U.S. patent application number 11/914522 was filed with the patent office on 2008-12-11 for composite nanomaterials for photocatalytic hydrogen production and method of their use.
Invention is credited to Trevor Douglas, Timothy E. Elgren, John W. Peters, Mark J. Young.
Application Number | 20080302669 11/914522 |
Document ID | / |
Family ID | 38309659 |
Filed Date | 2008-12-11 |
United States Patent
Application |
20080302669 |
Kind Code |
A1 |
Peters; John W. ; et
al. |
December 11, 2008 |
Composite Nanomaterials for Photocatalytic Hydrogen Production and
Method of Their Use
Abstract
The present invention is directed to a composite material for
photocatalytic H.sub.2 production comprising: 1) a polymer gel; 2)
a photocatalyst; and a protein based H.sub.2 catalyst. The
invention also relates to a method to produce H.sub.2, comprising
reacting an electron donor with a composite material comprising 1)
a polymer gel, 2) a photocatalyst, and 3) a protein based H.sub.2
catalyst.
Inventors: |
Peters; John W.; (Bozeman,
MT) ; Young; Mark J.; (Bozeman, MT) ; Douglas;
Trevor; (Bozeman, MT) ; Elgren; Timothy E.;
(Bozeman, MT) |
Correspondence
Address: |
COOLEY GODWARD KRONISH LLP;ATTN: Patent Group
Suite 1100, 777 - 6th Street, NW
WASHINGTON
DC
20001
US
|
Family ID: |
38309659 |
Appl. No.: |
11/914522 |
Filed: |
May 16, 2006 |
PCT Filed: |
May 16, 2006 |
PCT NO: |
PCT/US06/18900 |
371 Date: |
June 26, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60681174 |
May 16, 2005 |
|
|
|
Current U.S.
Class: |
205/340 |
Current CPC
Class: |
C12N 9/0067 20130101;
C12N 11/14 20130101; C12P 3/00 20130101 |
Class at
Publication: |
205/340 |
International
Class: |
C25B 1/02 20060101
C25B001/02 |
Goverment Interests
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
[0002] The invention disclosed herein was funded, in part, by the
National Science Foundation under grant number MCB-0328341 and the
Office of Naval Research under grant number 19-00-R0006.
Claims
1. A composite material for photocatalytic H.sub.2 production
comprising: 1) a polymer gel 2) a photocatalyst; and 3) a protein
based H.sub.2 catalyst.
2. The composite material of claim 1, wherein the H.sub.2 catalyst
is a hydrogenase.
3. The composite material of claim 2, wherein the hydrogenase is
encapsulated in the polymer gel.
4. The composite material of claim 3, wherein the gel is
porous.
5. The composite material of claim 4, wherein the gel is
sol-gel.
6. The composite material of claim 1, wherein the H.sub.2 catalyst
is a hydrogenase mimic.
7. The composite material of claim 6, wherein the hydrogenase mimic
is a nanoparticle.
8. The composite material of claim 7, wherein the nanoparticle is
in the form of a protein cage comprising a shell and a core.
9. The composite material of claim 8, wherein the shell comprises a
protein.
10. The composite material of claim 9, wherein the protein is a 24
subunit protein.
11. The composite material of claim 10, where the protein is small
heat shock protein (HSp).
12. The composite material of claim 8, wherein the core comprises a
metal.
13. The composite material of claim 12, wherein the metal is
selected from the group consisting of platinum, nickel, iron, and
cobalt.
14. The composite material of claim 13, wherein the metal is
platinum.
15. The composite material of claim 1, further comprising a redox
mediator.
16. The composite material of claim 15, wherein the redox mediator
comprises poly viologen.
17. The composite material of claim 16, further comprising an
oxygen scavenger.
18. The composite material of claim 17, wherein the oxygen
scavenger comprises Cu(0).
19. The composite material of claim 1 wherein the photocatalyst is
formulated as a nanoparticle.
20. The composite material of claim 1 or claim 19, wherein the
photocatalyst is encapsulated in a protein cage architecture.
21. The composite material of claim 1, wherein the hydrogenase
enzyme is derived from Clostridium pasteurianum, Lapiobacter
modestogalophilus, Thiocapsa reseopericina, or a combination
thereof.
22. The composite material of claim 17, wherein the material
comprises: i) an inner layer further comprising the photocatalyst
and the hydrogenase enzyme, and ii) an outer layer further
comprising the photocatalyst, the redox mediator, and the oxygen
scavenger.
23. A method to produce H.sub.2, comprising reacting an electron
donor with a composite material comprising 1) a polymer gel, 2) a
photocatalyst, and 3) a protein based H.sub.2 catalyst.
24. The method of claim 23, wherein the H.sub.2 catalyst is a
hydrogenase.
25. The method of claim 24, wherein the hydrogenase enzyme is
derived from Clostridium pasteurianum, Laprobacter
modestogalophilus, Thiocapsa reseopericina, or a combination
thereof.
26. The method of claim 24, wherein the hydrogenase is encapsulated
in the polymer gel.
27. The method of claim 26, wherein the gel is porous.
28. The method of claim 27, wherein the gel is sol-gel.
29. The method of claim 23, wherein the H.sub.2 catalyst is a
hydrogenase mimic.
30. The method of claim 29, wherein the hydrogenase mimic is a
nanoparticle.
31. The method of claim 30, wherein the nanoparticle is in the form
of a protein cage comprising a shell and a core.
32. The method of claim 31, wherein the shell comprises a
protein.
33. The method of claim 32, wherein the protein is a 24 subunit
protein.
34. The method of claim 33, where the protein is small heat shock
protein (HSp).
35. The method of claim 31, wherein the core comprises a metal.
36. The method of claim 35, wherein the metal is selected from the
group consisting of platinum, nickel, iron, and cobalt.
37. The method of claim 36, wherein the metal is platinum.
38. The method of claim 23, wherein the photocatalyst is formulated
as a nanoparticle.
39. The method of claim 23 or claim 38, wherein the photocatalyst
is encapsulated in a protein cage architecture.
40. The method of claim 23, wherein the electron donor is one of
the group consisting of acetic acid, citric acid, tartaric acid,
ethanol, EDTA, hydroxylamine, and mixtures thereof.
41. The method of claim 23, wherein the electron donor is one of a
group consisting of sulfite, thiosulfate, and dithionite.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is entitled to priority pursuant to 35
U.S.C. .sctn.119(e) to U.S. provisional patent application No.
60/681,174, which was filed on May 16, 2005, which is incorporated
herein in its entirety for all purposes.
FIELD OF THE INVENTION
[0003] The present invention relates to composite materials
containing hydrogenase enzymes and hydrogen-producing nanoparticles
with photocatalysts for hydrogen production from renewable
sources.
BACKGROUND OF THE INVENTION
[0004] There is considerable recent interest in the production of
hydrogen gas as an alternative 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
(Armor 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.
[0005] Hydrogenases are highly evolved catalysts that produce
hydrogen gas at rates that are the envy of synthetic chemists.
Metal-containing hydrogenases (H.sub.2ases) (Vignais et al. 2001;
Peters et al. 1998, 1999; Adams et al. 1990) are produced by a
variety of microorganisms where they function either in hydrogen
oxidation or proton reduction according to the following
reaction:
##STR00001##
[0006] Hydrogen oxidation is coupled to the generation of reducing
equivalents to drive energy yielding or biosynthetic processes.
During anaerobic fermentation some microorganisms 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 known enzymes that
utilize these normally 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).
[0007] 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 9000H.sub.2 per enzyme per second
have been observed (Adams et al. 1990; Cammack et al. 1999) from
these enzymes, the extreme sensitivity of these enzymes to oxygen,
limited expression, and difficult isolation have hindered their use
as a practical means of hydrogen production.
[0008] Attempts to develop methods and systems to produce hydrogen
on a commercial scale using the hydrogenase enzyme have been
deficient. U.S. Pat. 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 form molecular hydrogen.
[0009] 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 comprises 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 decompose the accumulated material by photosynthesis to
evolve hydrogen. This method uses a nitrogen gas purge technique to
remove oxygen from the culture.
[0010] 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
medium 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 CO.sub.2 and atmospheric O.sub.2.
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 H.sub.2-producing cultures
with inert gases has allowed for the sustained production of
H.sub.2, such purging is expensive and impractical for large-scale
mass cultures of algae.
[0011] 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 H.sub.2 formation, photocatalytic nanomaterials, and
electro/photo-chromic polymer gel technology.
SUMMARY OF THE INVENTION
[0012] 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 H.sub.2 production
comprising: 1) a polymer gel, 2) a photocatalyst, and 3) a protein
based H.sub.2 catalyst. The H.sub.2 catalyst may be an enzyme such
as a hydrogenase enzyme, which can be derived from a variety of
organisms, such as microorganisms including but not limited to
Clostridium pasteurianum, Laprobacter modestogalophilus, Thiocapsa
reseopericina, or combinations thereof. The composite material can
further comprise a redox mediator, such as poly viologen, and an
oxygen scavenger, such as Cu(0). The photocatalyst can be
formulated as a nanoparticle and can be encapsulated in a protein
cage architecture.
[0013] The H.sub.2 catalyst may also be an artificial enzyme such
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
platinum, nickel, iron, and cobalt.
[0014] The present invention also relates to a method for producing
H.sub.2 using the composite materials of the present invention. For
example, the present invention provides methods of producing
H.sub.2 comprising reacting an electron donor with a composite
material comprising 1) a polymer gel, 2) a photocatalyst, and 3)
protein based H.sub.2 catalyst. The electron donor can be obtained
from a variety of sources, such as but not limited to acetic acid,
citric acid, tartaric acid, ethanol, EDTA, hydroxylamine, and
mixtures thereof. The electron donor can also be sulfite,
thiosulfate, and dithionite.
BRIEF DESCRIPTION OF THE DRAWINGS
[0015] For a more complete understanding of the present invention
and for further advantages thereof, reference is made to the
following drawings:
[0016] FIG. 1 is a drawing of a composite material used in a
hydrogen producing device.
[0017] FIG. 2 is a graph showing hydrogen production activity of C.
pasteurianum (CpI) and L. modestogalophilus (Lm) hydrogenases in
solution and encapsulated in sol-gel: .diamond-solid., CpI in
solution; , CpI in sol-gel; .tangle-solidup., Lm in solution;
.box-solid., Lm in sol-gel.
[0018] FIG. 3. is a graph showing temperature dependence of
hydrogen production activity of C. pasteurianum (CpI) and L.
modestogalophilus (Lm) hydrogenases in solution and encapsulated in
sol-gel: .diamond-solid., CpI in solution; , CpI in sol-gel;
.tangle-solidup., Lm in solution; .box-solid., Lm in sol-gel.
[0019] FIG. 4. is a graph showing hydrogen production activity of
solution and sol-gel encapsulated hydrogenase from C. pasteurianum
in the presence (open) and absence (shaded) of protease.
[0020] FIG. 5 is (A) a space filling representation of the small
heat shock protein (Hsp) cage from Methanococcus jannaschii (pdb: 1
shs) and (B) a cut-away view of Hsp showing the interior cavity of
the cage.
[0021] FIG. 6 is a size exclusion chromatography of Hsp: (A)
umineralized 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).
[0022] FIG. 7 is an image showing (A) TEM of Hsp 1000 Pt unstained.
The inset shows electron diffraction of Pt.sup.0 from Hsp 1000 Pt.
(B) TEM of Hsp 1000 Pt stained with 2% uranyl acetate. (C)
Histogram of Pt particle diameters in Hsp 1000 Pt. Average 2.2 (0.7
nm. Scale bars) 20 nm.
[0023] FIG. 8 is an image showing (A) TEM of Hsp 250 Pt stained
with 2% uranyl acetate. (B) Histogram of Pt particle diameters in
Hsp 250 Pt. Scale bar) 20 nm.
[0024] FIG. 9 is a schematic presentation of the light-mediated
H.sub.2 production from Pt-Hsp. Methyl viologen (MV.sup.2+) is used
as an electron-transfer mediator between the Ru(bpy).sub.3.sup.2+
photocatalyst and the Pt-Hsp responsible for H.sub.2
production.
[0025] FIG. 10 is a graph showing H.sub.2 production from Pt-Hsp in
0.2 mM Ru(bpy).sub.3.sup.2+, 0.5 mM methyl viologen, 200 mM EDTA,
and 500 mM acetate pH 5.0: .tangle-solidup., 1000 Pt/Hsp
5.1.times.10.sup.-10 mol Pt; .box-solid., 250 Pt/Hsp
8.2.times.10.sup.-10 mol Pt.
DETAILED DESCRIPTION OF THE INVENTION
[0026] 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.
[0027] 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.
[0028] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention 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 preferred methods and materials are described.
[0029] The present invention is related to enzymatic H.sub.2
formation, photocatalytic nanomaterials, and electro/photo-chromic
polymer gel technology. The invention provides systems that
substantially increase the efficiency with which 1H.sub.2 can be
produced from very simple feedstocks, namely 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 enzyme
and produce H.sub.2. To optimize these results, hydrogenase enzyme
is immobilized and encapsulated into a polymer gel matrix and the
resulting enzyme gel pellets are incubated with an electron donor
such as dithionite and an electron transfer mediator such as methyl
viologen to generate H.sub.2. This approach utilizes the efficiency
and specificity of the hydrogenase enzyme, and the synthetic
control exerted in the polymer formulation to construct robust
composite materials that will meet many of the targets and
objectives of economic hydrogen gas production.
[0030] 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, which 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
thereof. 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.
[0031] One of metal catalyst commonly used for chemical reaction is
platinum. 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
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.
[0032] 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 number 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;
Flenniken 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).
[0033] 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
H.sub.2ase. 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 forming 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 H.sub.2ase: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 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
[0034] The purified hydrogenase from Clostridium pasteurianum is a
highly efficient catalyst for the reduction of H.sup.+ to form
H.sub.2. The long-term stability of this enzyme is significantly
enhanced by modification of the protein through attachment of a
thin polymer coating to the exterior surface of the protein or
through encapsulation in polyelectrolyte multilayers. Additionally,
the enzyme is immobilized within a 3-D cross-linked poly-viologen
gel matrix for enhanced electron transfer efficiency.
Material Engineering
[0035] 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
[0036] The efficient H.sub.2 production from sunlight is accessed
by controlling the amount of light and directly analyzing the
amount of H.sub.2 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.sub.2 Production
[0037] The rate of hydrogen production can be measured directly in
enzymatic assay as it has been previously described. The rate of
H.sub.2 is measured under conditions where mass transport of redox
partners is minimized in the formulation of the composite materials
described in detail in the Examples.
Hydrogenase and O.sub.2 Inhibition
[0038] Incorporation of the hydrogenase into an electroactive
polymer 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 O.sub.2 reactive Cu colloid
generated by photolysis of the catalyst. Cu is commonly used as an
O.sub.2 scavenger and the high surface area of the shell makes this
very attractive for this purpose. Thus, the outer layer may act as
an O.sub.2 scrubbing layer to protect the hydrogenase present
within the inner layer. The inner layer may comprise the
photocatalyst, the hydrogenase, redox mediators, and electron
donors. This engineered approach may significantly enhance the
overall stability of the hydrogenase towards O.sub.2.
[0039] 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 term 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.
[0040] 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 Clostridium pasteurianum, Lamprobacter
modestogalophilus, and Thiocapsa roseopersicina can be immobilized
with an apparent activity at least 65-70% of that of the enzyme in
solution measured in the reaction of hydrogen evolution.
Encapsulated hydrogenases show some enhanced stability under
storage and increased temperature. The immobilized NiFe hydrogenase
from L. modestogalophilus 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 form, however, represents a major step
in addressing the practicality of utilizing hydrogenases in solid
phase hydrogen producing materials by heterogenous catalysis.
H.sub.2 Producing Materials
Hydrogenase Stability
Immobilization of Hydrogen Producing Enzymes in Electroactive
Polymer Gel Matrixes
[0041] The present invention provides an immobilized hydrogenase
system that consists of using various synthetic polymers to
encapsulate molecules of hydrogenase. Various methods of
encapsulation results in increased stability of the enzyme. Other
factors with respect to optimization of the polymer 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). Immobilization within
these porous polymers allows for high-throughput heterogeneous
catalysis.
[0042] The immobilized 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
[0043] The present invention further provides a method of
controllably expressing heterologous hydrogenases in host cells.
Controlled heterologous expression of hydrogenase from Shewanella
oneldensis has been achieved in the host E. coli. 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.
Photocatalytic H.sub.2 Generation
[0044] The present invention further includes a method of utilizing
photocatalyst in the process of generating hydrogen gas. The
addition of photocatalysts adds an additional element of control to
the system and potentially allows the use of lower potential
electron transfer agents/mediators as a source of reducing
equivalents. The catalyst systems operates through the application
of either an electrical or chemical oxidation/reduction potential
across the catalyst itself. Key components of the photocatalysts
and their specific synthesis include: [0045] 1) Synthesis of
catalytic nanoparticles with controlled size and composition [0046]
2) Electron transfer from photo-activated nanoparticles to electron
transfer mediators [0047] 3) Electron transfer from photo-activated
light harvesting molecules to protein cage encapsulated catalyst
nanoparticles
Synthesis of Nanoparticles with Controlled Size and Composition
[0048] 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
materials include, but not limited to, Fe.sub.2O.sub.3,
Fe.sub.3O.sub.4, Mn.sub.2O.sub.3, Mn.sub.3O.sub.4, CO.sub.2O.sub.3,
CO.sub.3O.sub.4, TiO.sub.2.nH.sub.2O as well Fe- and Co-based
materials doped with varying amounts of Ni(II), ZnSe, CdSe, CdS,
ZnS, and MoS.sub.2. These materials have all been synthesized and
structurally characterized.
O.sub.2 Scavenging
[0049] In one embodiment, the present invention employs O.sub.2
scavenger in the construction of Nanoparticles. For example, Cu(0),
which are highly O.sub.2 reactive and can act as scavengers of
O.sub.2 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 O.sub.2 scavenging
system 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.sup.+ can be used to drive
the turnover of the hydrogenase system. The reduced MV.sup.+ is not
able to reduce Cu(II) to Cu(0) so these two products of the
photoreduction are naturally independent of each other.
Photo-Catalysts and Light Harvesting Chromophores
[0050] In another embodiment, the present invention provides a
method of using protein case architecture as a platform 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 attachment of light harvesting molecules,
which can be used to drive the photochemical 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 from the oxidized methyl viologen (MV)
through the photochemical oxidation of organic species (EDTA, for
example) with Ru(bpy).sub.3.sup.2+ as the catalyst. In addition,
light harvesting chromophores can be directly attached to redox
active enzymes, such as hydrogenase, and potentially eliminate mass
transport limitations and the need for electron transfer mediators
such as methyl viologen.
Protein Cage Encapsulated Catalyst Nanoparticles
[0051] 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
H.sub.2 formation through reduction of H.sup.+. 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, H.sub.2 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.
[0052] 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
phage-display) onto the inside of the protein cage architectures.
These peptides have been shown to direct the nucleation and
particle growth of a particular inorganic solid and are also able
to direct polymorph selection. Thus, the synthesis of a number of
inorganic phases can be directed to screen for an optimal balance
of long-term catalyst stability and activity in the reduction of
H.sup.+ to form H.sub.2.
[0053] 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
[0054] The present invention further includes reductants for
reduced methyl viologen (and other) mediator formation. 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 (SO.sub.3.sup.2-) is that this
is a polluting by-product of petroleum refining
(SO.sub.2+H.sub.2O.fwdarw.HSO.sub.3.sup.-). The oxidation of
sulfite results in the formation of sulfate (SO.sub.4.sup.2-).
Thus, utilizing sulfite as a reductant also overcomes the problem
of CO.sub.2 generation caused by oxidation of organic species.
Composite Materials
[0055] 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 from electroactive matrixes.
A variety of bioelectronic glasses have been reported, including
Sn-doped silica [Sn/SiO.sub.2], V.sub.2O.sub.5, MoO.sub.3, and
MnO.sub.2.
Coupled Enzyme Systems for Hydrogen Production from Sulfite
[0056] Hydrogenase can be coupled to the enzyme sulfite oxidase
either 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 (SO.sub.3.sup.2-) is that this is a
polluting byproduct of petroleum refining
(SO.sub.2+H.sub.2O.fwdarw.HSO.sub.3.sup.-). The oxidation of
sulfite results in the formation of sulfate (SO.sub.4.sup.2-).
Thus, utilizing sulfite as a reductant also overcomes the problem
of CO.sub.2 generation caused by oxidation of organic species.
Coupling Photocatalysts to Hydrogenase
[0057] Hydrogenase enzymes is coupled to photocatalysts (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(0) serves to protect the oxygen sensitive
hydrogenases from oxygen inactivation.
[0058] 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 limiting in any way the remainder of the
disclosure.
EXAMPLE 1
Encapsulation of Hydrogenases in Polymer Gel
[0059] This example is focused specifically on the hydrogen
production activity of H.sub.2ase:sol-gel materials. Hydrogenases
are bi-directional enzymes which also catalyze the oxidation of
hydrogen. H.sub.2ase: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 methyl viologen. H.sub.2ase:sol-gel
pellets with either the NiFe (Lamprobacter modestogalophilus (Lm)
and Thiocapsa roseopersicina (Tr)) or Fe-only (Clostridium
pasteurianum (CpI)) forms of H.sub.2ase 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.; Gorlenko, V. M.
Biochemistry (Mosc) 2004, 69, 164-169 (b) T. roseopersicina:
Sheiman, M. B.; Orlova, E. V.; Smirnova, E. A.; Hovmoller, S.;
Zorin, N. A. J Bacteriol. 1991, 173, 2576-2580. (c) C.
pasteurianum: Chen, J. S.; Mortenson, L. E. Biochim. Biophys. Acta
1974, 371, 283-298).
TABLE-US-00001 TABLE 1 Hydrogen Production Activity.sup.a of
Sol-Gel Encapsulated Hydrogenases hydrogenase solution gel
solution/gel (%) C. pasteurianum 12550 7581 60.4 .+-. 16 L.
modestogalophilus 9150 6175 67.5 .+-. 9 T. roseopersicina 12600
8834 70.1 .+-. 3 .sup.aThe activity measure at 25.degree. C. is
indicated in nmol/min/mg protein. The values represent average rate
over a four-hour period.
[0060] Blank gels assayed under identical conditions showed no
hydrogen production. Partially 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.degree. C. for 10 min, overnight precipitation at
4.degree. C. and removal of cellular particulate and precipitated
protein by centrifugation at 20 000 g) of the Fe-only hydrogenase
CpI from C. pasteurianum 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 laborious, and the preparation of bulk purified materials very
costly. To date, CpI is purified directly from cells of C.
pasteurianum grown under anaerobic conditions and the enzyme is
purified in the absence of oxygen and in the presence of reducing
agents.
[0061] Long term stability and temperature compatibility are
essential features of potential high throughput hydrogengenerating
biocatalysts. Activated hydrogenase enzymes are sensitive to
oxygen, and typically, dilute enzyme preparations can have a
limited shelf life at room temperature. These properties are
barriers that need to be overcome for the utility of these
biocatalysts to be realized. Detailed studies on shelf life (FIG.
2) and thermal stability (FIG. 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 (FIG. 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 function comparably to those in solution (FIG.
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.
[0062] It is unclear at this point what factors contribute to the
time dependent decline in activity of the H.sub.2ase: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 H.sub.2ase:sol-gel over time.
[0063] Solution and sol-gel encapsulated samples of CpI H.sub.2ase
were treated with protease (Samples of C. pasteurianum 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 (FIG. 4) (Tr and Lm hydrogenases
are not susceptible to proteolytic digestion). Solution CpI
H.sub.2ase activity is reduced to nearly zero after 25 min exposure
to protease. CpI H.sub.2ase: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 H.sub.2ase. These results clearly indicate that the
majority of the active H.sub.2ase is embedded within the gel and is
protected against proteolysis. Furthermore, these results show that
there is a high 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.
[0064] Nanoscopic confinement of H.sub.2ase 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
[0065] 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
Methanococcus jannaschii 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
environment (FIG. 5) (Kim et al. 1998).
[0066] Briefly outlined, purified Hsp was incubated with
PtCl4.sup.2- at 65-C for 15 min. The protein cage was incubated
with either 150, 250, or 1000 Pt per protein cage. Subsequent
reduction with dimethylamine 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 (FIG. 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 (FIG. 7A
inset) and intact 12 nm protein cages when negatively stained (FIG.
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 (FIG. 7C). At theoretical loadings of 250
Pt/cage, particles of 1 (0.2 nm were observed (FIG. 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 only a
fraction of the Pt remained in solution with a wide distribution of
particle sizes (3-120 nm) when observed by TEM.
[0067] The Pt-Hsp protein cage composites are highly active
artificial catalysts able to reduce H.sup.+ to form H.sub.2 at
rates comparable to the highly efficient hydrogenase enzymes.
Typical of hydrogenase assays, reduced methyl viologen (MV.sup.+)
was used as a source of reducing equivalents to drive the reaction.
Unlike most in Vitro hydrogenase assays, which use dithionite to
generate MV.sup.+, visible light and a cocatalyst
(Ru(bpy).sub.3.sup.2+) have been used to generate MV.sup.+ through
oxidation of simple organics such as EDTA (Brugger et al. 1981;
Jiang et al. 2004) (FIG. 9). In this modified assay, the solution
was illuminated at 25.degree. 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 .mu.M) was illuminated in the presence of MV.sup.2+ (0.5 mM),
Ru(bpy).sub.3.sup.2+ (0.2 mM), and EDTA (200 mM) at pH 5.0, and the
resulting H.sub.2 was quantified by gas chromatography. When
calculated on a per cage basis, the initial rate of H.sub.2
formation was 4.47.times.10.sup.3H.sub.2/s ((394H.sub.2/s) for a
loading factor of 1000 Pt per Hsp and 7.63.times.10.sup.2 H.sub.2/s
((405H.sub.2/s) for a loading factor of 250 (FIG. 6). These rates
are comparable to those reported for hydrogenase enzymes
(4.times.10.sup.3 to 9.times.10.sup.3 H.sub.2/s per protein
molecule) (Adams et al. 1990).
[0068] No H.sub.2-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.
[0069] When the H.sub.2 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
268H.sub.2/Pt/min, which is significantly better than reported
literature values (20H.sub.2/Pt/min (Brugger et al. 1981),
16H.sub.2/Pt/min (Keller et al. 1980), and 6.5H.sub.2/Pt/min (Song
et al., 2004), where comparisons are possible. In addition, initial
H.sub.2 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 photochemical reaction to produce H.sub.2 has not been
optimized, and a significant slowing down of the reaction is
observed after the first 20 min (FIG. 10). The H.sub.2 production
decay may be mainly due to the degradation of the photocatalyst
Ru(bpy).sub.3.sup.2+, and the electron mediator (MV.sup.2+), which
is subject to Pt-catalyzed hydrogenation (Keller et al. 1980).
[0070] Unlike the hydrogenase enzymes, the artificial Pt-Hsp
systems are not sensitive to O.sub.2 and show no significant
inhibition of H.sub.2 production by CO but are poisoned by thiols.
The Pt-Hsp catalyzed reaction was driven by the presence of the
reduced viologen (MV.sup.+). The MV.sup.+ could be generated either
by the photoreduction described above or by using the Jones
redactor (Harris et al. 1999) (Zn amalgam), which yielded rates for
H.sub.2 production approximately 40% slower than the coupled
photoreduction reactions. Also, the Pt-Hsp is able to catalyze the
reverse reaction (H.sub.2--2H.sup.++2e.sup.-) as monitored by the
iii situ reduction and bleaching of methylene blue (Seeffeldt et
al. 1989). Importantly for the utility of this artificial system,
the Pt-Hsp construct is remarkably stable and can be heated to
85.degree. C. without precipitation of the composite or loss of the
catalytic activity.
[0071] A well-defined thermally 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.sup.+ to form H.sub.2. 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.degree. C. illustrating the
utility of using protein architectures for the design and
implementation of functional nanomaterials.
[0072] The foregoing detailed description has been given for
clearness of understanding only and no unnecessary limitations
should be understood therefrom as modifications will be obvious to
those skilled in the art.
[0073] 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 known 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.
[0074] 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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