Cascade reactions in an all-enzyme nanoreactor

Article abstract of DOI:10.1002/chem.200902063

The composed of horseradish peroxidase-polystyrene (HRP-PS) and horseradish peroxidase-poly (methyl methacrylate) (HRP-PMMA) giant amphiphiles, which contain a guest enzyme (glucose oxidase, GOx) inside the self- assembled structures has been reported. The polymer can be attached to the cofactor, the protoporphyrin IX (PPIX), either by the use of an initiator-modified derivative followed by polymerization or by the coupling of an acetylene end-capped polymer to an azido-derived cofactor by click chemistry. The heme-PMMA-based giant amphiphile gave spherical aggregates with diameters of 50-100 nm. Enzymatic activities were measured by using an ABTS/H₂O₂ assay. The cofactor reconstitution appears to be the technique of choice, since the same cofactor can serve for the reconstitution of several different apoproteins and thus different combinations inside the shell can also be envisioned.

Full text of DOI:10.1002/chem.200902063

DOI: 10.1002/chem.200902063
Cascade Reactions in an All-Enzyme Nanoreactor
[
a]
[a]
[a, b]
Guillaume Delaittre, Irene C. Reynhout, Jeroen J. L. M. Cornelissen,*
and
[
a]
Roeland J. M. Nolte*
Currently, there is great interest in the development of
[
1]
self-assembled nanoreactors for application in catalysis.
[2]
[3]
For this purpose, liposomes, polymersomes, and even
[4]
virus particles have been employed. In previous papers, we
have shown that nanoreactor systems can be constructed
from a new class of amphiphiles, which are composed of a
protein or enzyme as the head group and a hydrophobic po-
lymer as the tail. For these biohybrid compounds the name
giant amphiphiles was coined (Figure 1a). They can be pre-
pared according to different routes, for example, by making
[5]
use of the biotin–streptavidin couple, by cofactor reconsti-
[6]
tution, or by covalent coupling of the polymer to a specific
location on the surface of the protein. The covalent coupling
can be performed by the attachment of a preformed func-
[7]
tional polymer to the protein (by click chemistry or Mi-
Figure 1. a) Schematic representation of a HRP giant amphiphile. b) A
nanocontainer composed of a bilayer of HRP giant amphiphiles contain-
ing glucose oxidase in the inner aqueous compartment. c) Cascade reac-
tion studied in this paper.
[8]
chael-type addition ) or by growing the polymer directly
from the protein with an attached initiator or transfer
[9]
agent. In particular, controlled radical polymerization is a
very suitable technique for the bioconjugation of polymers
[10]
to proteins.
[11]
Herein, we present our work on nanoreactors that are
composed of horseradish peroxidase–polystyrene (HRP–PS)
and horseradish peroxidase–poly(methyl methacrylate)
based nanoreactor presented very recently, our system has
the advantage of being self-reactive. These all-enzyme as-
semblies are able to catalyze a two-step cascade reaction,
see Figure 1c. Although their activity can be slightly altered
(
HRP–PMMA) giant amphiphiles (Figure 1a), which con-
[12]
tain a guest enzyme (glucose oxidase, GOx) inside the self-
assembled structures (Figure 1b). Compared with a protein-
by this process, encapsulating enzymes in confined spaces
can be of particular interest to protect them from proteases
and to prolong their lifetime by slowing down the denatura-
[13,14]
tion processes caused by aggregation.
[
a] Dr. G. Delaittre, I. C. Reynhout, Prof. J. J. L. M. Cornelissen,
Prof. R. J. M. Nolte
Institute for Molecules and Materials, Radboud University Nijmegen
Heyendaalseweg 135, 6525 AJ Nijmegen (The Netherlands)
Fax : (+31)243-652-143
For the preparation of the giant amphiphiles, we used the
reconstitution method previously described by our group
[6]
(
Scheme 1). Several examples of cofactor reconstitution
are described in the literature showing that, although it is
usually not the case, the activity of the enzyme can some-
E-mail: r.nolte@science.ru.nl
[15]
[
b] Prof. J. J. L. M. Cornelissen
times be enhanced by modification of its cofactor. The po-
lymer can be attached to the cofactor, the protoporphyri-
n IX (PPIX), either by the use of an initiator-modified de-
Present address: Laboratory for Biomolecular Nanotechnology
MESA+ Institute, University of Twente
PO Box 217, 7500 AE, Enschede (The Netherlands)
Fax : (+31)534894645
[16]
rivative followed by polymerization or by the coupling of
an acetylene end-capped polymer to an azido-derived cofac-
E-mail: j.j.l.m.cornelissen@tnw.utwente.nl
[17]
tor by click chemistry.
In the present study, we used a
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.200902063.
third strategy, which is an amidation reaction between an
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Chem. Eur. J. 2009, 15, 12600 – 12603

COMMUNICATION
Figure 2. TEM images showing assemblies of HRP–PS30 giant amphi-
philes without GOx (a) and with encapsulated GOx (b).
Scheme 1. Synthetic route to HRP–polymer giant amphiphiles.
3
a) i) PyBOP/DIPEA, THF/DMF 1:1(v/v); ii) TFA, CH Cl iii) FeCl ,
THF/DMF 1:3 (v/v), 658C; b) THF, apo-HRP in buffer, pH 7.5.
PyBOP=(benzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluoro-
phosphate; DIPEA=diisopropylethylamine, TFA=trifluoroacetic acid.
Previously, we have demonstrated that in the case of
HRP–PS90 the enzymatic activity dropped dramatically after
reconstitution and self-assembly in water. A turnover fre-
A
H
U
G
R
N
U
2
2
;
À1
quency (TOF) of 360 h was measured, compared with
À1
7
6000 h
for the native enzyme under the same con-
[
6a]
AHCTUNGTRENNUNGd itions. To see if the polymer length and the nature of the
polymer had an effect on the HRP activity, we conducted
activity experiments with assemblies of HRP–PS and HRP–
PMMA by using the ABTS/H O assay (ABTS=2,2’-azido-
amino-functionalized polymer and a mono-tert-butyl-pro-
tected PPIX (2; Scheme 1). Three polymers, PS20 (1a), PS30
2
2
(
1b), and PMMA120 (1c) were synthesized by atom transfer
bis(3-ethylbenzthiazoline-6-sulfonic acid)). In this assay the
conversion of ABTS into the green, oxidized radical cation
form is followed by UV/Vis spectroscopy at 420 nm. As pre-
viously observed, the introduction of a polymeric chain onto
the cofactor part of the enzyme partly suppressed the
[18]
radical polymerization (ATRP) by using a protected ester
bromo-initiator and, after deprotection, an a-amino group
was attached to the resulting compounds. These polymers
were subsequently coupled, by an amidation reaction, to 2.
After deprotection and insertion of iron, the heme-function-
alized polymers 3 were reconstituted with HRP to generate
the giant amphiphile 4. The a-heme polymers were thus dis-
solved in THF and injected into an aqueous buffered solu-
tion of apo-HRP. In the case of giant amphiphile 4b, de-
rived from PS , vesicular aggregates with a dark interior
were obtained as observed by TEM (Figure 2a). The aggre-
gates were less well defined for the giant amphiphile 4a,
which was prepared from the shorter heme–PS20 polymer
enzyme activity, however, the smaller polystyrene blocks,
À1
with TOF of 4360 and 1200 h (PS20 and PS , respectively),
30
led to a lesser attenuation of the activity than that of the
larger one (PS ). The HRP–PMMA aggregates displayed a
9
0
[6a]
À1
higher activity (5540 h ) than the HRP–PS aggregates de-
spite the fact that the degree of polymerization of the
PMMA block was larger. Thus, one can assume that this de-
crease in activity, compared with the native HRP, is not only
related to the steric shielding of the active site of the
enzyme by the polymer chains in the aggregates, but also to
the intrinsic nature of the specific polymer acting as a hy-
drophobic barrier to transport. Being less hydrophobic than
their PS counterparts, PMMA chains probably allow for
better access to the active site of the enzyme. Altogether,
these experiments demonstrate that the HRP-containing
nanoaggregates do retain their enzymatic activity. This
30
(
see the Supporting Information). We can assume that the
more hydrophilic nature of 4a is responsible for the less-fa-
vorable aggregation behavior. The heme–PMMA120-based
giant amphiphile 4c gave spherical aggregates with diame-
ters of 50–100 nm. Effective reconstitution of the heme–
polymers with HRP was also proven by UV/Vis spectro-
scopic analysis (see the Supporting Information).
Chem. Eur. J. 2009, 15, 12600 – 12603
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J. J. L. M. Cornelissen, R. J. M. Nolte et al.
stimulated us to use them as catalytically active nanocon-
tainers in which another enzyme can be encapsulated to per-
form a fully enzymatically catalyzed cascade reaction. Con-
sidering the fact that the immobilization of a single enzyme
has already proven to be efficient at performing cascade re-
absence of GOx and the enzyme was then added before di-
alysis to determine whether it adhered to the surface of the
aggregates and to prove its effective encapsulation in the
test experiments. No significant catalytic activity was ob-
served in this case (Figure 3, line e). In a second blank reac-
tion, neither the HRP–polymer conjugate nor GOx were
present in the solution and no conversion of ABTS was
measured at all (Figure 3, line f). In a final blank reconstitu-
tion, we used a Zn-containing heme–PS, instead of an Fe-
containing one, to show that active Fe–heme–HRP is
needed to catalyze the cascade reaction. Again no relevant
activity was measured (Figure 3, line d). When the Fe-con-
taining HRP–polymer and GOx were used together there
was significant formation of oxidized ABTS, which proved
that hydrogen peroxide (formed in the HRP catalytic cycle)
was produced (Figure 3, lines a and b). Since this oxidant
was not present initially and can only be generated during
the catalytic action of GOx on glucose, the formation of oxi-
dized ABTS demonstrated that a cascade reaction had
indeed occurred. Cascade catalysis by using the PMMA-
based assemblies (Figure 3, line a) revealed higher conver-
sion rates than the PS-based ones (Figure 3, line b). This is
without any doubt related to the higher activity of HRP
within the vesicle shell composed of PMMA reported
herein.
[19]
actions with other nonenzymatic catalysts,
we believed
that an all-enzyme nanoreactor would be a significant ad-
vance.
We decided to use GOx as the encapsulated enzyme be-
cause, in the presence of molecular oxygen, it catalyzes the
oxidation of b-d-glucose into d-glucono-1,5-lactone produc-
ing hydrogen peroxide as a byproduct. The latter can then
take part as a reagent in the catalytic action of HRP to pro-
vide a two-enzyme cascade reaction. Our group previously
studied this cascade reaction with encapsulated enzymes by
using polymersomes as containers. We observed that the
substrate was able to pass through the membrane, whereas
[3]
the enzymes were not. To encapsulate GOx into the inner
aqueous compartment of the HRP–polymer nanoreactors,
we performed reconstitution experiments as described
above, but with GOx present in the initial aqueous apo-
HRP solution. Since the highest GOx activity is reached at
pH 6 and the optimal HRP reconstitution is carried out at
pH 7.5, the best conditions for this step had to be deter-
mined. These were found to be the following: reconstitution
of the HRP at pH 7.5, followed by dialysis at pH 7.0
In summary, we have presented a novel approach to con-
struct all-enzyme nanoreactors and to perform enzymatic re-
actions in a confined and protected space. Given the wide
variety of possible enzyme combinations and the fact that
enzyme nanoreactors can be constructed in different ways
(see above), the methodology presented herein opens new
routes to synthesize compounds by enzyme cascade catalysis
in the future. In particular, cofactor reconstitution appears
(
300 kDa cut off) to remove nonencapsulated species. A
TEM picture of the obtained system evidencing the un-
changed nature of the aggregates in the presence of GOx is
shown in Figure 2b.
To perform the cascade reaction, we added glucose and
ABTS to the solution containing the two-enzyme nanoreac-
tors. The reaction was followed by measuring the formation
of the oxidized ABTS species by UV/Vis spectroscopy. Sev-
eral experiments were carried out to establish the occur-
rence of a stepwise catalytic cascade reaction (Figure 3). In
each experiment (both tests and blanks), UV/Vis spectrosco-
py confirmed that reconstitution had effectively taken place
[15]
to be the technique of choice, since the same cofactor can
serve for the reconstitution of several different apoproteins
and thus different combinations inside the shell can also be
envisioned.
(
not shown). A blank reconstitution was carried out in the
Experimental Section
Synthesis of HRP-functionalized polymers 4 by the cofactor reconstitu-
tion method: Reconstitutions were carried out at 228C by injecting a so-
À5
lution of the modified cofactor in THF (1.0 mL, 4.4ꢁ10 mmol) into a
glass vial containing an aqueous phosphate-buffered solution (10 mL,
2
1
0 mm, pH 7.5) and a 2.5-fold excess of the HRP apoenzyme (1.1ꢁ
À4
0
mmol). The solutions were stirred vigorously for four days. The
excess apoenzyme was removed by using a 100 kDa dialysis bag, which
allows the enzyme (18 kDa for HRP) and the cofactors to pass through,
while retaining any aggregates formed by the modified enzymes.
Enzymatic activity test: Enzymatic activities were measured by using an
ABTS/H O assay. An aqueous solution of H O (12 mL, 0.07%) was
2 2 2 2
added to a solution of ABTS in phosphate buffer (20mm, pH 7) (400 mL,
À5
2
.3ꢁ10 mm). Subsequently, the respective reconstituted mixture (10 mL)
2 2
was brought to room temperature and added to the ABTS/H O mixture,
upon which the enzymatic activity was immediately monitored for 20 min
by using UV/Vis spectroscopy (l=420 nm). The enzymatic activity of
apo-HRP and HRP were taken as references. To study the catalytic activ-
ity of the modified cofactors in aqueous solution in the absence of apo-
HRP, a solution of the cofactor in THF (10 mL) was injected into an
Figure 3. Activity curves of HRP–GOx cascade reactions and blank ex-
periments. a) PMMA120-HRP; b) PS30-H30-HRP; c) no polymer/no GOx;
d) Zn-containing polymer; e) PS30-HRP/no GOx; f) no polymer/no
HRP.
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Chem. Eur. J. 2009, 15, 12600 – 12603

Cascade Reactions in an All-Enzyme Nanoreactor
COMMUNICATION
aqueous phosphate-buffered solution (10 mL). The modified cofactors
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did not show any catalytic activity, as determined from the ABTS/H
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2 2
O
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À6
À4
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[
[
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57 mL, 1.8 mm), in a phosphate buffer (771 mL) was added to a solution
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lowed by UV/Vis spectroscopy; quantitative comparison is possible be-
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6
Keywords: bioconjugates · cascade reactions · cofactors ·
enzyme catalysis · vesicles
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Received: July 24, 2009
Published online: October 26, 2009
Chem. Eur. J. 2009, 15, 12600 – 12603
ꢀ 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
12603