CA2608870A1 - Composite nanomaterials for photocatalytic hydrogen production and methods of their use - Google Patents

Abstract

The present invention is directed to a composite material for photocatalytic H2 production comprising: 1) a polymer gel; 2) a photocatalyst; and 3) a protein based H2 catalyst. The invention also relates to a method to produce H2, comprising reacting an electron donor with a composite material comprising 1) a polymer gel, 2) a photocatalyst, and 3) a protein based H2 catalyst.

Description

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;

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 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 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.

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 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 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 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 ~

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 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.

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 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 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 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 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 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 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 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 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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Claims (41)

1. A composite material for photocatalytic H2 production comprising:
1) a polymer gel

2) a photocatalyst; and

3) a protein based H2 catalyst.

2. The composite material of claim 1, wherein the H2 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 H2 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, Laprobacter 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 H2, comprising reacting an electron donor with a composite material comprising 1) a polymer gel, 2) a photocatalyst, and 3) a protein based H2 catalyst.

24. The method of claim 23, wherein the H2 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 H2 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.