Enzyme-based nanoscale composites for use as active decontamination surfaces

Article abstract of DOI:10.1002/adfm.200901388

Perhydrolase S54V (AcT) effectively catalyzes the perhydrolysis of propylene glycol diacetate (PCD) to generate peracetic acid (PAA). PAA is a potent oxidant used for sanitization and disinfection, with broad effectiveness against bacteria, yeasts, fungi, and spores. In this study, active and stable composites are developed by incorporating AcT-carbon nanotube conjugates into polymer and latex-based paint At a conjugate loading of 0.16% (w/v), the composite generated 11mM PAA in 20 min, capable of killing more than 99% spores initially charged at 10⁶ colony-forming units per milliliter.

Full text of DOI:10.1002/adfm.200901388

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Enzyme-Based Nanoscale Composites for Use as Active
Decontamination Surfaces
By Cerasela Zoica Dinu, Guangyu Zhu, Shyam Sundhar Bale, Gaurav Anand,
Philippa J. Reeder, Karl Sanford, Gregg Whited, Ravi S. Kane,* and
Jonathan S. Dordick*
corrosive sulfuric acid in the product are
high.[10] As an alternative to chemical
synthesis, several biocatalytic routes have
been derived, including the use of lipases,
esterases, and cholinesterases. These
enzymes catalyze perhydrolysis of acyl
substrates in the presence of H2O2 to
generate peracids under mild reaction
conditions.[11–13] Lipases in particular are
well known to generate peracids in non-
aqueous media, which are then able to
oxidize alkenes stoichiometrically to gen-
Perhydrolase S54V (AcT) effectively catalyzes the perhydrolysis of propylene
glycol diacetate (PGD) to generate peracetic acid (PAA). PAA is a potent
oxidant used for sanitization and disinfection, with broad effectiveness
against bacteria, yeasts, fungi, and spores. In this study, active and stable
composites are developed by incorporating AcT–carbon nanotube conjugates
into polymer and latex-based paint. At a conjugate loading of 0.16% (w/v), the
composite generated 11 mM PAA in 20 min, capable of killing more than 99%
spores initially charged at 106 colony-forming units per milliliter.
erate epoxides and peracids.[14–16] For example, Novozyme 435
(lipaseBfromCandidaantarcticaimmobilizedonacrylicresin)has
been used to generate peracids either by direct synthesis from
carboxylic acids and H2O2 or by perhydrolysis of carboxylic acid
esters.[17] The enzymatically produced PAA was found to have
sporicidal activity similar to that of commercial PAA. However, the
major drawback of using hydrolases is their very low perhydrolytic
activity in aqueous solutions and fast deactivation by high
concentrations of H2O2 and the resulting PAA.
Significant efforts have been made to address the generation of
PAA from an aqueous environment through identification of
perhydrolases with greater reactivity on H2O2 than on water as the
acyl acceptor. In particular, perhydrolase S54V (denoted as AcT)
from Mycobacterium smegmatis is active on various acyl donor
substrates and exhibits a perhydrolysis to hydrolysis ratio greater
than one. This results in perhydrolase activity 50-fold higher than
that of the best lipase tested.[18,19]
1. Introduction
Peracetic acid (PAA) is a potent oxidant that exhibits excellent and
rapid disinfection activity against a broad spectrum of pathogens,
such as bacteria, yeasts, molds, fungi, and spores.[1–3] As a
disinfectant PAA is more effective and is needed at lower
concentrations than H2O2,[4] while as a sanitizer PAA has been
found to be more effective than chlorine to deactivate biofilms on
stainless-steel surfaces.[5] PAA can be used over a wide range of
temperatures (0–40 8C) and pH (3.0–7.5)[6] and it decomposes into
nontoxic oxygen, acetic acid, and water. As a result, PAA has been
approved by the U.S. EPA as a pesticide and by the FDA for direct
food contact and food contact surfaces. PAA has also been used to
disinfect medical supplies,[7,8] for waste water treatment, and for
pulp and textile bleaching.[9]
Commercial PAA is generally produced by reacting acetic acid
with H2O2 using sulfuric acid as the catalyst. However, this
reaction is slow (requiring up to several days to yield high amounts
of PAA); moreover, residual levels of acetic acid, H2O2, and
In the current work, we have exploited AcT interaction with
multiwalled carbon nanotubes (MWNTs) to produce bioactive
composites that generate PAAby perhydrolysis of propylene glycol
diacetate (PGD) in the presence of H2O2 (Fig. 1). Our strategy
involved covalent attachment of AcT to MWNTs and subsequent
incorporation of the resulting conjugates into polymers (poly-
(methyl methacrylate) (PMMA) and poly(vinyl acetate) (PVAc)),
and latex-based paint. MWNTs were chosen as support material
due to several intrinsic advantages, including: i) ease of surface
[*] Prof. R. S. Kane, Prof. J. S. Dordick, Dr. C. Z. Dinu, Dr. G. Zhu,
S. S. Bale, G. Anand, Dr. P. J. Reeder
Department of Chemical and Biological Engineering
Rensselaer Nanotechnology Center and
Center for Biotechnology & Interdisciplinary Studies
Rensselaer Polytechnic Institute
Troy, NY 12180 (USA)
E-mail: kaner@rpi.edu; dordick@rpi.edu
Dr. K. Sanford, Dr. G. Whited
Genencor International
Palo Alto, CA 94304 (USA)
Figure 1. AcT-catalyzed perhydrolysis of PGD to generate peracetic acid
(PAA).
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functionalization,[20] ii) very high surface-area-to-volume ratios
that afford high enzyme loading without diffusional limitations
and facile isolation and purification of enzyme-MWNT con-
jugates,[21] iii)lightweightandyethighmechanicalstrength[22] that
make them excellent filling materials to reinforce polymers[23–25]
andceramics,[20,26] andiv)ensuredentrapmentoftheenzyme, and
thus enzyme retention within the composite (with no enzyme
leaching). The incorporation of AcT–MWNT conjugates into
polymers and paints is the first step in the preparation of bioactive
composites with enhanced strength and extended lifetime. These
composites may be applied as decontaminating coatings for
buildings and environmental remediation or for medical settings
where effective killing of a variety of infectious organisms is
critical.
of MWNTs, as determined by the bicinchoninic acid (BCA) protein
assay and elemental analysis. The resulting AcT–MWNT
conjugates were soluble to at least 2.5 mg mLꢀ1 in aqueous
buffer (50 mM potassium phosphate, pH 7.1). This solubility was
deemed sufficient to provide further uniform dispersion of the
conjugates into polymericandpaint composites, which is expected
to lead to uniform activity per unit area of the composites by
distributing the enzyme conjugates throughout the
material.[23,31,32]
Contrary to their excellent water dispersity, the AcT–MWNT
conjugates only retained about 7% of the native AcT activity.
Changing the conditions for AcT immobilization, such as varying
the pH of the buffer and using different ratios of AcT/nanotube or
EDC/NHS, did not improve bound enzyme activity. This activity
was substantially lower than that for other enzymes immobilized
onto carbon nanotubes. For example, glucose oxidase (GOx)
immobilized onto carbon nanotube retained 68% of the free GOx
activity [33] while soybean peroxidase (SBP) covalently attached to
MWNTs retained around 55% of the free SBP activity.[21]
AcT is a large molecule (an octamer, Mw ¼ 184 kDa) with
2. Results and Discussion
2.1. AcT–Carbon-Nanotube Conjugates
˚
dimensions of 72 ꢂ 72 ꢂ 60 A (Fig. 3a) formed through tight
association of pairs of dimers. Bioinformatic calculation (per-
formed using ProtParam and images created on MOE, Chemical
Computing Group Inc.) revealed that ꢃ60% of the amino acid
residues that constitute the monomer are hydrophobic and the
average hydropathicity of the monomer is 0.117 (Supporting
Information Fig. S1). Specifically, there are four insertions:
residues 17–27, residues 59–69, residues 122–130, and residues
142–156 in the AcT structure (Fig. 3b), which form loops at the
dimer interfaces and contribute to stabilization of the octameric
structure. These loops enable formation of a hydrophobic channel
that extends to the exterior of the octameric surface (Supporting
Information Fig. S2). The regions forming the hydrophobic
channel lead to the active sites of the AcT being somewhat buried
and thus having restricted substrate accessibility.[18] Moreover, the
large block-like structure and extensive hydrophobicity of AcT
would presumably lead to substantial nonspe-
High activity and good solubility/dispersibility of enzyme–
nanotube conjugates are required to construct practically useful
bioactive composites. Aggregation of pristine carbon nanotubes in
both aqueous and organic solvents due to surface–surface van der
Waals interactions reduces available surface areas for biomolecule
attachment and also prevents their efficient dispersion in a
polymer or paint composite.[27,28] To that end, we used func-
tionalized MWNTs that have been oxidized via acid treatment[29]
yielding free carboxylic acid groups. The acid-functionalized
MWNTs were soluble in water up to at least 5 mg mLꢀ1 following
brief sonication.
Covalent attachment of AcT to the water-soluble MWNTs was
performed via EDC/NHS chemistry[30] (Fig. 2a; see Experimental
Section for more details) providing AcT loadings of
(0.12 ꢁ 0.01) mg (mean ꢁ standard deviation) AcT per milligram
cific hydrophobic interactions between the AcT
surface and the non-functionalized hydropho-
bic regions of the MWNTs. These nonspecific
hydrophobic interactions determine close
packing of AcT molecules onto the MWNT
surface and potentially bury the hydrophobic
catalytic site (Fig. 2a, inset; as a comparison the
bare acid-treated MWNTs are also shown).
Consequently, the attached AcT molecules
would have limited flexibility and their strong
interaction with nanotube surface (via hydro-
phobic–hydrophobic interactions) would
reduce the substrate accessibility to the active
site and thus the enzyme activity.
Amphiphilic poly(ethylene glycol) (PEG) is a
particularly effective linker, which is known to
reduce nonspecific interactions,[34,35] will not
decrease the solubility of the carbon nano-
tubes,[36,37] and can enhance enzyme activity
Figure 2. Covalent attachment of AcT onto multiwalled carbon nanotubes (MWNTs). a) Direct
attachment of AcT onto MWNTs. In addition to covalent binding, nonspecific hydrophobic
interaction also occurs due to the large size and hydrophobic nature of AcT. Inset: TEM image of
AcT–MWNT conjugates. b) Attachment of AcT onto MWNTs using dPEG as spacer.
due to improved surface hydrophilicity.[38] To
this end, a bifunctional amino-dPEG12-acid
(dPEG, 4.7 nm in contour length) spacer was
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catalytic turnover as that of the free enzyme,
indicating that the PEG linker markedly
improved the reactivity of the enzyme when
compared to the direct covalent attachment of
the enzyme to the MWNTs. The Km values were
115 and 123 mM for free AcT and AcT–dPEG–
MWNT conjugates, respectively. Therefore,
attachment of AcT onto functionalized
MWNT via dPEG spacer did not significantly
alter substrate-binding affinity. The good
kinetic properties of the AcT–dPEG–MWNT
conjugates led us to use this formulation for
preparation of the polymer and paint
composites.
2.2. AcT–dPEG–MWNT Composites
Figure 3. Structure of AcT. a) AcT is an octamer with the catalytic triad Ser11, Asp192, and His195
shown in filled space; all other residues are shown with lines where the colored residues indicates
green: hydrophobic, pale blue: hydrophilic, dark blue: basic, and red: acidic. b) Molecular surface
of monomeric AcT; colored residues, green: hydrophobic, pink: hydrogen bonding, and blue: mild
polar.
Having established that the AcT–dPEG–
MWNT conjugates retained relatively high
intrinsic catalytic activity and had high water
dispersity, we incorporated the conjugates into
two industrially relevant polymers—poly-
(methyl methacrylate) (PMMA) and poly(vinyl
first covalently attached to the acid treated MWNTs and
subsequently AcT was attached to the free end of the spacer—
both attachments being performed via EDC/NHS amide forma-
tion (Fig. 2b; Supporting information Fig. S3). Attenuated total-
reflection Fourier infrared spectroscopy (ATR-FTIR) was used to
determine the presence of amide bonds before and after the
protein was chemically grafted onto nanotubes (Supporting
information Fig. S4). Covalent attachment of PEG to the
MWNT surface was confirmed by the presence of amide I band
(1600–1700 cmꢀ1, centered at 1656 cmꢀ1). Further functionaliza-
tion of the dPEG–MWNTs with AcT led to additional vibrational
stretch(1656 cmꢀ1)andꢃ50%increaseintheamidebondsarising
primarily from carbonyl bond stretching vibrations that formed
the protein linkages.[39]
The dPEG spacer was effective in increasing the specific activity
of the resulting AcT–dPEG–MWNTconjugates to ꢃ24% of that of
free AcTand also in blocking the hydrophobic non-functionalized
nanotubes walls thus preventing non-specific enzyme attach-
ment[35] (Fig. 4a). Other PEG spacers could be also employed;
however, similar effects in terms of blocking non-specific protein
binding should be observed.[40] Longer spacers (>2000), however,
are to be avoided, since they could potentially create porous
composites and thus alter their mechanical properties.[41] When
0.2 mg mLꢀ1 nanotube and 0.4 mg mLꢀ1 AcT were used in the
coupling reaction, the resulting AcT–dPEG–MWNT conjugates
had an enzyme loading of (0.06 ꢁ 0.02) mg AcT per mg of
nanotube as determined by elemental analysis. These conjugates
were soluble up to 2.5 mg mLꢀ1 in aqueous buffer. Kinetic studies
were performed for both free AcT and AcT–dPEG–MWNT
conjugates by varying the concentration of H2O2 from 0.1 to
400 mM whilemaintainingthePGDconcentrationat200 mM.Both
free AcTand AcT–dPEG–MWNT conjugates followed Michaelis–
Menten kinetics (Fig. 4b) with kcat values of 4.6 ꢂ 105 and
Figure 4. a) Specific activity of AcT–nanotube conjugates compared to free
AcT. b) Kinetics of free AcT (filled circles) and AcT–dPEG–NT (filled
squares) conjugates.
1.3 ꢂ 105 minꢀ1 for free AcT and AcT–dPEG–MWNT conjugates,
respectively. Thus, the conjugate possessed ꢃ28% of the intrinsic
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acetate) (PVAc)—and into a latex-based paint. Incorporating
biocatalysts into materials has been of interest for more than a
decade.[42–48] In addition to direct crosslinking of biomolecules
into the composite matrix, polymeric composites have also been
prepared by interacting enzymes with a third component such as a
different polymer,[49] activated carbon,[50] or carbon nano-
tubes.[51,52] With regard to the latter, we have shown that both
single- and multiwalled carbon nanotubes are able to stabilize
enzymes in polymer composites,[51,52] eliminating the need to
crosslink the enzymes within the network.
To prepare polymeric composites, water solutions of AcT–
dPEG–MWNT conjugates were added to acetone solutions of
PMMA or PVAc at a volume ratio of 1:15 (enzyme-based
conjugates:polymer) and mixed by vortexing. The composites
were then formed either by direct evaporation of the acetone and
water in a glass vial or by spin-coating the solution onto a glass
cover slide. The use of acetone as the solvent avoided phase
separation encountered by more hydrophobic solvents such as
toluene and chloroform, while the initial solubilization of the
conjugates in water aided their subsequent dispersion in the
polymer solutions without sonication. In the case of paint
composites, a 1:10 volume ratio of water solution of AcT–
dPEG–MWNTconjugates to latex was used. Visually there was no
phase separation for either the polymer or latex-based composites.
The two-step process applied in this work also made it convenient
to control the composite activity simply by varying the loading of
AcT–dPEG–MWNT conjugates.
AcT activity in the PMMA and PVAc composites obtained by
direct evaporation was quite low, <10% of that of the AcT–dPEG–
MWNT conjugates in aqueous solution (Fig. 5a). However, these
polymer films had a thickness of ꢃ200 mm, which could have
severely limited the diffusion of the two substrates PGD and H2O2
totheimmobilizedAcT. Indeed, estimationof theThiele Modulus,
f2 (Equation 1), for H2O2 revealed a value of 230 indicating strong
diffusional limitations (Thiele Modulus represents the ratio of the
intrinsic reaction rate in the absence of mass transfer limitation to
the rate of diffusion through the medium).[53]
ꢀ ꢁ
2
h
vmax
f2 ¼
(1)
2
Deff Km
In Equation 1, h is the film thickness (200 mm) and vmax is the
maximal enzyme reaction rate (¼ kcat ꢂ enzyme concentration).
Weused thevalueofkcat ¼ 1.3 ꢂ 105 minꢀ1 (asfoundin ourkinetic
studies above), a Km of 123 mM, and the enzyme concentration was
obtained from the loading (40 mg conjugate or 2.4 mg of AcT,
184 kDa enzyme molecular weight) in PMMA. The effective
diffusivity (Deff) was estimated to be 10ꢀ10 cm2 sꢀ1. This value was
based on water diffusion in a PMMA (Mw ¼ 834 kDa) film at a
thickness of 200 mm.[54] In addition to the diffusional limitations
causedbythethickfilms, thelargemolecularsizeandhydrophobic
character of AcT could lead to extensive interaction with the
surrounding hydrophobic PMMA and PVAc molecules, which
would further limit the accessibility of the substrates to the AcT
active sites.
Figure 5. a) Specific activity of the composites relative to that of AcT–
dPEG–MWNT conjugates. Black bars indicated thick films obtained by
direct evaporation (200 mm for polymeric composites and 400 mm for paint
composite) and grey bars indicated thin films obtained by spin-coating
(ꢃ2 mm for polymeric composites). b) Stability of the paint composite
tested under different conditions: (ꢄ) dry state at room temperature;
(!) immersed in water at room temperature; (&) dry state at 508C;
(^) immersed in water at 50 8C. c) PAA generated in 20 min by paint
composite at different loadings of AcT–dPEG–MWNT conjugates. The
paint has a surface area of 5 cm2 and a thickness of around 400 mm.
Reactions were conducted in 1 mL potassium phosphate buffer (50 mM,
pH7.1) containing 100 mM H2O2 and 100 mM PGD.
To overcome mass transfer limitations in the polymeric
composites, we dramatically reduced the film thickness from
200 mm to ꢃ2 mm by spin-coating the mixed solution of
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AcT–dPEG–MWNT conjugate and polymer onto a cover glass
(Supporting Information Fig. S5). The corresponding activity of
the AcT–dPEG–MWNT polymer films increased to ꢃ40% and
more than 90% of the conjugate activity for PMMA and PVAc,
respectively (Fig. 5a). The relatively hydrophilic nature of PVAc
film may contribute to its higher activity when compared to the
more hydrophobic and dense PMMA film. The more porous latex
paint composite was not nearly as diffusionally limited as the
polymers with ꢃ40% of the conjugate activity even at a thickness
of 400 mm.
incubated in the reaction solution for 1, 2, and 3 h after a typical 20-
minreaction. Thepaintswerethenrinsedwithwaterandair-dried.
After 24 h, the paints retained 75%, 60%, and 30% of their original
activity, respectively. This indicates that PAA is able to cause
enzyme deactivation. In addition, to evaluate sample-handling
effects (e.g., drying and washing) on the activity loss, we also
compared the activity of the paint immersed in buffer with that of
the paint immersed in the reaction solution after five reaction
cycles (one day per cycle). The paint incubated in buffer showed no
activitylosswhilethepaintincubatedinreactionsolutionshoweda
60% activity loss. We reasoned that the residual PAA was able to
diffuse easily out of the paint stored in buffer, and hence cause less
deactivation of immobilized AcT. The PVAc and PMMA thin films
showed similar stability trends as that of the paint under all testing
conditions. When immersed in water, after five days and four
reaction cycles the PVAc film retained 53% of its original activity,
while after four days and three reaction cycles the PMMA film
retained 62% of its original activity.
The ability of these composites to generate PAA is of interest in
the development of decontaminating coatings. PVAc thin films
(thickness ꢅ 2 mm, surface area of 5 cm2) containing a conjugate
loading of 0.06 wt% (0.004 wt% of AcT) generated 0.2 mM PAA in
20 min; under the same conditions the thin PMMA film generated
0.05 mM of PAA at a conjugate loading of 0.08 wt% (0.005 wt% of
AcT). Moreover, the paint composite (thickness ꢅ 400 mm)
generated > 11 mM PAA at a conjugate loading of 0.16%
(0.01 wt% of AcT; Fig. 5c). Even though the polymeric thick films
obtained by direct evaporation were diffusion limited, the larger
amount of catalyst present (as compared to the spin-coated films)
wasabletoyieldhigherlevelsofPAA.IthasbeenreportedthatPAA
is bactericidal at 0.13 mM and fungicidal at 0.39 mM.[56] In other
tests, PAA was shown to effectively kill bacteria at concentration as
low as 0.05 mM[57] and reduce spore colony-forming units (CFUs)
103.5-fold at a PAA concentration of 4 mM.[6] Hence, the AcT-based
paints would be expected to be highly microbicidal/sporicidal.
Indeed, following 20-min incubation of the AcT-containing paint,
the supernatant was capable of killing > 99% of Bacillus cereus
spores initially charged at 106 CFU mLꢀ1. Further investigation is
underway to assess the killing efficiency of these composites on
different pathogens.
The MWNT-based composites formulation enabled high
retention of the active and stable enzyme in the polymeric film
orinpaintswithnoleachingfromthecomposite.Incontrols,when
the enzyme is entrapped in the composite without the MWNT
support, >50% of the enzyme is lost from the composite (PMMA,
PVAc, or paint) due to leaching in less than 1 h. In other controls,
we have immobilized AcT onto silica-based nanoparticles
(15 ꢁ 5 nm) by physical adsorption or by covalent binding. For
physical adsorption, we incubated directly AcT to nanoparticles
with or without n-octadecyltrimethoxy silane (n-ODMS) function-
alization, while for covalent binding we used carboxyl-
functionalized nanoparticles and similar immobilization condi-
tions as for MWNTs (Supporting Information). Physical attach-
ment led to high loadings of AcT (ꢃ0.05 mg AcT/mg nanoparticle
basedontheBCAassay);lowerloadingswereobtainedforcovalent
attachment (ꢃ0.03 mg AcT/mg nanoparticles based on the BCA
assay). However the physically attached enzyme quickly leached
fromthepolymerorpaintcomposites, presumably dueto theweak
enzyme–nanoparticle interaction and high porosity of the
resultant films. The covalently attached enzyme did not leach
out; however, it showed only ꢃ25% of the activity of the AcT–PEG–
MWNTs and significant loss of composite enzyme activity
occurred. This is in agreement with previous reports that have
shown that binding of the enzymes to nanoparticles is strongly
affected by size, and hence the surface curvature of the support
with thephysical propertyat the nanoscale influencing thestability
and unfolding of adsorbed proteins.[55] Thus, MWNTs provide a
unique material for enzyme incorporation into the polymeric or
paint films and lead to composites with active and stable
biocatalytic activity, with no enzyme leaching observed.
In addition to composite activity and leaching experiments, we
also examined composite stability and reusability. The spin-coated
polymer films and the latex paint composites were stored under
different conditions and their activity was measured every 24 h.
The storage conditions were selected to mimic the environment
that could be encountered in real-life applications, and thus
included storage in dry state at room temperature and 508C, and
storage in the hydrated state (by immersing the composites in
water) at room temperature and 508C respectively. The paint
exhibited high stability when immersed in water at room
temperature (Fig. 5b). After a six-day incubation and five reaction
cycles, the paint retained >50% of its initial activity. Surprisingly,
when stored in dry state at room temperature the paint only
enabled ꢃ20% of its original activity after six days and five reaction
cycles. A similar trend was also observed at 508Cwith a more rapid
loss in activity. The unusual result whereby lower enzyme stability
was observed in the dry state was rationalized as being due to
residual PAA from the enzymatic reaction being retained in the
AcT-containing paint. To test this hypothesis, the paints were
3. Conclusions
In summary, highly water-soluble AcT–carbon nanotube con-
jugates were prepared and uniformly incorporated into polymer
films and latex paint. AcTisperhaps the largest enzyme attached to
carbon nanotubes (an octameric enzyme with a molecular
˚
weight > 180 kDa and dimensions of 72 ꢂ 72 ꢂ 60 A) to show
activity and stability. Large proteins have an increased surface area
that can result in multiple interactions with the support and thus
maycauseconsiderable lossof activity. The stability andreusability
of the composites was shown to be sufficient for use as a
microbicidal and sporicidal decontaminating surface. Moreover,
the entanglement of MWNTs in the polymer/paint composite
enabled full retention of the nanoscale support. This, in turn,
enabled active enzyme to be retained in the composite and is
expectedtoreduceoreliminatepotentialadversehealtheffectsthat
could result from release of MWNTs into the environment. Finally,
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composites were prepared by adding water suspension of AcT–nanotube
conjugates into latex-based paint (typically 0.2 mL) in a glass vial (2.5-cm
diameter). The two components were mixed thoroughly using a pipette tip
and the mixture was air-dried.
thecapabilityofgeneratingsufficientlyhighamountofpotentPAA
makes these composites useful as coating materials for
disinfection.
Enzyme Loading: The concentration of AcT in solutions was measured
using the standard BCA protein assay. Briefly, the working reagent was
prepared by mixing 50 parts of reagent A with 1 part of reagent B. AcT
solution (50 mL) was added to the working reagent (1 mL) and the mixture
was incubated at 37 8C for 30 min followed by measuring the absorbance at
562 nm on a UV–vis spectrophotometer (Shimadzu UV-2401). Serial
dilution of AcT was performed to create the calibration curve. The amount
of AcT attached on MWNTs was determined by subtracting the amount of
enzyme washed out in the filtrates from the amount of AcT initially added.
Alternatively, the AcT loading on nanotube was determined by elemental
analysis (analyzed by Galbraith Laboratories Inc., Knoxville, TN).
Activity Assays: The activity of AcT was determined by measuring the
PAA generated [58]. In a typical reaction, H2O2 stock solution (10.6 mL, final
concentration 100 mM) was added to a mixture of PGD solution (0.8 mL,
final concentration 100 mM in potassium phosphate buffer, 50 mM, pH 7.1)
and AcT solution (0.2 mL, 2.0 mg mLꢀ1 final concentration for free AcT or
equivalent concentration of AcT for AcT–nanotube conjugates). The
mixture was shaken at 200 rpm for 20 min at room temperature. PAA assay
was conducted by diluting the reaction solution (25 mL) 100 times in
deionized water and subsequently mixing the diluted solution (25 mL) with
deionized water (75 mL) and assay reagent (0.9 mL; the assay reagent
was prepared by mixing potassium citrate buffer (5 mL, 125 mM, pH 5.0)
with ABTS water solution (50 mL, 100 mM) and KI water solution (10 mL,
25 mM)). The mixture was then incubated at room temperature for 3 min
and the absorbance at 420 nm was measured on a UV–vis spectro-
photometer. PAA concentration was calculated by [Peracetic Acid] (mM) ¼
A420nm ꢂ 0.242 ꢂ 400 (0.242 is the correlation coefficient between the
concentration of PAA and the absorbance at 420 nm, and 400 is the dilution
factor). The specific activity of AcT–nanotube conjugates was calculated
as the ratio of the normalized activity of the conjugates to that of the
native AcT.
The activity of the composites (polymer films and paint) was measured
by adding PGD solution (0.8 mL, final concentration 100 mM), buffer
(0.2 mL), and H2O2 solution (10.6 mL, final concentration 100 mM) into the
container containing the polymer film or paint. After incubation at room
temperature for 20 min, the reaction solution (25 mL) was withdrawn and
PAA assay was conducted as described above. Kinetics of AcT (free AcT and
AcT–nanotube conjugates) was studied by measuring the initial reaction
rates at different substrate concentrations. The concentration of hydrogen
peroxide was varied from 0.1 mM to 428 mM while maintaining the PGD
concentration at 200 mM.
Dispersity Analysis: The dispersity of functionalized MWNT and AcT–
nanotube conjugates in water was determined by centrifuging the
corresponding water suspension (initial concentration 8 mg mLꢀ1 for
MWNT and 4 mg mLꢀ1 for AcT–nanotube conjugates) at 3000 rpm for
5 min and then filtering the supernatant (0.8 mL) through a 0.2-mm
membrane. After complete drying under vacuum, the amount of MWNT or
AcT–nanotube conjugates on the membrane was measured and the
dispersity was calculated based on the volume. It should be noted that the
obtained values did not reflect the saturation dispersity, which is actually
the corresponding solubility.
4. Experimental
Materials: Perhydrolase S54V (AcT) solution was provided by Genencor
International, Inc. (Palo Alto, CA). MWNTs (purity > 95%, outer diameter
15 ꢁ 5 nm, length 5–20 mm) were purchased from NanoLab, Inc. (Newton,
MA). Sulfuric acid (H2SO4, 95–98%), nitric acid (HNO3, 68%–70%), and
cover glass (circular, 25 mm) were purchased from Fisher Scientific
(Hampton, NH). Propylene glycol diacetate (PGD), 2-(N-morpholino)etha-
nesulfonic acid sodium salt (MES), hydrogen peroxide solution (30%), and
uranyl acetate were purchased from Sigma (St. Louis, MO). 1-Ethyl-3-[3-
dimethylaminopropyl] carbodiimide hydrochloride (EDC) was purchased
from Acros Organics (Morris Plains, NJ). BCA protein assay kit, N-
hydroxysuccinimide (NHS), and (2,20-azinobis [3-ethylbenzothiazoline-6-
sulfonic acid] (ABTS) were purchased from Pierce (Rockford, IL). Isopore
filter membrane (pore size 0.2 mm, type GTTP, polycarbonate) was
purchased from Millipore (Billerica, MA). Amino-dPEG12-acid was
purchased from Quanta Biodesign (Powell, OH). Poly(methyl methacry-
late) (PMMA, average Mw ¼ 996 000) and poly(vinyl acetate) (PVAc,
average Mw ¼ 500 000)) were purchased from Aldrich (Milwaukee, WI).
Latex enamel (gloss white, manufactured by Yenkin-Majestic Paint
Corporation, Columbus, OH) was purchased from a local store.
Functionalization of Carbon Nanotubes: Carboxylic acid groups were
created on MWNTs by acid treatment. Typically, MWNTs (100 mg) were
added to an acid mixture of H2SO4 and HNO3 (45 mL and 15 mL,
respectively; H2SO4:HNO3 ¼ 3:1, v/v) and the suspension was sonicated
at room temperature for 6 h in a VWR ultrasonic cleaner (model 50T,
frequency from 38.5 to 40.5 kHz, average power of 45 W). The suspension
of functionalized MWNTs was then diluted in Milli-Q water (200 mL) and
filtered through a 0.2-mm filter membrane. The nanotubes on the
membrane were redispersed in Milli-Q water (200 mL) by sonication and
filtered again. This process was repeated at least six times to remove
residual acids and any solubilized impurities. The functionalized MWNTs
were dried under vacuum and stored at room temperature.
Preparation of AcT–MWNT Conjugates: AcT was covalently attached to
functionalized MWNTs via a two-step process involving EDC/NHS
activation followed by enzyme coupling [30]. In a further study, amino-
dPEG12-acid was used as a spacer between AcT and MWNT. Typically,
functionalized MWNTs (2 mg) were dispersed in MES buffer (2 mL, 50 mM,
pH 4.7) containing EDC and NHS (160 mM and 80 mM, respectively) by
brief sonication. After 15-min shaking at 200 rpm and room temperature,
the NHS-activated MWNTs were filtered through 0.2-mm filter membrane
and washed thoroughly with MES buffer. NHS–MWNTs were used
immediately in the enzyme coupling reaction. For direct enzyme
immobilization, NHS–MWNTs (2 mg) were dispersed in potassium
phosphate buffer solution of AcT (0.4 mg mLꢀ1 in 10 mL buffer, 50 mM,
pH 7.1) and the enzyme coupling was allowed to proceed for 3 h at room
temperature by shaking at 200 rpm. The AcT–MWNT conjugates were
filtered and washed extensively with potassium phosphate buffer to remove
free enzymes. The AcT–dPEG–MWNT conjugates were prepared by first
covalently attaching amino-dPEG12-acid (1 mg mLꢀ1) to MWNTs and then
attaching AcT to dPEG–MWNTs following the two-step process previously
described.
Preparation of Polymer and Paint Composites: Thin AcT–nanotube
polymer films were prepared by spin-coating (spin processor model: WS-
400E-6NPP-LITE, Laurell Technologies Corporation, North Wales, PA) the
conjugate–polymer solution at 4500 rpm for 50 s on a cover glass. The
conjugate–polymer solution was prepared by mixing water suspension of
AcT–dPEG–MWNT conjugates and acetone solution of PMMA (0.08 g
mLꢀ1) or PVAc (0.1 g mLꢀ1) by vortexing. The thickness of the polymer
films was measured using a profilometer (Dektak 8, Veeco Instruments
Inc., Plainview, NY). To prepare thick films, the conjugate–polymer
solutions (1 mL) were added in a glass vial (2.5-cm diameter) and the
solvents were evaporated under vacuum. The AcT–nanotube-paint
Sample Imaging: The morphology of AcT–MWNT conjugates was
observed by transmission electron microscopy (TEM) with a field emission
gun at 120 kV (Phillips, CM-12). Typically, the conjugate solution in water
(10 mL) was dropped on a Formvar carbon-coated grid (from Electron
Microscopy Sciences, Hatfield, PA) and then exposed to a 0.5% solution of
uranyl acetate for ꢃ3 s. The samples were vacuum-dried overnight prior to
TEM imaging.
Acknowledgements
C. Z. Dinu and G. Zhu contributed equally to this work. This work was
supported by a contract (W911SR-05-0038) from the U.S. Army under a
subcontract from Genencor International, and a contract from DTRA
Adv. Funct. Mater. 2010, 20, 392–398
ß 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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