An enzyme-copper nanoparticle hybrid catalyst prepared from disassembly of an enzyme-inorganic nanocrystal three-dimensional nanostructure

Article abstract of DOI:10.1039/c5ra27904f

Enzyme-copper phosphate composites with highly porous three-dimentional (3D) structures were first prepared by self-assembly of protein molecules with copper ions in phosphate buffer solution. The self-assembled enzyme-copper phosphate 3D nanostructures w

Full text of DOI:10.1039/c5ra27904f

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An enzyme–copper nanoparticle hybrid catalyst
prepared from disassembly of an enzyme–
inorganic nanocrystal three-dimensional
nanostructure†
Cite this: RSC Adv., 2016, 6, 20772
Received 28th December 2015
Accepted 16th February 2016
a
a
a
a
b
a
Zhixian Li,‡ Yi Ding,‡ Xiaoling Wu, Jun Ge,* Pingkai Ouyang and Zheng Liu
DOI: 10.1039/c5ra27904f
www.rsc.org/advances
Enzyme–copper phosphate composites with highly porous three-
On the other hand, nanostructures can be categorized into
10
dimentional (3D) structures were first prepared by self-assembly of zero-, one-, two- and three-dimensional nanostructures.
protein molecules with copper ions in phosphate buffer solution. The Enzyme–metal nanoparticle composites which are in the form
self-assembled enzyme–copper phosphate 3D nanostructures were of nanoparticles are considered as zero-dimensional nano-
then disassembled by sodium borohydride reduction to produce structures (0D nanostructures). Different from the above-
enzyme-bound copper nanoparticles, which are regarded as 0D mentioned traditionally prepared enzyme–metal nanoparticle
nanostructures. Based on the enzyme–copper phosphate composite, composites, enzyme-incorporated more complexed 3D nano-
11–13
which has a high enzymatic activity, the direct transformation of structures
prepared by self-assembly usually display high
copper phosphate into copper nanoparticles produced an enzyme– enzyme loading amounts and highly retained enzyme activities,
copper nanoparticle hybrid catalyst with both high enzymatic activity because these structures are generally formed by specic and
and copper-catalytic activity, serving as a novel type of hybrid catalyst spontaneous interactions between enzyme molecules and
for chemo-enzymatic cascade reactions.
chemical components at mild conditions. The self-assembly
processes guarantee the high enzyme activities. For example,
the facile self-assembly approach to prepare the enzyme–inor-
ganic nanocrystal porous 3D nanostructures with high enzyme
Hybrid catalysts consisting of enzymes and metal nanoparticles
with chemical catalysis capability are of great interest in chemo-
loadings and highly preserved enzyme activities has been re-
1,2
enzymatic cascade reactions. The traditional way of preparing
enzyme–metal nanoparticle hybrid catalysts relies on the
physical adsorption or chemical conjugation of enzyme mole-
14–17
ported by several groups.
We propose that if we could
prepare the enzyme–metal nanoparticle composites (0D nano-
structure) from the enzyme-incorporated 3D nanostructures,
high enzyme loading and high enzyme activity might be easily
achieved.
Therefore, as shown in Scheme 1, instead of the traditional
way of direct synthesis of enzyme–metal nanoparticle hybrid 0D
nanostructures by the ‘bottom-up’ approach from molecular
level, we propose a ‘bottom-up’–‘top-down’ integrated approach
in this study. This approach starts from the self-assembly of
enzyme-incorporated 3D nanostructures, followed by the
disassembly to produce the enzyme–metal nanoparticle 0D
nanostructures. We chose Candida antarctica lipase B (CALB) as
3–7
cules on as-prepared metal nanoparticles. However, some-
times the physical adsorption or chemical conjugation of
enzyme molecules on metal nanoparticles results in reduced
8,9
enzyme activity. In addition, the low affinity between protein
and metal nanoparticles oen results in low efficiency of
enzyme loading. Therefore, the facile synthesis of enzyme–
metal nanoparticle hybrid catalysts with high enzyme activity is
still a big challenge.
a
Key Lab for Industrial Biocatalysis, Ministry of Education, Department of Chemical the model enzyme due to its wide applications in industrial
Engineering, Tsinghua University, Beijing 100084, China. E-mail: junge@mail.
18–21
biocatalysis.
The CALB–copper nanoparticle hybrid catalyst
tsinghua.edu.cn
combing the enzyme catalyst and copper catalyst was prepared
by the new approach and was applied in the chemo-enzymatic
cascade reaction of converting p-nitrophenyl butyrate to the
b
College of Biotechnology and Pharmaceutical Engineering, Nanjing University of
Technology, Nanjing 211816, China
†
Electronic supplementary information (ESI) available: Experimental details for

nal product p-aminophenol which is an important interme-
the synthesis of enzyme–copper nanoparticle hybrid catalyst, enzymatic activity
22,23
assays, catalytic activity of copper, cascade reaction, electron microscope diate for the manufacture of analgesic and antipyretic drugs.
analysis (SEM & TEM), X-ray diffraction (XRD) analysis and dynamic light
More specically, as shown in Scheme 1, by adding cupric
sulfate to the phosphate buffer saline solution containing
scattering (DLS) analysis are included in ESI. See DOI: 10.1039/c5ra27904f
‡
These authors contribute equally to this work.
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structures and are incorporated with enzyme. As investigated by
14
our previous work, in this self-assembly process, protein
molecules induced the nucleation of copper phosphate and
served as a “glue” to bind the nanocrystals of copper phosphate
together based on the coordination interaction between copper
ions and amino acids of protein to construct the highly porous
protein–inorganic hybrid 3D nanostructures. In this study, the
disassembly of the self-assembled nanostructures was triggered
4
by adding sodium borohydride (NaBH ) to eliminate the coor-
2
+
dination interaction by reducing Cu to Cu. Aer reducing the
copper phosphate component, particles with the average size
around 3 mm were obtained (Fig. 1b). The addition of PVP
resulted in shaping the particles with a much smaller average
Scheme
1
The self-assembly–disassembly approach to prepare size of around 50 nm (Fig. 1c). Moreover, the PVP assisted the
enzyme–copper nanoparticle 0D nanostructure.
formation of nanoparticles with less aggregates. It was
supposed that the PVP as an amphiphilic polymer can bind with
the protein–copper phosphate composites in aqueous solution
and therefore result in a steric hindrance to prevent Cu nano-
particles from accumulating during the reducing reaction. The
transmission electron microscope (TEM) image and dynamic
light scattering (DLS) showed that the average size of
CALB&PVP@Cu was around 50 nm (Fig. 1d and e). The darker
grey spots presented in TEM image represent the
CALB&PVP@Cu nanoparticles while the lighter grey spots
might be due to the excess of PVP added in the synthesis.
For both CALB@Cu and CALB&PVP@Cu samples, three
enzyme, the enzyme–copper phosphate composites with highly
porous 3D nanostructures (CALB@CP) were prepared by self-
assembly based on the driving force of coordination interac-
tion between protein and metal ions. Then, the self-assembled
enzyme–copper phosphate nanocrystal 3D nanostructures were
disassembled by eliminating the driving force of coordination
interaction through the chemical reduction of Cu to Cu in the
presence of sodium borohydride. The chemical-triggered
disassembly produced the enzyme–copper nanoparticle hybrid
catalysts (CALB@Cu). In order to prevent the aggregation of Cu
nanoparticles, polyvinyl pyrrolidone (PVP) which is a macro-
molecular surfactant usually used in the solution synthesis of
nanoparticles as the protecting agent can be added during the
preparation of enzyme–copper nanoparticle hybrid catalysts
2
+
ꢀ
ꢀ
ꢀ
diffraction peaks at 43.3 , 50.4 , 74.1 , appeared in the X-ray
diffraction (XRD) patterns (Fig. 2), corresponding to (111),
(200) and (220) lattice planes of copper, respectively, agreed well
with the crystal structure of copper (JCPDS04-0836), indicating
the successful preparation and high purity of copper nano-
particles. The XRD pattern of CALB@CP was presented in
Fig. S2.†
(CALB&PVP@Cu). Detailed procedures for the preparation of
CALB@Cu and CALB&PVP@Cu can be found in ESI.†
The scanning electron microscope (SEM) images showed
that the CALB@CP presented as microspheres (Fig. 1a). Our
Aer
washing
the
CALB@CP,
CALB@Cu
and
CALB&PVP@Cu samples with water for three times to remove
unbound enzyme molecules, the amounts of enzyme bound
on nanoparticles were determined as ꢁ2%, ꢁ5% and ꢁ5%
respectively, by the method described in ESI.† Then, the
specic activity of CALB in CALB@CP, CALB@Cu and
CALB&PVP@Cu samples was measured by using p-nitrophenyl
14
previous work proved that these microspheres have porous
Fig.
1 (a) SEM image of CALB–copper phosphate composites
(
CALB@CP) (inset is the SEM image showing the 3D nanostructure of
composites at high resolution); (b) SEM image of CALB–copper
nanoparticles without PVP (CALB@Cu); (c) SEM image of CALB–
copper nanoparticles with PVP (CALB&PVP@Cu); (d) TEM image of
CALB–copper nanoparticles with PVP (CALB&PVP@Cu); (e) DLS of
CALB–copper nanoparticles with PVP (CALB&PVP@Cu).
Fig. 2 XRD patterns of CALB@Cu and CALB&PVP@Cu samples.
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2
4
butyrate (p-NPB) as the substrate at standard condition
remained to be above 90% aer 5 consecutive reactions in the
(
please see ESI† of the detailed measurement of enzyme case of CALB&PVP@Cu (Fig. 3b). For CALB@Cu, above 80% of
activity). As shown in Fig. 3a, with the same enzyme concen- activity can be retained (Fig. 3b). Aer each run of the enzymatic
À1
tration (0.25 mg mL ) in the activity assay, compared with reaction, the hybrid catalyst was washed with deionized water
free enzyme, CALB@CP retained 95% of catalytic activity. Aer and subjected to the next run, suggesting that the binding
the reducing reaction, CALB@Cu kept 63% of activity. The between enzyme molecules and Cu nanoparticles was stable
addition of PVP might have an effect of protecting enzyme and no obvious leaking of enzyme or loss of enzyme activity was
activity during the reducing process, because 82% of enzyme occurred during the repeated use.
activity was preserved for CALB&PVP@Cu. As a control, the Cu
Aer evaluating the enzyme activity and reusability of the
nanoparticles without CALB didn't show any activity for the hybrid catalyst, it was subjected to the chemo-enzymatic
hydrolysis of p-NPB. Reported in literature, CALB@Pd nano- cascade reaction of converting p-nitrophenyl butyrate (p-NPB)
particles were successfully prepared by directly using enzyme to p-aminophenol (p-AP). As shown in Fig. 4a, the cascade
8
as the reducing agent, and the highest activity was ꢁ47% reaction consists of two steps, rst of which is the hydrolysis of
compared with free enzyme. In another study, alcohol dehy- p-nitrophenyl butyrate (p-NPB) by CALB to form p-nitrophenol
drogenase (ADH) was immobilized on the modied noble (p-NP), and second of which is the reduction of p-nitrophenol
9
metal (Au, Ag) nanoparticles by chemical conjugation, and (p-NP) to p-aminophenol (p-AP) catalyzed by Cu. Therefore, the
the highest activity of about 46% was obtained compared to hybrid catalyst made of CALB and Cu nanoparticles can catalyze
free enzyme. Compared with the above studies, in our study the cascade reaction by using only one catalyst, to simplify the
the enzyme was rstly incorporated in the copper phosphate process. In the presence of NaBH4 which was used at the
porous 3D structures with a very high enzyme activity retained. reducing agent in the Cu-catalyzed reaction, p-NP has the
Therefore, aer reducing copper ions to prepare the enzyme– maximum absorbance at 400 nm. Therefore, by calculating the
copper nanoparticle composite (0D structure), the enzyme ratio of absorbance (400 nm) at t min (A
t
) and the original
activity was highly preserved. The strategy of disassembly of absorbance (A ), followed by plotting the reaction time t with
0
enzyme–copper phosphate nanocrystal 3D structures to the natural logarithm of the absorbance ratio ln(A
enzyme–copper nanoparticle 0D nanostructures in the current
study presented a more effective way to achieve high enzyme
activity.
t 0
/A ), the
Five consecutive runs of the enzymatic reaction suggested
the good reusability of the enzyme–copper nanoparticle hybrid
catalyst. Compared to its original activity, the enzymatic activity
Fig. 4 (a) Cascade reaction catalyzed by the hybrid composite; (b) the
Fig. 3 (a) Relative enzyme activities of different hybrid catalysts reaction rate constants of the reducing reaction catalysed by
(
setting free enzyme as 100%); (b) residual activity of the hybrid cata- CALB&PVP@Cu, CALB@Cu, the mixture of CALB and copper powder,
lysts during five consecutive runs. CALB@CP and CP.
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Table 1 Comparison of CALB&PVP@Cu and CALB@Cu in cascade catalyst enabled chemo-enzymatic cascade reactions, with lots
reactions
of promising applications in the elds of industrial catalysis,
organic synthesis and biosensors if various types of enzymes
and metal nanoparticles employed.
Cu activity
À1
À1
Catalyst
Enzyme activity
k (min
)
TOF (min )
CALB@Cu
CALB&PVP@Cu
63%
82%
0.65
1.29
56
126
Acknowledgements
This work was supported by the National High Technology
Research and Development Program (“863” Program) of China
reaction rate constant (k) was determined from the absolute under the grant number of 2014AA020507, the National Natural
value of the slope (Fig. 4b).
Science Foundation of China under the grant number
For the cascade reactions catalyzed by CALB&PVP@Cu and 51573085, the Beijing Natural Science Foundation under the
CALB@Cu, the reaction rate constants (k) for the second step grant number of 2144050 and the Tsinghua University Initiative
À1
(
reduction reaction) were calculated as 1.29 min and 0.65 Scientic Research Program under the grant number of
À1
min respectively. As a control, free CALB without Cu nano- 20131089191.
particles didn't show any activity for the reduction reaction. In
addition, the same reaction catalyzed by the mixture of CALB
and normal copper powder gave a reaction rate constant of 0.17
min . As control, CALB@CP and CP (copper phosphate crys-
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