Lipase polystyrene giant amphiphiles

Article abstract of DOI:10.1021/ja017809b

A new type of giant amphiphilic molecule has been synthesized by covalently connecting a lipase enzyme headgroup to a maleimide-functionalized polystyrene tail (40 repeat units). The resulting biohybrid forms catalytic micellar rods in water. Copyright

Full text of DOI:10.1021/ja017809b

Published on Web 03/29/2002
Lipase Polystyrene Giant Amphiphiles
Kelly Velonia, Alan E. Rowan, and Roeland J. M. Nolte*
Department of Organic Chemistry, NSR Center, UniVersity of Nijmegen, ToernooiVeld 1,
NL-6525 ED Nijmegen, The Netherlands
Received December 19, 2001
Amphiphilic molecules, often also called surfactants because of
their surface active properties, have been the subject of intensive
studies for many years.1 As part of our studies on the design of
new building blocks for the construction of nanosized self-
assembled systems we have developed a new class of amphiphilic
molecules, that is, giant amphiphiles, which consist of an enzyme
headgroup and a single covalently connected hydrophobic polymeric
tail (Figure 1).2
In the case of low-molecular weight amphiphiles (e.g., phos-
pholipids) studies have shown that the shape of the individual
amphiphilic molecule very often determines the structure of the
resulting aggregate.1 Similar rules with respect to shape and
structure seem to hold for the more recently introduced class of
super-amphiphiles,3 which are composed of hydrophilic-hydro-
phobic diblock copolymers. These block copolymers have also been
shown to form highly ordered aggregates with morphologies
dependent on the block lengths or the presence of inorganic salts4
or both. In terms of both molecular volume and molecular weight
the here-reported giant amphiphiles are considerably larger than
their low-molecular weight and polymeric counterparts. We thought
it to be of interest to investigate whether this next generation of
amphiphiles would behave in the same manner as their super- and
low-molecular weight analogues and also form well-defined as-
semblies in aqueous solution.
In the past a wide variety of polymer/enzyme adducts have been
synthesized by more or less randomly coupling an enzyme to a
polymeric matrix. To the best of our knowledge the synthesis of a
precisely defined, nearly monodisperse enzyme-polymer hybrid,
aimed at developing surfactants, has not been reported before. In
the following we describe such a hybrid, derived from a lipase and
polystyrene, and report on its self-assembling properties in water.
The enzyme headgroup chosen was the lipase B from Candida
antarctica (CAL B, EC 3.1.1.3, triacylglycerol hydrolase).5,6 This
well-studied 33 kDa lipase catalyzes in aqueous solutions the
hydrolysis of esters, and in anhydrous organic solvents, the reverse
esterification reaction.7 CAL B is very stable even in various organic
solvents and at high temperatures. A maleimide-functionalized
polystyrene of 40 repeat units (PDI ) 1.04) was used as the
hydrophobic tail. The specific attachment of this polymer to the
enzyme was achieved by functionalizing the single disulfide bridge
(Cys293-Cys311), which is exposed on the outer surface of the
native lipase. The first step of the synthesis of the giant amphiphile
was the complete reduction of this disulfide bridge with dithio-
threitol (DTT), which was confirmed by titrating the reduced
enzyme with Ellman’s reagent (see Scheme 1, Supporting
Information).8-12 To study the coupling of maleimides to the
resultant free thiol groups, the reduced CAL B (0.25 mM) was
incubated with an excess of maleimide (0.6 mM maleimide in 20
Figure 1. Computer-generated models of (a) a phospholipid representing
the class of molecular amphiphiles (molecular volume ∼0.5 nm3, molecular
weight ∼1 kDa), (b) a diblock polymer of polystyrene and a polyisocyano-
peptide representing the class of super-amphiphiles (molecular volume ∼6.5
nm3, molecular weight ∼6 kDa), and (c) the lipase-polystyrene (n ) 40)
biohybrid representing the new class of giant amphiphiles (molecular volume
∼25 nm3, molecular weight ∼40 kDa).
mM phosphate buffer, pH 6.8). Titration of the product with
Ellman’s reagent revealed the presence of one remaining free thiol
group, indicating the coupling of only one maleimide per lipase
molecule (see Figure 1, Supporting Information).13
The coupling of the maleimide-functionalized polystyrene (0.05
mM, prepared from the carboxy-terminated polystyrene (n ) 40)
by reaction with SOCl2 and then with maleimide and base, see
Supporting Information) to the reduced protein (0.03 mM) was
initially performed in aqueous solution (100 mM phosphate buffer,
pH 6.8, containing 2 mM EDTA and 150 mM NaCl); however,
only ill-defined architectures were observed by transmission electron
microscopy (TEM, see Figure 3, Supporting Information).14
To obtain more defined structures and carry out the coupling in
a more controlled manner, the reaction was carried out at the air-
water interface using a Langmuir-Blodgett trough. A chloroform
solution of the maleimide-funtionalized polystyrene was spread at
the air-water interface, and the reduced enzyme was added to the
sub-phase. The reaction was allowed to proceed for 4 h. After
compression of the monolayer the pressure-surface area isotherm
of the reaction mixture revealed a lift-off area of ∼23 nm2 (Figure
2, III). When compared to the lift-off areas of the native lipase (12
nm2, Figure 2, I) and the maleimide-functionalized polystyrene (∼5
nm2), this increase of molecular area is in agreement with the
formation of the giant amphiphile. A blank experiment using
carboxy-terminated polystyrene and native CAL B was also
performed to ascertain that the lift-off area increase was not due to
nonspecific adsorption of the protein to the polymer monolayer.
(This experiment resulted in a lift-off area of 7.5 nm2, Figure 2,
II).
A coupling reaction between nonaggregated components was
subsequently tried by using a THF/water mixture (90/10 v/v). It
has been previously reported that polystyrene (n ) 40) remains
* To whom correspondence should be addressed. Fax: (+31) 24-3652929.
E-mail: nolte@sci.kun.nl.
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J. AM. CHEM. SOC. 2002, 124, 4224-4225
10.1021/ja017809b CCC: $22.00 © 2002 American Chemical Society

C O M M U N I C A T I O N S
hydrophobic polystyrene tail, which is attached without the help
of a hydrophilic spacer. In addition, upon aggregation of the giant
amphiphiles from fibers to bundles of fibers, fewer active sites of
the enzyme molecules will remain accessible to substrate mol-
ecules.18
In conclusion, we have shown that monodispersed and precisely
defined giant amphiphiles consisting of an enzyme covalently
connected to a single hydrophobic polymeric tail can be prepared
and induced to self-assemble in a fashion similar to that of low-
molecular weight amphiphiles. Further studies are directed at
investigating the influence of different polymer tails (i.e., poly-
styrenes of different lengths with different hydrophilic spacer groups
for attachment to the enzyme) upon the activity and the self-
assembling behavior of the giant amphiphiles.
Figure 2. Surface pressure/surface area isotherms recorded in a Langmuir-
Blodgett trough using a subphase containing 20 mM phosphate buffer (pH
6.8), T ) 20 °C. (I) CAL B, (II) reaction mixture of polystyrene-maleimide
and native CALB, (III) the giant amphiphile formed in the monolayer, and
(IV) the giant amphiphile formed in THF/water system.
Acknowledgment. This research was supported by the EC TMR
Sisitomas and Smarton programs. We thank Dr. J. J. L. M.
Cornelissen and Dr. N. A. J. M. Sommerdijk for initial discussions
and Novo Nordisk A/S for the generous donation of the lipase.
Supporting Information Available: Experimental Section (PDF).
This material is available free of charge via the Internet at
http://pubs.acs.org.
References
(1) Feiters, M. C.; Nolte, R. J. M. AdVances in Supramolecular Chemistry;
Gokel, G. W., Ed.; Jai Press Inc.: Stamford CT, 2000; Vol. 6, pp 41-
156.
(2) Hannink, J. M.; Cornelissen, J. J. L. M.; Farrera, J. A.; Foubert, P.; De
Schryver, F. C.; Sommerdijk, N. A. J. M.; Nolte, R. J. M. Angew. Chem.,
Int. Ed. 2001, 40, 4732-4734.
Figure 3. TEM images (Pt shadowing) of the aggregates obtained after
mixing reduced CAL B in water with maleimide-terminated polystyrene,
in THF/water (9/1 v/v) mixture (A, B). The insert (B) shows µm long
bundles of fibers, with the smallest fiber (C) having a diameter of ∼ 25
nm corresponding to a micellar rod (D).
(3) Shei, H.; Eisenberg, A. Angew. Chem., Int. Ed. 2000, 39, 3310-3312.
(4) (a) Gitsov, I.; Wooley, K. L.; Fre´chet, J. M. J. Angew. Chem., Int. Ed.
Engl. 1992, 31, 1200. (b) Cornelissen, J. J. L. M.; Fisher, M.; Sommerdijk,
N. A. J. M.; Nolte, R. J. M. Science 1998, 280, 1427-1430. (c) Hest, J.
C. M. v.; Baars, M. W. P. L.; Delnoye, D. A. P.; Genderen, M. H. P. v.;
Meijer, E. W. Science 1995, 268, 1592-1595.
monodispersed in THF solutions with up to 10% water content.15
The reaction was performed in a THF solution of maleimide-
polystyrene (0.03 mM) to which the reduced enzyme in water (0.015
mM, 100 mM phosphate buffer, pH 6.8, 2 mM EDTA, 150 mM
NaCl) was added to give a final water content of 8%. The reaction
was allowed to proceed overnight, and then additional water was
slowly added over a period of ∼2 h.16 TEM studies of the resulting
reaction mixture revealed the presence of well-defined µm-long
fibers (Figure 3A,B). Closer examination of these fibers showed
that they were built up from bundles of rods, with the smallest rod
having a diameter between 25 and 30 nm (Figure 3C). The size of
the smallest rod corresponds closely to the predicted diameter of a
micellar architecture formed by the self-assembly of the giant
amphiphile (Figure 3D).17 It is remarkable that these giant am-
phiphiles exhibit self-assembling properties similar to those of their
low-molecular weight counterparts.
These giant amphiphiles were also spread at the air-water
interface of a Langmuir-Blodgett trough. The ∼28 nm2 lift-off
area measured after compression of the monolayer (Figure 2, IV),
further verifies the formation of the biohybrids.
Preliminary catalytic studies were carried out on the self-
assembled fibers using the pro-fluorescent substrate 6,8-difluoro-
4-methylumbelliferyl octanoate (DiFMU-octanoate). The activities
of the native lipase and the reduced lipase were also determined
for comparison (see Supporting Information). The lipase retained
∼70% of its initial activity after the disulfide reduction step. In
contrast, the fibers exhibited only 6-7% of this activity. This
decrease in activity can probably be ascribed to destabilization of
the active conformation of the enzyme due to the presence of the
(5) (a) Schulze, B.; Wubbolts, M. G. Curr. Opin. Biotechnol. 1999, 10, 609-
615. (b) Schmid, R. D.; Verger, R. Angew. Chem., Int. Ed. 1998, 37,
1608-1633.
(6) Uppenberg, J.; Hansen, M. T.; Patkar, S.; Jones, T. A.; Structure (London),
1994, 2, 293-308.
(7) Rotticci, D.; Ottosson, J.; Norin, T.; Hult K. Methods in Biotechnology;
Halling, P., Vulfson, E., Woodley, J., Holland, B., Eds.; Humana Press:
Totowa, NJ, 2001; Vol. 15, pp 261-276.
(8) The native enzyme contains additional disulfide bridges, which are buried
in the inner shell.
(9) Ellman, G. L. Arch. Biochem. Biophys. 1959, 82, 70-77.
(10) Trost, B. M.; Kallander, L. S. J. Org. Chem. 1999, 64, 5427-5435.
(11) Kempin, U.; Hennig, L.; Knoll, D.; Welzel, P.; Muller, D.; Markus, A.;
Heijenoort J. v. Tetrahedron 1997, 53, 17669-17690.
(12) Thionitrobenzoate (TNB-), which is formed upon reaction of Ellman’s
reagent with a sulfhydryl group, has an intense absorption band at 412
nm (ꢀ ) 14150 M-1 cm-1) which allowed an accurate detection of the
free sulfhydryls after the reduction step (see Supporting Information).
(13) The addition of only one maleimide is thought to be the result of steric
hindrance.
(14) Unlike in the case of low-molecular weight amphiphiles where sonification
is often used to aid self-aggregation, this approach could not be used for
these molecules, since sonification destroys the enzyme.
(15) Yu, Y.; Zhang, L.; Eisenberg, A. Macromolecules 1998, 31, 1144-1154.
(16) The formation of the giant amphiphile was confirmed by SEC chroma-
tography, electrophoresis, and UV titrations (see Supporting Information).
(17) Calculations using the Israellachvilli’s rules predict for the assembly a
spherical micellar architecture (P ) 0.04). These calculations, however,
do not allow one to discriminate between spherical and rodlike micelles
because the precise volume of the polystyrene tail and the diameter of
the enzyme group are not known.
(18) According to simple calculations, if a hexagonal packing is assumed, seven
rods when assembled have only 40% of their surface area accessible to
substrate molecules. If larger assemblies are formed, this value drops
further. Hence, it is not unreasonable to assume that upon aggregation
the catalytic activity drops considerably.
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