Note: Descriptions are shown in the official language in which they were submitted.
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COMPOSITE NANOMATERIALS FOR PHOTOCATALYTIC
HYDROGEN PRODUCTION AND METHOD OF THEIR USE
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is entitled to priority pursuant to 35 U.S.C. 119(e) to U.S.
provisional patent application No. 60/681,174, whicli was filed on May 16,
2005, whicli is
incorporated herein in its entirety for all purposes.
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR
DEVELOPMENT
The invention disclosed herein was funded, in part, by the National Science
Foundation under grant nuinber MCB-0328341 and the Office of Naval Research
under grant
number 19-00-R0006.
FIELD OF THE INVENTION
The present invention relates to composite materials containing hydrogenase
enzyines and hydrogen-producing nanoparticles with photocatalysts for hydrogen
production
from renewable sources.
BACKGROUND OF THE INVENTION
There is considerable recent interest in the production of hydrogen gas as an
altenlative source of energy. The practicality of the increased use of
hydrogen fuel cell
technologies is dependent on the ability to produce stores of hydrogen gas in
an efficient,
economically feasible, and environmentally sound manner (Arznor et al. 2005;
Speigel et al.
2004; Tseng et al. 2005; Winter et al. 2005). The majority of hydrogen
produced for energy
yielding applications is generated by the process of reforming methane or
fossil fuels and
thus a hydrogen energy economy based on these approaches does little to reduce
the
dependence on nonrenewable fossils fuels. Therefore, the development of
catalysts for
chemical reactions that can produce hydrogen gas efficiently from renewable
sources, such as
feedstocks, and without the production of greenhouse gases or other
environmental pollutants
is of paramount importance.
Hydrogenases are highly evolved catalysts that produce hydrogen gas at rates
that are
the envy of synthetic chemists. Metal-containing hydrogenases (H2ases)
(Vignais et al. 2001;
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Peters et al. 1998, 1999; Adains et al. 1990) are produced by a variety of
microorganisins
where they function either in hydrogen oxidation or proton reduction according
to the
following reaction:
2H+(aq) + 2e H*--'H2(g)
2ase
Hydrogen oxidation is coupled to the generation of reducing equivalents to
drive
energy yielding or biosynthetic processes. During anaerobic fermentation some
inicroorganisms are capable of coupling the oxidization and regeneration of
electron carriers
necessary for sugar oxidation to proton reduction and the production of
hydrogen gas. The
catalytic sites of most metal-containing hydrogenases consist of either di-Fe
or heterometallic
NiFe sites with diatomic ligands of carbon monoxide and cyanide to Fe.
Hydrogenases are
the only 1clZown enzyines that utilize these nornially toxic compounds as
integral parts of an
active state of an enzyme. Hydrogenases; like enzymes in general, are
assembled to display
precise organizational motifs that use their protein architecture to position
and chemically
poise an active site in which pathways for substrate access and product
removal are key
"design" features. Substrate, reductant, and product must have access to and
from the
catalytic site. Furthermore, continuous cycling of the catalyst requires
ongoing addition
of reactants and removal of products. The catalytic site of hydrogenase
enzymes consists of
unique biological metal clusters (Fe or NiFe) with carbon monoxide and cyanide
ligands
(Peters et al. 1998; Happe et al. 1997; Nicolet et al. 2000; Pierik et al.
1998; Volbeda et al.
1995).
Biological production of hydrogen is likely to play a key role in the emerging
hydrogen economy (Varfolomeyev et al. 2004; Wunschiers et al. 2002; Chum et
al. 2001).
There is a growing interest in using enzyme catalysts in materials for a
variety of
applications. In the context of utilizing enzymes in applications, there are
several
considerations that typically need to be addressed. Substrate, reductant, and
product must have
access to and from the catalytic site. Furthermore, continuous cycling of the
catalyst requires
ongoing addition of reactants and removal of products. While rates of up to
9000 H2 per
enzyme per second have been observed (Adams et al. 1990; Cammack et al. 1999)
from
these enzymes, the extreine sensitivity of these enzymes to oxygen, limited
expression, and
difficult isolation have hindered their use as a practical means of hydrogen
production.
Attempts to develop methods and systems to produce hydrogen on a commercial
scale
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using the hydrogenase enzyine have been deficient. U.S. Patent No. 6,858,718
discloses a
gene encoding for hydrogenase and a method for using the gene product for the
microbial
production of molecular hydrogen. More specifically, the invention discloses
isolated nucleic
acid sequences encoding a stable hydrogenase enzyme (HydA) that will catalyze
the
reduction of protons to forin molecular hydrogen.
U.S. Pat. No. 4,532,210 discloses the biological production of hydrogen in an
algal
culture using an alternating light and dark cycle. The process coinprises
alternating a step for
cultivating the alga in water under aerobic conditions in the presence of
light to accumulate
photosynthetic products (starch) in the alga, and a step for cultivating the
alga in water under
microaerobic conditions in the dark to decoinpose the accumulated material by
photosynthesis to evolve hydrogen. This method uses a nitrogen gas purge
teclinique to
remove oxygen from the culture. -
U.S. Pat. No. 4,442,211 discloses that the efficiency of a process for
producing
hydrogen, by subjecting algae in an aqueous phase to light irradiation, is
increased by
culturing algae which has been bleached during a first period of irradiation
in a culture
mediuin in an aerobic atmosphere until it has regained color and then
subjecting this algae to
a second period of irradiation wherein hydrogen is produced at an enhanced
rate. A reaction
cell is used wherein light irradiates the culture in an environment which is
substantially free
of CO2 and atmospheric 02. This environment is maintained by passing an inert
gas (e.g.
helium) through the cell to remove all hydrogen and oxygen generated by the
splitting of
water molecules in the aqueous medium. Although continuous purging of H2 -
producing
cultures with inert gases has allowed for the sustained production of H2, such
purging is
expensive and impractical for large-scale mass cultures of algae.
Accordingly, the present invention satisfies a long felt need to produce
hydrogen on
a commercial scale that has been achieved by combining knowledge from the
disparate fields
of enzymatic H2 formation, photocatalytic nanomaterials, and electro/photo-
chromic polymer
gel teclmology.
SUMMARY OF THE INVENTION
The present invention is directed to composite materials and methods of
producing
hydrogen from renewable sources, such as simple feedstocks. In one aspect, the
present
invention relates to a composite material for photocatalytic H2 production
comprising: 1) a
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polymer gel, 2) a photocatalyst, and 3) a protein based H2 catalyst. The H2
catalyst may be
an enzyine such as a hydrogenase enzyme, which can be derived from a variety
of organisms,
such as microorganisms including but not limited to Clostridium pasteurianunz,
Laprobacter-
modestogalophilus, Tlaiocapsa reseopericina, or coinbinations tllereof. The
composite
material can furtller comprise a redox mediator, such as poly viologen, and an
oxygen
scavenger, sucli as Cu(O). The photocatalyst can be fonnulated as a
nanoparticle and can be
encapsulated in a protein cage architecture.
The H2 catalyst may also be an artificial enzyme sucli as a hydrogenase mimic
in the
form of a protein cage comprising a shell and a core. The shell of the protein
cage may
comprise a protein wherein the protein is a 24 subunit protein such as a small
heat shock
protein (HSp). The core of the protein cage may comprise a metal wherein the
metal is
selected from the group consisting of platinuin, nickel, iron, and cobalt.
The present invention also relates to a method for producing H2 using the
cornposite
materials of the present invention. For exanple, the present invention
provides methods of
producing H2 comprising reacting an electron donor with a composite material
comprising 1)
a polymer gel, 2) a photocatalyst, and 3) protein based H2 catalyst. The
electron donor can be
obtained from a variety of sources, such as but not limited to acetic acid,
citric acid, tartaric
acid, etllanol, EDTA, hydioxylainine, and mixtures thereof. The electron donor
can also be
sulfite, thiosulfate, and dithionite.
BRIEF DESCRIPTION OF THE DRAWINGS
For a more coinplete understanding of the present invention and for further
advantages thereof, reference is made to the following drawings:
Figure 1 is a drawing of a composite material used in a hydrogen producing
device.
Figure 2 is a graph showing hydrogen production activity of C. pasteut ianuzna
(CpI)
and L. modestogalophilus (Lm) hydrogenases in solution and encapsulated in sol-
gel: =,
CpI in solution; =, CpI in sol-gel; =, Lni in solution; ~, Lm in sol-gel.
Figure 3. is a graph showing temperature dependence of hydrogen production
activity
of C. pasteur ianuTn (CpI) and L. naodestogalophilus (Lm) hydrogenases in
solution and
encapsulated in sol-gel: =, Cp1 in solution; =, CpI in sol-gel; =, Lm in
solution; ~, Lm in
sol-gel.
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Figure 4. is a graph sllowing hydrogen production activity of solution and sol-
gel
encapsulated hydrogenase from C. pasteurianum in the presence (open) and
absence
(shaded) of protease.
Figure 5 is (A) a space filling representation of the small heat shock protein
(Hsp)
cage from Methaizococcus jaTZnasclaii (pdb: 1 shs) and (B) a cut-away view of
Hsp
showing the interior cavity of the cage.
Figure 6 is a size exclusion chromatography of Hsp: (A) unmineralized Hsp; (B)
Hsp
mineralized with 250 Pt/Hsp showing coelution of protein (280 nm) and mineral
(350 nm); (C)
Hsp mineralized with 1000 Pt/Hsp showing coelution of protein (280 nm) and
mineral
(350 nm).
Figure 7 is an image showing (A) TEM of Hsp 1000 Pt unstained. The inset shows
electron diffraction of Pt from Hsp 1000 Pt. (B) TEM of Hsp 1000 Pt stained
with 2% uranyl
acetate. (C) Histograin of Pt particle diameters in Hsp 1000 Pt. Average 2.2
(Q.7 nm. Scale
bars) 20 nm.
Figure 8 is an image showing (A) TEM of Hsp 250 Pt stained with 2% uranyl
acetate. (B) Histogram of Pt particle diaineters in Hsp 250 Pt. Scale bar) 20
nm.
Figure 9 is a schematic presentation of the light-mediated H2 production from
Pt-
Hsp. Methyl viologen (MV2+) is used as an electron-transfer mediator between
the
Ru(bpy)32+ photocatalyst and the Pt-Hsp responsible for H2 production.
Figure 10 is a graph showing H2 production from Pt-Hsp in 0.2 mM Ru(bpy)32+,
0.5
mM methyl viologen, 200 mM EDTA, and 500 mM acetate pH 5.0: =, 1000 Pt/Hsp 5.1
x
10"10 mol Pt; ~, 250 Pt/Hsp 8.2 x 10-10 mol Pt.
DETAILED DESCRIPTION OF THE INVENTION
All publications and patent applications herein are incorporated by reference
to the
same extent as if each individual publication or patent application was
specifically and
individually indicated to be incorporated by reference.
The following description includes information that may be useful in
understanding
the present invention. It is not an admission that any of the information
provided herein is
prior art or relevant to the presently claimed inventions, or that any
publication specifically or
implicitly referenced is prior art.
Unless defined otherwise, all technical and scientific tenns used herein have
the saine
meaning as commonly understood by one of ordinary skill in the art to which
this invention
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belongs. Although any methods and materials similar or equivalent to those
described herein
can be used in the practice or testing of the present invention, the prefei7ed
methods and
materials are described.
The present invention is related to enzyinatic H2 foi7nation, photocatalytic
nanoinaterials, and electro/photo-chromic polyiner gel technology. The
invention provides
systems that substantially increase the efficiency with whicll H2 can be
produced from very
siinple feedstoclcs, nainely visible light and simple organic acids (such as
acetate - vinegar).
In one aspect, the inventors have demonstrated that using simple organic
acids, visible light,
and a photocatalyst, one can generate enough reducing equivalents to initiate
activity of the
hydrogenase enzyine and produce H2. To optimize these results, hydrogenase
enzyme is
immobilized and encapsulated into a polymer gel matrix and the resulting
enzyine gel pellets
are incubated with an electron donor such as dithionite and an electron
transfer mediator such
as methyl viologen to generate H2. This approach utilizes the efficiency and
specificity of the
hydrogenase enzyine, and the synthetic control exei-ted in the polymer
formulation to
construct robust coinposite materials that will meet niany of the targets and
objectives of
economic hydrogen gas production.
In another aspect, the present invention is directed to the use of novel
protein cages
or nanoparticles as hydrogenase mimics-to produce hydrogen gas. The
nanoparticles, whieh
comprise both a protein "shell" and a"core", can be mixed together to form
novel
compositions of either complete nanoparticle or core mixtures. In addition,
the shells can be
loaded to form the complete nanoparticles with any number of different
materials, including
organic, inorganic and metallorganic materials, and mixtures tliereof.
Particularly preferred
embodiments utilize metal catalyst such as platinum, to allow for efficient
reduction of
protons. Furthermore, as the shells are proteinaceous, they can be altered to
alter any number
of physical or chemical properties by a variety of methods, including but not
limited to
covalent and non-covalent derivatization as well as recombinant methods.
One of metal catalyst commonly used for chemical reaction is platinuin.
However,
platinum is known for its high cost and limited supply. To maximize the use of
this catalyst,
it is necessary to explore ways of maximizing the catalytic efficiency of Pt
on a per atom
basis in order to develop economically feasible catalysts (Spiegel et al.
2004). In a particle
based approach toward developing a Pt catalyst, it is necessary to minimize
the diameter of
the particle and thus increase the surface area (i.e., the number of exposed
Pt atoms per
particle). A number of different synthetic approaches have been used to
synthesize platinum
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nanoparticles with different passivating layers (Brugger et al. 1981; Chen et
al. 2000; Chen
et al. 1999; Eklund et al. 2004; Gomez et al. 2001; Jiang et al. 2004; Keller
et al. 1980;
Narayanan et al. 2004; Song et al. 2004; Teranishi et al. 2000; Tu et al.
2000; Zhao et al.
2002). The passivating layer generally interferes with the exposed Pt atoms
and reduces
efficiency (Brugger et al. 1981). In the present invention, the inventors have
employed a
protein cage as a synthetic platform which, unlike a passivating layer, does
not coat the entire
surface of the nanoparticles but still isolates the particle in solution and
prevents aggregation.
Protein cage architectures have been used as biotemplates to create interfaces
between
proteins and metals (Allen et al., 2003; Flenniken et al. 2003). Cage-like
architectures have
previously been shown to act as a molecular container for the encapsulation of
both organic
and inorganic materials (Flenniken et al. 2003; McMillan et al. 2002). Protein
cage
architectures are self-assembled from a limited nuinber of protein subunits to
create well-
defined, container-like morphologies in which the interior and exterior
surfaces can be
chemically distinct (Douglas and Young, 1998; Flenniken et al. 2003). In
addition,
molecular access to the interior can be controlled by pores at the subunit
interfaces
(Kim et al. 1998). The inventors have previously shown that protein cage
architectures can
be utilized as size- and shape-constrained reaction environments for
nanomaterials synthesis.
(Douglas and Young, 1998; Usselman et al. 2005; Allen et al. 2002, 2003;
Fleimiken et al.
2003). These cages have been shown to stabilize inorganic nanoparticles in
defined sizes and
crystal forms. In addition, through genetic and chemical modifications, active
sites can be
created at precise locations within the cage architecture to create
dramatically new
functionality (Klem et al. 2005).
Using the interior of the cage for spatially selective mineralization, the
inventors of the
present application have successfully constructed a protein cage architecture
that mimics the
controlled molecular access to the active site displayed in H2ase. Briefly,
Xero- and hydro-
gels containing hydrogenase were prepared using tetramethoxysilane (TMOS)
starting
material. Sonication of TMOS with water and HCl generates tetrahydroxysilane.
Addition
of the buffered (pH 8.0) protein solution (1:1 v:v) initiates condensation
fonning the Si-O-Si
network. Sol-gel pellets were cast from this mixture in Teflon wells or
directly in reaction
vials. Gels were rinsed repeatedly with anaerobic buffer to remove
unencapsulated protein.
All H2ase:sol-gel samples were prepared under anaerobic conditions. Hydrogen
production
was initiated by adding methyl viologen and dithionite to the gel suspended in
buffer solution
contained in sealed, anaerobic vials as previously described. The headspace of
these reaction
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vials was sampled and analyzed by gas chromatography for quantification of
hydrogen
produced. Various aspects of the present invention are further provided below.
Material Durability
The purified hydrogenase from Clostricliuna pasteuf-ianurn is a highly
efficient
catalyst for the reduction of H+ to fonn H2. The long-tenn stability of this
enzyine is
significantly enhanced by modification of the protein through attaclunent of a
thin polymer
coating to the exterior surface of the protein or through encapsulation in
polyelectrolyte
multilayers. Additionally, the enzyme is iinmobilized within a 3-D cross-
linked poly-
viologen gel matrix for enhanced electron transfer efficiency.
Material Engineering
The ability to control nanoparticle composition and morphology, and therefore
the
ability to tune the photocatalytic properties of the nanomaterials, has been
exploited to
optimize the nanoparticles for efficient electron transfer to the hydrogenase
system. In
addition, this synthetic control utilizes electron donors that are cheap and
readily available
such as simple organic acids (acetate, tartrate, citrate). In addition,
expertise in the
formulation of electroactive poly-viologen gels allows the inventors to
incorporate the
nanoparticle photocatalyst and hydrogenase into a solid matrix, which enhances
and
optimizes the electron transfer efficiency between the electron donor and the
active
hydrogenase catalyst through control of the stoichiometric and spatial
relationship between
these two components.
Efficiency
The efficient H2 production from sunlight is accessed by controlling the
ainount of
light and directly analyzing the amount of H2 produced as a function of
hydrogenase,
photocatalyst, redox mediator, and electron donor. A Xe arc lamp, which mimics
the solar
spectrum, has been utilized as a light source for these studies.
Rate of H,,production
The rate of hydrogen production can be measured directly in enzymatic assay as
it
has been previously described. The rate of H2 is measured under conditions
where mass
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transport of redox partners is minimized in the fonnulation of the composite
materials
described in detail in the Exalnples.
Hydrogenase and Oa iifllibition
Incorporation of the hydrogenase into an electroactive polyiner gel allows
creation
of a material having a core-shell structure. The outer layer may be
incorporate with a
photocatalyst, a redox mediator (viologen) and an 02 reactive Cu colloid
generated by
photolysis of the catalyst. Cu is commonly used as an 02 scavenger and the
higli surface area
of the shell makes this very attractive for this purpose. Thus, the outer
layer may act as an 02
scrubbing layer to protect the hydrogenase present within the inner layer. The
inner layer
may coinprise the photocatalyst, the hydrogenase, redox mediators, and
electron donors. This
engineered approach may significantly enhance the overall stability of the
hydrogenase
towards OZ.
It has been demonstrated previously that various enzymes can be immobilized in
silica-oxide gel matrices (Sol-Gel) and remain fully active. In fact, in many
instances the
enzyme stability and long tenn durability or half-life of certain enzymes is
increased. The
ability to encapsulate enzymes has tremendous promise in both basic science
and
biotechnological applications. In some cases, immobilization of enzymes may
prolong the
stabilization of intermediates that are very short lived in solution. For
biotechnology
encapsulation can result in generating durable heterogenous catalyst for
potential industrial
applications.
Encapsulation of purified active hydrogenases in tetramethyl ortho silicate
derived
sol-gels has been demonstrated. The inventors have shown that a high
percentage of the
overall hydrogenase activity of both hydrogen oxidation and proton reduction
is retained
when these enzymes are embedded in these porous silica oxide polymeric gels.
The activity
of encapsulated hydrogenases from Clostridiurn pastef ianuin, LanapYobacter-
modestogalophilus, and Thiocapsa roseopersicilaa can be iminobilized with an
apparent
activity at least 65-70% of that of the enzyine in solution measured in the
reaction of
hydrogen evolution. Encapsulated hydrogenases show some enhanced stability
under storage
and increased temperature. The iminobilized NiFe hydrogenase from L.
naodestogalophilus
retains 85% of its hydrogen producing activity over a ten day period when
stored at room
temperature under nitrogen atmosphere. The results that hydrogenase enzymes
can be
immobilized in an active fonn, however, represents a major step in addressing
the practicality
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of utilizing hydrogenases in solid phase hydrogen producing materials by
heterogenous
catalysis.
Hz PRODUCING MATERIALS
Hydrogenase Stability
Iinmobilization of hydrogen producing enzyines in electroactive polrner gel
matrixes
The present invention provides an immobilized hydrogenase systein that
consists of
using various synthetic polyiners to encapsulate molecules of hydrogenase.
Various inethods
of encapsulation results in increased stability of the enzyine. Other factors
witli respect to
optimization of the polyiner matrix in which the hydrogenase enzyme are
embedded are 1)
the ability to dope the polymers with various electron transfer
agents/mediators and 2)
permeability of the polymer to substrates and products of the reaction. The
following
hydrogen producing enzymes have been successfully encapsulated: 1) Fe-only
hydrogenase,
2) NiFe bidirectional hydrogenase, and 3) Alkaline phosphatase (oxidation of
phosphate
coupled to hydrogen production). linmobilization within these porous polymers
allows for
high-throughput heterogeneous catalysis.
The iminobilized catalyst systems allow reducing potential to be obtained
through
chemical or electrical means. The doping of the three-dimensional catalyst
with electron
transfer agents/mediators (either from a synthetic chemical or biological
source) allows the
external source of reducing power to be applied at the surface of the three-
dimensional
immobilized catalyst system.
Controlled hydrogenase heterologous expression
The present invention further provides a method of controllably expressing
heterologous hydragenases in host cells. Controlled heterologous expression of
hydrogenase
from Slaewanella oneldensis has been achieved in the host E. coil. This was
accomplished by
the simultaneous expression of hydrogenase structural genes and putative
accessory gene
products involved in hydrogenase maturation. Maximal hydrogenase expression
may be
achieved by optimization of the current system and by substitution of
hydrogenase genes
from various sources. The controlled expression allows genetic engineering of
hydrogenase
enzymes for enhancing stability, catalytic activity, and derivatization for
the construction of
composite materials.
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Photocatalytic H2 generation
The present invention further includes a method of utilizing photocatalyst in
the
process of generating hydrogen gas. The addition of photocatalysts adds an
additional
eleinent of control to the system and potentially allows the use of lower
potential electron
transfer agents/niediators as a source of reducing equivalents. The catalyst
systems operates
through the application of either an electrical or cheinical
oxidation/reduction potential across
the catalyst itself. Key components of the photocatalysts and their specific
synthesis include:
1) Synthesis of catalytic nanoparticles with controlled size and composition
2) Electron transfer froin photo-activated nanoparticles to electron transfer
mediators
3) Electron transfer fronl photo-activated light harvesting molecules to
protein cage
encapsulated catalyst nanoparticles
Synthesis of nanoparticles with controlled size and composition
The present invention further provides a method of synthesizing nanoparticles
with
controlled size and composition. A biomimetic approach has been adopted to
systematically
-alter the"composition 'of the metal oxide based nanomaterials to tune the
efficiency of the
photo-redox process. A range of protein encapsulated nanomaterials have been
generated to
evaluate the effect of composition of the effectiveness of MV generation.
These inaterials
include, but not limited to, Fe203, Fe304, Mn203, Mn304, Co203, Co304,
TiO2=nH2O as well
Fe- and Co- based materials doped with varying amounts of Ni(II), ZnSe, CdSe,
CdS, ZnS,
and MoS2. These materials have all been synthesized and structurally
characterized.
O Scaven ing
In one embodiment, the present invention employs 02 scavenger in the
construction
of Nanoparticles. For example, Cu(O), which are highly 02 reactive and can act
as
scavengers of 02 may be encapsulated within protein cage architectures to
protect the activity
of hydrogenase (or other) redox active enzyme. Thus, a hydrogenase protein
cage containing
copper oxide may have several advantages. First, Cu acts as an in situ 02
scavenging systein
and the high surface area of the nanoparticles makes this very attractive for
the purpose.
Second, the reducing equivalents generated by the photoreduction of MV+ can be
used to
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drive the turnover of the hydrogenase system. The reduced MV+ is not able to
reduce Cu(II)
to Cu(O) so these two products of the photoreduction are naturally independent
of each other.
Photo-catalysts and light harvesting chromophores
In another embodiment, the present invention provides a metllod of using
protein
case architecture as a platfonn for light harvesting molecules. For example,
the protein cage
architectures of CCMV (and other viruses), ferritin (and ferritin-like
proteins), small heat
shock proteins, Dps proteins can be used as a multivalent templates for
attachinent of light
harvesting molecules, whicli can be used to drive the photocheinical reduction
of electron
transfer mediators (like methyl viologen). These include molecules such as
Ru(II)bipyridine
and Ru(II)phenanthroline which can be attached to the cages in a site specific
manner. The
reduced methyl viologen can be generated fiom the oxidized methyl viologen
(MV) through
the photochemical oxidation of organic species (EDTA, for example) with
Ru(bpy)32+ as the
catalyst. In addition, light harvesting chromophores can be directly attached
to redox active
enzyines, such as hydrogenase, and potentially eliminate mass transport
limitations and the
need for electron transfer mediators such as inethyl viologen.
Protein cage encapsuTated cafalyst nanoparticles
The present invention further provides a composition comprising a protein cage
where catalyst nanoparticles are encapsulated. The inventors have demonstrated
that
nanoparticles of Pt can be efficiently encapsulated within the protein cage
architectures
(CCMV, ferritin, Hsp, Dps). These particles are size and shape constrained by
the protein
cage, giving rise to Pt colloids with very high relative surface areas, which
yields high H2
formation through reduction of H+. The required reducing equivalents for this
reaction can be
supplied by reduced methyl viologen (MV). Alternatively, the reduced viologen
can be
generated chemically by the oxidation of Zn (as Hg/Zn amalgam) in the presence
of EDTA.
Using this coupled system, H2 generation may be optimized through
investigation of the Pt
particle size dependence, nature of the protein cage architecture (i.e.,
diffusional access of the
reduced viologen to the Pt nanoparticle), inhibiting catalyst poisoning for
longevity,
composition of the nanoparticle catalyst (e.g., Pd, CoPt, FePt), and the
nature of the
photocatalyst couple.
Synthesis of nanoparticles of different composition and alloy particles in
particular
may take advantage of the inventor's success in incorporating small peptides
(derived from
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phage-display) onto the inside of the protein cage architectures. These
peptides have been
shown to direct the nucleation and particle growth of a par-ticular inorganic
solid and are also
able to direct polyinorph selection. Thus, the synthesis of a nuinber of
inorganic phases can
be directed to screen for an optimal balance of long-terin catalyst stability
and activity in the
reduction of H} to fonn H2.
Pt particles encapsulated within the protein cage as disclosed herein serve as
synthetic hydrogenase mimics. Such a system allows the incorporation of the
best
characteristics of colloidal catalysts with biological catalysts into a
synthetic material. While
both colloidal catalysts and biological catalysts have their own limitations
(sensitivity to
oxygen, poisoning, costs, and reaction conditions and longevity), a
combination of these two
types of catalyst circumvents these limitations.
Reductants
The present invention further includes reductants for reduced methyl viologen
(and
other) mediator forination. Organics such as ethylenediamine tetraacetic acid,
tartrate,
citrate, acetate, ethanol (and other alcohols). Inorganics such as
hydroxylamine, sulfite,
thiosulfate, dithionite, and Zn can all be used. The advantage of using
Sulfite (S032-) is that
this is a polluting by-product of petroleum refining (SOZ + H20--> HS03-). The
oxidation of
sulfite results in the fonnation of sulfate (S042-). Thus, utilizing sulfite
as a reductant also
overcomes the problem of COZ generation caused by oxidation of organic
species.
Composite materials
The present invention further provides a method of facilitating electron
transfer from
redox protein to electrode. The nanoscopic confinement of redox active
proteins in silica-
derived sol-gel materials requires mediators, such as methyl viologen, to
facilitate electron
transfer with the protein. The porous nature of these gels provides access to
the encapsulated
protein. Materials that facilitate direct electron transfer between an
electrode surface and
redox centers of hydrogenase and other redox active proteins will eliminate
the catalytic
dependence on chemical reductants. In the case of hydrogenase, these novel
materials will
facilitate the flow of electrons from the encapsulated enzyme to an electrode
during
enzymatic ally catalyzed generation and oxidation of H-2 (g). The materials
are derived froin
13
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WO 2007/086918 PCT/US2006/018900
electroactive matrixes. A variety of bioelectronic glasses have been reported,
including Sn-
doped silica [Sn/SiO2], V205, MoO3, and Mn02.
Coupled enz pne systems for hydrogen production from sitlfite
Hydrogenase can be coupled to the enzyine sulfite oxidase eitlier 1) in a
freely
diffusing solution based system 2) by covalent attachment (cross-linking of
sulfite oxidase
and hydrogenase or 3) by the incorporation of both components in an
electroactive porous
gel. In this system the reducing equivalents derived from the enzymatic
oxidation of sulfite
to sulfate can be directed to reduction of protons by hydrogenase. As
mentioned above the
advantage of using sulfite (S032-) is that this is a polluting byproduct of
petroleuin refining
(SO2 + H20 ---> HS03-). The oxidation of sulfite results in the forination of
sulfate (S042-).
Thus, utilizing sulfite as a reductant also overcomes the problem of CO2
generation caused by
oxidation of organic species.
Coupling photocatalysts to hydrogenase
Hydrogenase enzymes is coupled to photocataysts (including nanoparticle
photocatalysts) in the development of heterogenous catalysts that can harness
light energy to
produce hydrogen from abundant electron donor sources such as sulfite, organic
acids, or
perhaps ethanol. Coupled systems can work in either aqueous solution or in
immobilized gel.
To produce a heterogenous catalysis such that substrates and products can be
transferred in a
liquid phase, the components of the composite materials may be immobilized in
electroactive
gels (silica oxide or other polymers, for example, polyviologen). The addition
of oxygen
consuming catalytic nanoparticles such as Cu(O) serves to protect the oxygen
sensitive
hydrogenases from oxygen inactivation.
Without further description, it is believed that one of ordinary skill in the
art can,
using the preceding description and the following illustrative examples, make
and utilize the
compounds of the present invention and practice the claimed methods. The
following
working examples therefore, specifically point out the preferred embodiments
of the present
invention, and are not to be construed as liiniting in any way the remainder
of the disclosure.
Example 1: Encapsulation of Hydrogenases in Polymer Gel
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WO 2007/086918 PCT/US2006/018900
This example is focused specifically on the hydrogen production activity of
H2ase: sol-
gel materials. Hydrogenases are bi-directional enzymes whicll also catalyze
the oxidation of
hydrogen. H2ase: sol-gel pellets were assayed for hydrogen oxidation activity
by placing the
pellets in buffered (pH 8.0) solution under a head pressure of hydrogen.
Electron flow was
monitored by the reduction of inetliyl viologen. H2ase:sol-gel pellets with
either the NiFe
(Lamprobacter modestogalophilus (Lin) and Thiocapsa roseopetsicina (Ti),) or
Fe-only
(Clostridium pasteurianum (CpI)) forms of H2ase retain approximately 60-70% of
the
hydrogen evolution specific activity observed in solution (Table 1) (Isolation
of
hydrogenases: (a) L. modestogalophilus: Zadvorny, O. A.; Zorin, N. A.;
Gogotov, I. N.;
Gorlenlco, V. M. Biochemistry (Mosc) 2004, 69,164-169. (b) T. roseopersicina:
Shennan,
M. B.; Orlova, E. V.; Smirnova, E. A.; Hovmoller, S.; Zorin, N. A. JBacteriol.
1991,
173, 2576-2580. (c) C. pasteurianum: Chen, J. S.; Mortenson, L. E. Biochim.
Biophys.
Acta 1974, 371, 283-298).
Table 1 Hydrogen Production Activity of Sol-Gel Encapsulated Hydrogenases
hydrogenase solution gel solution/gel (%)
C. pasteuricrnurn 12550 7581 60.4 16
L. inoclestogaloplzilus9150 6175 _ 67.5 9 _
T. 7roseopersicina 12600 8834 70.1 ~ 3
a The activity measure at 25 C is indicated in nmol/min/mg protein.
The values represent average rate over a four-hour period.
Blank gels assayed under identical conditions showed no hydrogen production.
Par-tially purified preparations consisting of heat treated crude extract
preparations (treated
crude extracts were prepared by cell lysis by pressure cell treated followed
by incubation at
55 C for 10 min, overnight precipitation at 4 C and removal of cellular
particulate and
precipitated protein by centrifugation at 20 000g) of the Fe-only hydrogenase
CpI fiom C.
pasteusrianum retained similar levels of activity (70% and 60%, respectively)
following
encapsulated when compared to more highly purified preparations. This suggests
that the co-
encapsulation of additional protein macromolecular components of the extract
does not
significantly impact the activity. Although the encapsulation of crude
extracts results in a
more poorly defined material, the ability to encapsulate and immobilize crude
preparations of
enzyme while retaining a nearly equivalent level of activity is important as a
matter of
practicality since the heterologous expression of hydrogenase is not facile,
purification is
CA 02608870 2007-11-16
WO 2007/086918 PCT/US2006/018900
laborious, and the preparation of bulk purified materials very costly. To
date, CpI is purified
directly from cells of C. pasteurianurn grown under anaerobic conditions and
the enzyine is
purified in the absence of oxygen and in the presence of reducing agents.
Long term stability and temperature compatibility are essential features of
potential
high tllrougliput hydrogengenerating biocatalysts. Activated hydrogenase
enzymes are
sensitive to oxygen, and typically, dilute enzyme preparations can have a
limited shelf life at
rooin temperature. These properties are barriers that need to be overcome for
the utility of
these biocatalysts to be realized. Detailed studies on shelf life (Figure 2)
and thermal
stability (Figure 3) were conducted with purified Lm NiFe hydrogenase and CpI
heat-
treated extract. The gel-encapsulated enzyme can be stored at room temperature
under
anaerobic buffer while retaining approximately 80% of the activity of the
starting material
over a four-week period (Figure 2). The enhancement of the stability of the
encapsulated
hydrogenases is most pronounced at the longer time periods. Temperature
studies indicate
that the encapsulated enzymes fiu-ietion comparably to those in solution
(Figure 3). Future
studies will monitor longer storage periods; however, the results shown here
are encouraging
considering optimization of the conditions for encapsulation of these enzymes
has not been
attempted.
It is unclear at this point what factors contribute to the time dependent
decline in
activity of the H2ase:sol-gel. It appears that the reduced mediator binds to
the sol-gel
material after prolonged exposure. This is not surprising given that the Si-O
network of the
TMOS derived sol-gel and unreacted Si-OH moieties present strong ion pairing
potential
within the sol-gel material. This may contribute to the slow decrease in
activity of the
H2ase:sol-gel over time.
Solution and sol-gel encapsulated samples of CpI H2ase were treated with
protease
(Samples of C. pasteurianuna extract were treated with a protease cocktail for
a 25 min
period prior to activity assay) to ensure that the hydrogen producing activity
observed is
derived from enzyme embedded within the porous structure of the sol-gel
material
(Figure 4) (Tr and Lm hydrogenases are not susceptible to proteolytic
digestion). Solution
CpI H2ase activity is reduced to nearly zero after 25 lnin exposure to
protease. CpI H,,ase:sol-
gel activity decreases by less than 7% following similar protease treatment.
This small
decrease may result from protease digestion of accessible surface bound or
unencapsulated
H2ase. These results clearly indicate that the majority of the active H2ase is
embedded within
the gel and is protected against proteolysis. Furthermore, these results show
that there is a high
16
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WO 2007/086918 PCT/US2006/018900
efficiency of encapsulation and that observed reduction in the hydrogenase
activity is not due
to a partial encapsulation of protein. The loss of activity can therefore be
attributed to either
enzyme inactivation as a result of the encapsulation procedure or the overall
reduction in the
rate of mass transfer in the matrix.
Nanoscopic confinement of H2ase within a porous sol-gel has resulted in the
synthesis of a functional hydrogen producing biomaterial. As heterogeneous
catalysts, these
new materials have enormous potential and utility.
Example 2: Synthesis of Pt Nanoparticles Encapsulated within the Hsp Cage
This example describes the synthesis of Pt nanoparticles encapsulated within
the Hsp
cage. The self-assembled cage-like architecture of the small heat shock
protein (Hsp) from
Methanococcusjannaschii has been used to encapsulate metal clusters with a
defined spatial
arrangement (Flenniken et al. 2003). Hsp assembles from 24 subunits into a 12
nm cage
defining a 6.5 nm interior cavity with pores through the cage architecture, by
which
molecules can shuttle between the inside and outside environinent (Figure 5)
(Kim et al.
1998).
Briefly outlined, purified Hsp was incubated with PtC14Z- at 65 C for 15 min.
The
protein cage was incubated with either 150, 250, or 1000 Pt per protein cage.
Subsequent
reduction with diinetliylamine borane complex ((CH3)2NBH3) resulted in the
formation of a
brown solution. Characterization of the reaction product by size exclusion
chromatography revealed retention volumes identical with untreated Hsp and
showed
coelution of protein (280 nm) and Pt (350 nm) components (Figure 6). Dynamic
light
scattering indicated no change in the particle diameter after the reaction.
Visualization of the
Pt-treated Hsp (Pt-Hsp) by transmission electron microscopy (TEM) revealed
electron dense
cores identified as Pt metal by electron diffraction (Figure 7A inset) and
intact 12 iun protein
cages when negatively stained (Figure 7B). In the stained samples, the Pt
particles can
clearly be seen above the background stain and are localized within the cage
structure. For
average loadings of 1000 Pt/cage, metal particles of 2.2 ( 0.7 nm were
observed (Figure 7C).
At theoretical loadings of 250 Pt/cage, particles of 1( 0.2 nm were observed
(Figure 8), while
at loadings of 150 Pt/cage, no particles could be distinguished due to the
limitation of the
electron microscope. Hsp-free control reactions resulted in the formation of
aggregated Pt
colloids, which rapidly precipitated from solution. Control reactions using
bovine serum
albumin (BSA), at the same total protein concentration, also resulted in bulk
precipitation and
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WO 2007/086918 PCT/US2006/018900
only a fraction of the Pt remained in solution with a wide distribution of
particle sizes (3-120
nin) when observed by TEM.
The Pt-Hsp protein cage composites are highly active artificial catalysts able
to reduce
H4'to fonn H2 at rates coinparable to the highly efficient hydrogenase
enzymes. Typical of
hydrogenase assays, reduced inetliyl viologen (MV) was used as a source of
reducing
equivalents to drive the reaction. Unlike most in Vitro hydrogenase assays,
which use
dithionite to generate MV+, visible light and a cocatalyst (Ru(bpy)32) have
been used to
generate MV+ tlirough oxidation of simple organics such as EDTA (Biugger et
al. 1981;
Jiang et al. 2004) (Figure 9). In this modified assay, the solution was
illuminated at 25 C
with a 150 W Xe arc lamp equipped with an IR filter and a UV cutoff filter
(<360 nm).
The Pt-Hsp (0.51 M) was illuminated in the presence of MVZ+ (0.5 mM),
Ru(bpy)32} (0.2
mM), and EDTA (200 mM) at pH 5.0, and the resulting H2 was quantified by gas
chromatography. When calculated on a per cage basis, the initial rate of H2
formation was
4.47 x 103 H2/s ((394 H2/s) for a loading factor of 1000 Pt per Hsp and 7.63 x
102 H2/s
((405 H2/s) for a loading factor of 250 (Figure 6). These rates are
coinparable to those
reported for hydogenase enzymes (4 x 103 to 9 x 103 H2/s per protein molecule)
(Adams et
al. 1990).
No H2-producing activity was detected for the lowest Pt loading (150 Pt/Hsp).
This is
consistent with previous reports of size-dependent activity of Pt (Greenbaum
et al. 1988). It is
also consistent with our inability to detect discrete Pt particles by TEM in
these samples,
implying that the synthesized Pt particles are below some threshold limit
required for activity.
When the H2 production rates are calculated on a per Pt basis, they compare
very
favorably with other reported Pt nanoparticles. The initial rates for Pt-Hsp
with 1000 Pt/
cage are 268 H2/Pt/min, which is sigiiificantly better than reported
literature values (20
H2/Pt/min (Brugger et al. 1981), 16 H2/Pt/min (Keller et al. 1980), and 6.5
H2/Pt/min (Song
et al. 2004), where coinparisons are possible. In addition, initial H2
production rates for Pt-
Hsp are approximately 20-fold greater than those obtained for the Pt particles
produced in
protein-free control reactions. The long-term stability of the coupled
photocheinical reaction
to produce H? has not been optimized, and a sigiiificant slowing down of the
reaction is
observed after the first 20 min (Figure 10). The H2 production decay may be
mainly due to the
degradation of the photocatalyst Ru(bpy)32+, and the electron mediator (MV2+),
whicll is
subject to Pt-catalyzed hydrogenation (Keller et al. 1980).
Unlike the hydrogenase enzymes, the artificial Pt-Hsp systems are not
sensitive to 02
18
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WO 2007/086918 PCT/US2006/018900
and show no significant iiihibition of H2 production by CO but are poisoned by
thiols. The Pt-
Hsp catalyzed reaction was driven by the presence of the reduced viologen
(MV'). The MV+
could be generated either by the photoreduction described above or by using
the Jones
redactor (Harris et al. 1999) (Zn ainalgaln), which yielded rates for H2
production
approximately 40% slower than the coupled photoreduction reactions. Also, the
Pt-Hsp is able
to catalyze the reverse reaction (H2 -- 2H+ + 2e ) as monitored by the in situ
reduction and
bleaching of methylene blue (Seeffeldt et al. 1989). Importantly for the
utility of this
artificial system, the Pt-Hsp construct is reinarlcably stable and can be
heated to 85 C without
precipitation of the composite or loss of the catalytic activity.
A well-defined therinally stable protein cage architecture has been used to
generate an
artificial hydrogenase having many of the features common to those biological
catalysts.
small metal clusters in a spatially selective manner have been introduced to
the interior
of the cage-like structure of Hsp that act as active sites for the reduction
of H} to form H2.
The specific activities of these artificial enzymes are comparable to known
hydrogenase
enzymes and significantly better than previously described Pt nanoparticles.
The protein cage
architecture of Hsp acts to maintain the integrity of the small clusters,
preventing
agglomeration, and controlling access to these "active sites". The Pt-Hsp
composite is stable
up to 85 C illustrating the utility of using protein architectures for the
design and
impleinentation of fuiictional nanomaterials.
The foregoing detailed description has been given for clearness of
understanding
only and no umlecessary limitations should be understood therefiom as
inodifications will be
obvious to those skilled in the art.
While the invention has been described in connection with specific embodiments
thereof, it will be understood that it is capable of further modifications and
this application is
intended to cover any variations, uses, or adaptations of the invention
following, in general,
the principles of the invention and including such departures from the present
disclosure as
come within lalown or customary practice within the art to which the invention
pertains and
as may be applied to the essential features hereinbefore set forth and as
follows in the scope
of the appended claims.
All publications cited herein are incorporated herein by reference for the
purpose of
disclosing and describing specific aspects of the invention for which the
publication is cited.
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