintained in FTIR spectrum of nanoflowers. Furthermore, the curve of NPC3 [Fig. 2A(e)]
contained the characteristic absorptions of nanoflowers and PVAꢀchitosan hydrogels [Fig.
1
0

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2
A(d)]. These results indicated that the lipaseꢀCu (PO ) •3H O nanoflowers were successfully
3 4 2 2
introduced into the PVAꢀchitosan hydrogels.
Fig. 1. Proposed growth mechanism of schematic representation of the synthesis of NPC.
Fig. 2.(A) FTꢀIR spectra of (a) Cu (PO ) •3H O, (b) lipase, (c) lipaseꢀCu (PO ) •3H O
3
4 2
2
3
4 2
2
nanoflowers , (d) PVAꢀchitosan hydrogels, (e) NPC3. (B) XRD pattetrns of (a)
Cu (PO ) •3H O, (b) lipaseꢀCu (PO ) •3H O nanoflowers , (c) NPC3. (C) EDS pattern of
3
4 2
2
3
4 2
2
NPC3.
As shown in Fig. 2B, the XRD patterns illustrated that the crystallographic structure of the
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hybrid nanoflowers were Cu (PO ) •3H O, because all the diffraction peaks presented in Fig.
3
4 2
2
2
B(b) could be indexed to Cu (PO ) •3H O (JCPDS card no. 22ꢀ0548, Fig. 2B(a)). It suggested
3 4 2 2
that the lipaseꢀCu (PO ) •3H O nanoflowers were well crystallized and featured high
3
4 2
2
crystallinity after incorporating lipase. Meanwhile the lipaseꢀCu (PO ) •3H O nanoflowers also
3
4 2
2
maintained high crystallinity after being immobilized in the hydrogels [Fig. 2B(c) ]. The EDS
analysis of the NPC3 was shown in Fig. 2C. The NPC3 contained C, O, P and Cu element.
Among them, Cu element and P element were derived from Cu (PO ) •3H O, which could
3
4 2
2
prove that the lipaseꢀCu (PO ) •3H O nanoflowers were entrapped in PVAꢀchitosan hydrogels.
3
4 2
2
The microstructure of the lipaseꢀCu (PO ) •3H O nanoflowers , PVAꢀchitosan hydrogels,
3
4 2
2
and NPC3 were determined by scanning electron microscopy (SEM) after being freezeꢀdried,
respectively. As shown in Fig. 3a, the size of lipaseꢀCu (PO ) •3H O nanoflowers were uniform
3
4 2
2
in appearance with good monodispersity. As organicꢀmolecule complexing agents, lipase
completed strongly with metal ions. The obtained PVAꢀchitosan hydrogel showed irregular
micron pores by physical crossꢀlinking (Fig. 3b). The porosity of hydrogel plays a major role
for the transmission of substrate and products. After five freezingꢀthawing cycles, the
lipaseꢀCu (PO ) •3H O nanoflowers maintained a intact flowerlike structure in the internal
3
4 2
2
space of the hydrogel porous structure (Fig. 3c) and the analysis of the distribution of copper in
NPC3 by mapping (Fig. 3d) proved that the nanoflowers were uniformly distributed in the
composite hydrogel, which could provide a good catalytic environment. Moreover, the NPC
could be made into a variety of physical forms (Fig. 3e and 3f) to be suitable for different
application condition.
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Fig. 3. SEM images of (a) lipaseꢀCu (PO ) hybrid nanoflowers, (b) PVAꢀchitosan hydrogels,
3
4 2
(c) NPC3.(d) EDS mapping of NPC3. The photographs of (e) NPC3 membrane and (f) NPC3
spheres.
3
.2. Enzymatic activity, reusability and storage stability
In order to investigate the optimum ratio of nanoflowers to PVAꢀchitosan hydrogels, the
nanoflowers of same weight were put into the different volume of PVAꢀchitosan hydrogels
separately and NPC (NPC1 – NPC5) with different weight percentage of nanoflowers (1 wt% ꢀ
5
wt%) were obtained. The activities of free lipase, lipaseꢀCu (PO ) •3H O nanoflowers and
3 4 2 2
NPC were measured using pꢀNPP as substrate based on the same condition. The activity of free
lipase was determined to be 97.28 U/g. According to the lipase weight percentage in
lipaseꢀCu (PO ) •3H O
nanoflowers
(Table.
S1),
the
enzymatic
activity of
3
4 2
2
lipaseꢀCu (PO ) ·3H O nanoflowers was calculated to be 245.46 U/g, and the activity recovery
3
4 2
2
of hybrid nanoflower is 107.12% (for the first assembly immobilization). The enzyme solution
can be used for multiple immobilizations. After six immobilizations, the activity recovery of
hybrid nanoflower is 133.58 % (Table S2). Activity recovery was defined as the percentage,
1
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which the total activity of the immobilized lipase and the unimmobilized free lipase accounted
4
7
for the initial activity of the lipase introduced to immobilization reaction . As shown in Fig. 4a,
the enzymatic activity of NPC1, NPC2, NPC3, NPC4 and NPC5 were 130.77 U/g, 129.37 U/g,
1
46.12 U/g, 120.34 U/g and 82.42 U/g respectively (Table S3). Due to the same weight of
nanoflowers, NPC1 and NPC2 had similar enzymatic activity. With the increasing of the
concentration of nanoflowers, the volume of NPC continued to decrease. Therefore, the more
nanoflowers were dispersed in the surface layer, which was easier to contact with the substrate,
increasing the enzymatic activity. As the concentration of nanoflowers increased further, the
NPC volume became smaller and more nanoflowers were distributed inside the hydrogels. The
substrate was more difficult to contact with nanoflowers, decreasing the enzymatic activity. We
chose NPC3 as the optimum composite ratio.
The superiority of immobilized enzyme is to recycle and reuse. The reusability was
evaluated by examining the remaining activity of lipaseꢀCu (PO ) •3H O nanoflowers and the
3
4 2
2
NPC3 after each cycle, and the activity of free lipase was set as 100%. As shown in Fig.4b, the
enzymatic activity of lipaseꢀCu (PO ) •3H O nanoflowers decreased significantly to be 31.71
3
4 2
2
U/g after four cycles and lipaseꢀCu (PO ) •3H O nanoflowers were inactive after the fifth cycle
3
4 2
2
(
Table S4). However, enzymatic activity of NPC3 remained to be above 42% of free enzyme
activity even after seven cycle’s reactions. By comparing the images of nanoflowers and NPC3
before and after catalysis), it was observed that the appearance of nanoflowers in the four
cycles was seriously damaged (Fig. 4c), which directly led to the decrease of enzymatic
(
3
9
activity. The reason for the decrease of NPC3’ enzyme activity could be that the product was
1
4

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attached to the network of hydrogels, which reduced the exposure of the substrate to
nanoflowers (Fig. 4d).
Fig. 4. (a) Relative enzymatic activity of free lipase, lipaseꢀCu (PO ) •3H O nanoflowers and
3
4 2
2
NPC1ꢀNPC5, (b) Reusability of lipaseꢀCu (PO ) •3H O nanoflowers and NPC3. SEM images
3
4 2
2
of (c) lipaseꢀCu (PO ) •3H O nanoflowers and (d) NPC3 after continuous catalytic reaction.
3
4 2
2
Stabilization of the enzyme is compulsory for the application of the immobilized enzymes.
Therefore, the stability of free lipase, lipaseꢀCu (PO ) •3H O nanoflowers, and NPC3 against
3
4 2
2
soaking was compared. The free lipase, lipaseꢀCu (PO ) •3H O nanoflowers and NPC3 were
3
4 2
2
soaked in PBS at room temperature for 15 day and their enzyme activities were shown in Fig.5,
respectively. The nanoflowers and NPC3 still retains 79.31%, 82.22% of initial enzyme activity
after 15 day, far better than 15.83% of free lipase. The results showed that nanoflowers and
NPC3 had good storage stability after lipase being immobilized.
In addition, kinetic parameters (K and V ) of free lipase, lipaseꢀCu (PO ) •3H O
m
max
3
4 2
2
nanoflowers and NPC3, were assayed at substrate concentration from 0.1 mM to 1.0 mM and
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the enzyme concentration of 1 mg/mL by using the doubleꢀreciprocal plot method. Vmax is
defined as the highest possible velocity when the enzyme is saturated with substrate, therefore,
it reflects the effective characteristics of the immobilized enzyme, but may be affected by
diffusion constrains. K is defined as the substrate concentration that gives a reaction velocity
m
of 1/2 Vmax. This parameter reflects the intrinsic characteristics of the enzyme and depends
4
8
upon both partition and diffusion effects. Different enzymes have different K values, and the
m
same enzyme has different K values for different substrate. K values can approximate the
m
m
affinity between enzyme and substrate. Low K value indicates strong affinity and high Km
m
3
2
value indicates weak affinity. The K and V
values for the three forms of lipase were
max
m
obtained by calculating the intercept of the curves on the Xꢀaxis and Yꢀaxis (Fig. 5b), and
shown in Table 1. It was found that NPC3 and lipaseꢀCu (PO ) •3H O nanoflowers had the
3
4 2
2
lower K value, confirming that they had a better affinity of substrate toward lipase molecules
m
compared to free lipase. The decrease in the Km values is either due to the conformational
changes of the enzyme resulting in a higher possibility to form substrate–enzyme complex or
4
9
due to the higher accessibility of the substrate to the active site of the immobilized enzyme.
Moreover, the Vmax and Vmax/K of NPC3 and lipaseꢀCu (PO ) •3H O nanoflowers were
m
3
4 2
2
significantly increased. The results showed that catalytic efficiencies of both
lipaseꢀCu (PO ) •3H O nanoflowers and NPC3 were higher than free lipase. This result is
3
4 2
2
largely attributed to the presence of the large specific surface area of the nanoflower and the
porous network of hydrogels, which can create a more favorable interface for the mass transfer
of substrate or product to or from the active site of the enzyme.
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Fig. 5. (a) Storage stability and (b) Lineweaver–Burk plot of free lipase,
lipaseꢀCu (PO ) •3H O nanoflowers and NPC3.
3
4 2
2
Table. 1. The kinetic parameters of free lipase, lipaseꢀCu (PO ) •3H O nanoflowers and NPC3
3
4 2
2
Types of enzymes
freeꢀlipase
Km (mmol/L) Vmax (mmol/L·min)
Vmax/Km
0.0202
0.5696
.5429
0.5576
0.0115
0.0184
0.0150
lipaseꢀCu (PO ) •3H O
3
4 2
2
0
0.0339
0.0269
nanoflowers
NPC3
The simple and convenient separation of immobilized enzyme is very important for
industrial application. In the repeated catalytic reaction, the NPC3 could be processed into film,
and the product could be poured out directly without separation (Fig. 6). However the
lipaseꢀCu (PO ) •3H O nanoflowers must be separated from the reaction system by using high
3
4 2
2
speed centrifugation. This is the advantage of the large size composite hydrogels in catalytic
system.
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Fig. 6. The photographs of the catalysis procedure of pꢀNPP.
3
.3 Mechanical and antibacterial properties of NPC
In the repeated catalysis process, NPC is inevitable to withstand the external shear force, so it
must have excellent mechanical strength and toughness. A good hydrogel system can
effectively absorb external stress and transform into strain, thus protecting the rigid
nanoflowers. Fig.7a showed the tensile strain–stress curves of the NPC with various
nanoflowers content. The maximum stress of the NPC significantly increased at first and then
decreased with the increase of the nanoflowers content. A reasonable hypothesis could be that
nanoflowers provided a center of PVA crystallization, which could increase the number of
crystal regions in PVAꢀchitosan hydrogel. The crystal regions in the PVAꢀchitosan hydrogel
were helpful to enhance the stress of the NPC. However, too many crystal regions limited the
sliding of polymer segment, thus decreasing the elongation of the hydrogel. At the same time,
the NPC3 achieved a high compressive stress of more than 0.56 MPa when the compressive
strain was 80 % (Fig. 7b). These results showed that the NPC had good mechanical properties.
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Fig. 7. Mechanical properties of NPC (a) tensile stressꢀstrain curves, (b) compressive
stressꢀstrain curves.
PVA has been widely investigated for biomedical applications due to its excellent
biocompatibility. However, its intrinsical nature provides good support to microorganism
growth, which is not allowed in the actual catalytic process, especially in food. Therefore, the
5
0
chitosan was added to resist mycete growth . Antibacterial properties of NPC3 were detected
by plate culture method. The NPC3 was placed on a culture dish and the aspergillus niger spore
suspension was spread to the NPC3 surface. After being dried for 30 minutes, the culture dish
was placed in a 30 °C incubator. Using the composite hydrogels without chitosan as a control
group, the growth of colonies was observed. As shown in Fig. 8, the NPC3 significantly
inhibited the growth of aspergillus niger, which proved that the NPC3 had good antibacterial
properties.
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Fig. 8. Photographs of inhibition zone in cultures of Aspergillus niger (a) NPC3 film and (b)
composite hydrogel film without chitosan.
4
. Conclusions
We have immobilized lipaseꢀCu (PO ) •3H O nanoflowers into the PVAꢀchitosan hydrogels to
3
4 2
2
gain the nanoflowersꢀPVAꢀchitosan composite hydrogels with improved catalytic activity. The
porous network structures of NPC ensure that substrate can travel freely to the nanoflowers in
hydrogel. The nanoflowersꢀPVAꢀchitosan composite hydrogels have good reusability, storage
stability and good mechanical properties. Furthermore, they can be processed into various
physical forms. After the catalytic reaction, the nanoflowersꢀPVAꢀchitosan composite
hydrogels can be easily separated from the reaction system. Therefore, these
nanoflowersꢀPVAꢀchitosan composite hydrogels have great application prospects in industrial
catalysis.
Acknowledgements.
This work was supported by the National Natural Science Foundation of China (31771914,
2
2
1204007), Natural Science Foundation of Liaoning Province of China (20170540076,
0170540079), Research on science and technology of education department of Liaoning
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province project (2017J078).
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Graphical Abstract