A new nanobiocatalytic system based on allosteric effect with dramatically enhanced enzymatic performance

Article abstract of DOI:10.1021/ja3120136

We report a rational design of CaHPO₄-α-amylase hybrid nanobiocatalytic system based on allosteric effect and an explanation of the increase in catalytic activity when certain enzymes are immobilized in specific nanomaterials. Employing a calcification approach in aqueous solutions, we acquired such new nanobiocatalytic systems with three different morphologies, i.e., nanoflowers, nanoplates, and parallel hexahedrons. Through studying enzymatic performance of these systems and free α-amylase with/without Ca²⁺, we demonstrated how two factors, allosteric regulation and morphology of the as-synthesized nanostructures, predominantly influence enzymatic activity. Benefiting from both the allosteric modulation and its hierarchical structure, CaHPO₄-α-amylase hybrid nanoflowers exhibited dramatically enhanced enzymatic activity. As a bonus, the new system we devised was found to enjoy higher stability and durability than free α-amylase plus Ca²⁺.

Full text of DOI:10.1021/ja3120136

Communication
pubs.acs.org/JACS
A New Nanobiocatalytic System Based on Allosteric Effect with
Dramatically Enhanced Enzymatic Performance
†
†,‡
†
†
†
,†,§
Liang-Bing Wang, You-Cheng Wang, Rong He, Awei Zhuang, Xiaoping Wang, Jie Zeng,*
and J. G. Hou†
†
‡
§
Hefei National Laboratory for Physical Sciences at the Microscale, School of the Gifted Young, and Department of Chemical
Physics, University of Science and Technology of China, Hefei, Anhui 230026, P. R. China
*
S Supporting Information
nanomaterials during the synthesis of the nanobiocatalyst. Less
frequently, there are occasions which concerns activity retaining
of a majority of activity or even activity enhancement after
ABSTRACT: We report a rational design of CaHPO -α-
4
amylase hybrid nanobiocatalytic system based on allosteric
effect and an explanation of the increase in catalytic activity
when certain enzymes are immobilized in specific
nanomaterials. Employing a calcification approach in
aqueous solutions, we acquired such new nanobiocatalytic
systems with three different morphologies, i.e., nano-
flowers, nanoplates, and parallel hexahedrons. Through
studying enzymatic performance of these systems and free
3c,5
immobilization.
This apparently limits the possibility to
apply these nanobiocatalytic systems. Recently, an encouraging
breakthrough in preparation of immobilized enzymes with
greatly enhanced activities was achieved by Zare and co-
6
workers. They reported the synthesis of hybrid hierarchical
nanostructures comprising of copper phosphate (Cu (PO ) )
3
4 2
and enzymes via a coprecipitation method. Their success is in
part built around the environment (the synthesis was
conducted in an aqueous buffer solution without any organic
solvent involved) and high surface area of hierarchical
nanostructures. However, the mechanism exactly how activity
of immobilized enzymes improved has not been amply
interpreted and validated.
2
+
α-amylase with/without Ca , we demonstrated how two
factors, allosteric regulation and morphology of the as-
synthesized nanostructures, predominantly influence enzy-
matic activity. Benefiting from both the allosteric
modulation and its hierarchical structure, CaHPO -α-
4
amylase hybrid nanoflowers exhibited dramatically en-
hanced enzymatic activity. As a bonus, the new system we
devised was found to enjoy higher stability and durability
In biochemistry, allosteric effect denotes an interesting
phenomenon that binding of a ligand to one site (which we
designate as the allosteric site) on a protein molecule indirectly
manipulates the properties of another specific site (the
functional site and in enzymes called the active site) on the7
same protein as a consequence of conformational changes.
Some enzymes are allosteric proteins, and their activity is
regulated through the binding of an effector to an allosteric site.
An example of allosteric modulation is the heme−heme
interaction of hemoglobin in which heme is the allosteric or
2
+
than free α-amylase plus Ca .
anobiocatalysis, in which enzymes are immobilized in/on
nanostructured materials, has attracted ever increasing
N
attention due to its potential applications related to proteomic
1,2
analysis, antifouling, biofuel cells, and tissue engineering. In
general, enzyme immobilization is hopefully expected to result
in an increase in enzyme stability, which allows the industrial
reuse of enzymes for more reaction cycles and thus a massive
implementation of them in sustainable chemical processes.
Having manifested great efficiency in manipulating the local
environment of immobilized enzymes, nanomaterials, including
nanoporous silica, electrospun nanofibers, magnetic nano-
particles, carbon nanotubes, and polymer beads, serve as
competitive candidates in the immobilization of enzymes and
8
active site of an adjoining protein subunit. When oxygen
molecules, the effectors, attach to hemes, a conformational
change arises in those subunits which then interact with the
remaining active sites to promote oxygen affinity. Aside from
3
+
2+
2+
oxygen, metal ions, such as Fe , Ca , and Zn , are also known
9
to be effectors of various enzymes.
Here, we aim to establish a rational design of a nano-
biocatalytic system based on allosteric effect to cast light on the
mechanism of how enzymatic activity increases when
immobilized in nanomaterials. We selected α-amylase which
is capable of hydrolyzing α-bonds of α-linked polysaccharides,
such as starch and glycogen, yielding glucose and maltose to
3
many other areas of enzyme technology. Thanks to the efforts
from a number of research groups, nanomaterials have been
employed to effectively immobilize enzymes through a number
of strategies involving the “entrapment” approach and “single
10
2
,4
investigate this phenomenon. It manifests the allosteric effect
enzyme nanoparticles” technique. Most of these immobilized
enzymes exhibited enhanced stability compared to free
enzymes in solution. Nevertheless, their activities were reduced
after immobilization, which can be ascribed to three facts: (i)
the use of organic solvents (i.e., hexane) during the process of
immobilization, which inactivated part of the enzymes; (ii) the
significant mass transfer of substrate for the immobilized
enzymes; and (iii) the chemical reactions between proteins and
2
+
11
when interacted with Ca in aqueous solutions. In the
absence of Ca2+ in an aqueous solution, most α-amylases are
“
inactive” with the functional sites inhibited, whereas only a few
of them are “active” with the functional sites enabled. As shown
Received: December 10, 2012
Published: January 14, 2013
©
2013 American Chemical Society
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Journal of the American Chemical Society
Communication
2
+
in Figure 1A, with the involvement of Ca , allosteric sites
topographically separated from functional sites are combined
Figure 2. SEM images of (A) nanoflowers, (B) nanoplates, and (C)
parallel hexahedrons. (D) XRD patterns of nanoflowers, nanoplates,
and parallel hexahedrons, respectively (from top to bottom).
±
0.5 μm exhibit explicit hierarchical structure, and thickness of
the petals was only in the range of 20−25 nm (Figure S1B).
Further observation proved that all the petals were smooth with
no pores or rifts appearing on their surfaces. Figure 2B
characterized parallelogrammic nanoplates (∼500 nm in width
and ∼30 nm in thickness) we produced with a relatively high
concentration of PBS (≥15 mM). Finally, Figure 2C clearly
demonstrated parallel hexahedrons mixed with fragments which
were formed in the presence of a relatively high concentration
Figure 1. (A) Ca2+ binds to allosteric site in inactive α-amylase and
generates active α-amylase. (B,C) α-amylase immobilized in thinner or
thicker CaHPO4 nanocrystals. The ratio of embeded α-amylase
molecules to exposed ones increased with the thickness of CaHPO4
nanocrystals.
with Ca2+ ions, thus exciting previously inactive functional sites.
2
+
In other words, Ca acts as a switch to tune the catalytic
activity of α-amylase. This effect gave us the vital spark to
employ a calcification process to immobilize α-amylase in
of α-amylase (2 mg/mL). To corroborate the CaHPO -α-
4
amylase hybrid structures, we implemented XRD analysis
(Figure 2D). All the diffraction peaks can be indexed to the
calcium hydrogen phosphate (CaHPO ). Owing to positive
monoclinic CaHPO
the EDX analysis (Figure S2) of nanoflowers (as an example)
agreed well with the components of CaHPO ·2H O. We also
4 2
·2H O (JCPDS 72-0713). Furthermore,
4
2
+
modulation of the allosteric effect induced by Ca as effectors,
the immobilized α-amylase stays in an active form. Evidently,
the morphological characteristics of the nanocrystals, such as
thinness, interfere with the efficiency of the nanobiocatalytic
system, as illustrated in Figure 1B,C. A certain amount of α-
4
2
instanced TGA of nanoflowers to shape a clearer image of the
components of these systems. It is revealed that the weight
percentage of organic component (i.e., α-amylase) of the
untreated nanoflowers was 20.16% (Figure S3), signifying an
amylases was contained in CaHPO instead of being exposed
4
on the surface, considerably reducing the probability of
substrates to bind to active sites due to mass transfer limitation
and therefore diminishing overall catalytic activity. We
effective hybridization of CaHPO with α-amylase. The
4
encapsulation efficiency (defined as the ratio of the amount
of immobilized enzyme to the total amount of enzyme
employed) of α-amylase in nanoflowers, nanoplates, and
parallel hexahedrons was determined to be 96%, 95%, and
97%, respectively, by measuring the concentration of free α-
amylase in the supernatant of the solution using the Branford
eventually obtained CaHPO -α-amylase hybrid nanostructures
4
with diverse configuration, including nanoflowers, nanoplates,
and parallel hexahedrons, and then examined catalytic proper-
ties of these systems along with those of free α-amylases with/
without Ca2 in detail. The immobilized α-amylase exhibited
substantially enhanced enzymatic activity dependent on
morphology. We reached a point that both the allosteric effect
and morphology notably prominently influence catalytic
efficiency. The nanobiocatalytic systems we devised create a
feasible means of studying allosteric effects in the immobiliza-
tion of enzymes.
12
+
protein assay (see SI Experimental section).
To illuminate the impact of allosteric regulation and the
morphology of nanosturctures on catalysis, we used the
hydrolysis of 2-chloro-4-nitrophenylmaltotrioside (Cnp-G3)
by α-amylase as a model reaction to evaluate the catalytic
activities of free and immobilized α-amylase. Without catalysts,
Cnp-G3 is colorless. Upon the addition of α-amylase, Cnp-G3
was gradually hydrolyzed to generate 3-chloro-4-hydroxyl-
nitrobenzene (Chn), together with appearance of an
In a standard synthesis, an aqueous solution of CaCl (200
2
mM, 100 μL) was added into 5 mL of phosphate buffered
saline (PBS) solution (3 mM) containing 0.2 mg/mL α-
amylase at pH = 6.8. The reaction was then allowed to proceed
at rt (∼25 °C) for 12 h. By means of regulating the
concentrations of α-amylase, PBS, and CaCl2 (see SI
Experimental section), we accomplished formation of nano-
structures of distinctive morphology. Figure 2 shows SEM
images of the products, successively demonstrating nano-
flowers, nanoplates, and parallel hexahedrons. In Figures 2A
and S1A, the separated nanoflowers with an average size of ∼4
13
absorbance peak at 405 nm (see eq 1).
This phenomenon allows us to monitor the progress or kinetics
of the reaction by colorimetric method. The absorbance (in
positive correlation with the degree of reaction) of different
systems at Chn peak along time is demonstrated in Figure 3A.
1
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All the facts and statistics above proved that Ca2+ favors
catalytic activity, which can be accredited to allosteric
regulation, as mentioned beforehand. It is also confirmed that
immobilization of α-amylase in CaHPO strongly associates
4
with catalytic activity. In fact, when concentrations of α-amylase
2
+
were kept the same, two factors, the action of Ca (or the
allosteric effect it induces) and morphology of the structures,
dominate the system, as they constitute variables in our
experimental design. First, because α-amylase, like a variety of
proteins or enzymes, is endowed with the ability of allosteric
interaction, both addition of Ca2+ into free α-amylase and
immobilization of α-amylase in CaHPO nanocrystals lead to
4
allosteric effect where Ca2+ functions as effectors and bind to
specific functional sites. After this combination, α-amylase
experiences conformational changes and remains in the “active
form”. Conceivably, immobilization is seized at a higher
conversion of the inactive α-amylase to active α-amylase than
addition of Ca2+ directly into free α-amylase. On the basis of a
same total amount of enzyme, immobilization seems to hold
more α-amylase in “active form”. For that reason, the three
systems, nanoflowers, nanoplates, and parallel hexahedrons, are
primarily supposed to earn a more preferable activity than free
α-amylase with Ca2+ (although parallel hexahedrons make an
exception). Second, morphology is also an important factor that
contributes to the differences in activity. Nanoflowers and
nanoplates obviously enjoyed higher surface-to-volume ratio
than parallel hexahedrons as a result of less significant mass
transfer limitations. In consequence, a larger portion of
immobilized α-amylase rests on the surface (Figure 1B,C),
having a bigger possibility to meet the substrate molecules. On
the contrary, parallel hexahedrons conceal a great part of
immobilized α-amylase and eventually prevent them from
acting as enzymes. In our experiment, the compensation paid
by immobilization even fails to cover the hiding of α-amylase.
The reason why nanoflowers exceed nanoplates in activity
lies in that although nanoplates approximates petals of the
nanoflowers in thinness, they have a greater likelihood to self-
assemble, which is disastrous to activity. When they overlap,
nanoplates virtually lower the ratio of immobilized α-amylase
on the surface. However, nanoflowers consist of many
dispersive petals that expel rather than connect to each other
(Figure S1). They hold higher catalytic activity at a lower cost
of enveloping α-amylase inside the nanostructure. These
inspiring results indicate characteristic properties of nano-
biocatalytic systems with high catalytic activity: large surface-to-
volume ratio and hierarchical or isolated structure without
assembling.
Figure 3. (A) Absorbance at the peak position for Chn (405 nm) as a
function of time for six different catalytic systems. The concentration
of Cnp-G3 was 0.47 mM for each system. (B) Plots of −ln I vs time
sub
for five catalytic systems. The reaction rate constants (k) determined
−
3
−3
−3
−3
from these plots are 16.5 × 10 , 8.0 × 10 , 4.5 × 10 , 1.2 × 10 ,
−4
−1
and 4.4 × 10
s
for nanoflowers, nanoplates, free α-amylase with
2+
Ca , parallel hexahedron, and free α-amylase, respectively. (C)
Relative activity of nanoflowers over the course of eight rounds of
successive reaction. (D) Storage stabilities of nanoflowers and free α-
amylase in PBS (pH = 6.8) at rt.
The plot lies on the premise of setting equivalent initial
concentrations of Cnp-G3 to 0.47 mM. Without the addition of
free or immobilized α-amylase, no reaction was observed,
affirming that hydrolysis reaction was unable to occur by itself
under the experimental conditions. Upon the addition of free α-
2
+
amylase (0.04 mg/mL) without Ca , absorbance at 405 nm
2
+
increased over time. Then again, adding Ca (20 μL of 100
mM CaCl solution) to free α-amylase (0.04 mg/mL) brought
2
about an even higher absorbance. In CaHPO -α-amylase
4
systems (the concentrations of α-amylase put in were all 0.04
mg/mL), absorption enhanced significantly. Among them, the
catalytic reaction rate of nanoflowers ranked the first,
nanoplates the second, and parallel hexahedrons the last.
Compared to free α-amylase, nanoflowers and nanoplates
displayed stronger while parallel hexahedrons weaker absorb-
ance.
Note that in the reaction system, the concentration of H O
2
greatly exceeded that of Cnp-G3, so it is not unreasonable to
consider its concentration as a constant during the reaction.
The pseudofirst-order kinetics with respect to Cnp-G3 could be
applied to our experimental system. The approximately linear
shape of the plot of −ln I (Isub is the value obtained by
The attractive catalytic activity of CaHPO -α-amylase hybrid
sub
4
subtracting the real-time absorbance from the saturated one,
i.e., 0.26) vs time, as shown in Figure 3B, supports the
pseudofirst-order assumption. Based on the linear relationship,
the average reaction rate constants (k) were calculated to be 4.4
nanoflowers makes it plausible to use them as a nanobiocatalyst.
To substantiate this, their stability and durability were tested.
Figure 3C shows the plot of relative activity against the number
of successive catalytic reactions that repeatedly used the α-
amylase-incorporated nanoflowers as the catalyst. It is clear that
the nanoflowers only lost 16% of their catalytic activity over the
course of eight rounds of reaction, demonstrating a high
durability. As for the stability test (Figure 3D), the free enzyme
(adding Ca2+ when tested) lost 67% of its initial activity within
25 days when incubated in PBS (pH = 6.8) at rt, whereas under
the same condition, nanoflowers maintained most of their
initial activity (∼90%). It is worth pointing out that all the tests
did not result in any obvious morphological change for the
nanoflowers (Figure S5), indicating that the nanoflowers were
highly stable in the chemical environment of the catalytic
−4
−3
−3
−3
−3 −1
×
10 , 4.5 × 10 , 16.5 × 10 , 8.0 × 10 , and 1.2 × 10
s
2
+
2+
when the free α-amylase without Ca /with Ca and those
after incorporation in nanoflowers, parallelogrammic plates, and
parallel hexahedrons were employed as the catalysts,
respectively. To support, we calculated Michaelis constant
14
(K ) of each system (data shown in Figure S4). It was found
m
that nanoflowers undertook the lowest Km value, confirming
that they have a higher efficiency to convert Cnp-G3 into
products. All tests concerning catalysis were performed after
concentrations of α-amylase (whether immobilized or not) in
all systems were adjusted to be identical.
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Journal of the American Chemical Society
Communication
reaction although they have a large surface area and thereby a
high surface energy.
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In conclusion, to investigate the previously observed
phenomenon that certain enzymes exhibited increased activity
after immobilized in specific nanomaterials and to verify the
impact of allosteric regulation, we designed a series of novel
nanobiocatlytic systems, i.e., nanoflowers, nanoplates, and
parallel hexahedrons, the enzymatic activities of which were
(
(
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2
+
factors: allosteric effect of Ca ions with the amine groups of
α-amylase and morphology of nanostructures. Among these
systems, the hierarchically structured hybrid nanoflowers
exhibited the best enzymatic performance. Our design also
provides a convenient and environmentally benign route to
large-scale production because the process does not involve
high temperature or organic solvents. Plus with the
biocompatibility of CaHPO4 and the high stability and
durability, this new biocatalytic system based on allosteric
effects is promising to find widespread use in applications
related to biomedicine, biofuel cells, biosensor, and tissue
engineering.
(
10) Uitdehaag, J. C. M.; Mosi, R.; Kalk, K. H.; van der Veen, B. A.;
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ASSOCIATED CONTENT
Supporting Information
Experimental details, SEM images, EDX spectrum, and TGA
data. This material is available free of charge via the Internet at
http://pubs.acs.org.
■
*
S
AUTHOR INFORMATION
Corresponding Author
zengj@ustc.edu.cn
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
This work was supported by MOST of China
2011CB921403), NSFC under grant nos. 21203173 and
■
(
2
1121003, 51132007 and J1030412, and CAS. J.Z. also thanks
the University of Science and Technology of China for the
startup funds.
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