JP2008539786A - Composite nanomaterials for photocatalytic hydrogen generation and methods for their use - Google Patents

Abstract

The present invention relates to a composite material for producing photocatalyst H ₂ comprising 1) a polymer gel, 2) a photocatalyst, and 3) a protein-based H ₂ catalyst. The present invention also relates to a method for producing H ₂ comprising reacting an electron donor with a composite material comprising 1) a polymer gel, 2) a photocatalyst, and 3) a protein-based H ₂ catalyst.

Description

CROSS REFERENCE TO RELATED APPLICATIONS This application is a US Provisional Patent Application No. 60 / 681,174 filed May 16, 2005, which is incorporated herein in its entirety for all purposes. Are given priority in accordance with US Patent Act 119 (e).

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT The invention disclosed herein was in part made by the National Science Foundation under Grant No. MCB-0328341 and the Navy Research Office under Grant No. 19-00-R0006, Funding was provided.

FIELD OF THE INVENTION The present invention relates to composite materials containing hydrogen producing nanoparticles with hydrogenase enzymes and photocatalysts for hydrogen production from renewable sources.

BACKGROUND OF THE INVENTION There has been considerable interest recently in the production of hydrogen gas as an alternative energy source. The utility of increased use of hydrogen fuel cell technology depends on its ability to cause hydrogen gas accumulation in an efficient, economically viable and environmentally unfriendly manner (Armor) 2005; Spiegel et al., 2004; Tseng et al., 2005; Winter et al., 2005). Most of the hydrogen produced for energy-producing applications is generated by processes that reform methane or fossil fuels, and therefore, depending on the hydrogen energy economy based on these approaches, dependence on non-renewed fossil fuels Almost no decrease. Therefore, the development of a catalyst for chemical reaction that can generate hydrogen gas from renewable sources such as feedstocks efficiently and without generating greenhouse gases or other environmental pollutants is extremely important. .

に従って水素酸化またはプロトン還元のいずれかにおいて機能する種々の微生物により産生される。 Hydrogenases are highly developed catalysts that generate hydrogen gas at a rate that is envy of synthetic chemists. Metal-containing hydrogenase such _{(H 2} ase acids) (Biguneizu (Vignais) et al., 2001; Peters (Peters) et al., 1998, 1999; Adams (Adams) et al., 1990) has the following formula:

Produced by various microorganisms that function in either hydrogen oxidation or proton reduction.

  Hydrogen oxidation is linked to the production of reducing equivalents that cause energy generation or biosynthetic processes. During anaerobic fermentation, some microorganisms can link the proton reduction by sugar oxidation and the oxidation and regeneration of the electron carrier necessary for the production of hydrogen gas. The catalytic site of most metal-containing hydrogenases consists of a di-Fe or heterometallic NiFe site with a diatomic ligand of carbon monoxide and a cyanide for Fe. Hydrogenases are the only known enzymes that utilize these normally toxic compounds as an integral part of the active state of the enzyme. Similar to common enzymes, hydrogenases display precise tissue motifs that utilize protein structures that place and chemically maintain active sites where pathways for substrate access and product removal are important “design” features. Built for. Substrates, reductants, and products must be accessible to and from the catalytic site. Furthermore, continuous circulation of the catalyst requires ongoing reactant addition and product removal. The catalytic site of the hydrogenase enzyme consists of a unique biological metal cluster (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 an important role in the emerging hydrogen economy (Varforomeyev et al., 2004; Wunschiers et al., 2002; Chum et al., 2001). There is an increasing interest in the use of enzyme catalysts among materials for various applications. In the context of enzyme utilization in applications, there are some considerations that generally need to be addressed. Substrates, reductants, and products must be accessible to and from the catalytic site. Furthermore, continuous circulation of the catalyst requires ongoing reactant addition and product removal. Although rates up to 9000 H ₂ per second per enzyme were observed from these enzymes (Adams et al., 1990; Camack et al., 1999), the extreme sensitivity of these enzymes to oxygen, the expression of Limitations and difficulty of isolation prevent their use as a practical means of hydrogen production.

  There are insufficient attempts to develop methods and systems for generating hydrogen on a commercial scale using hydrogenase enzymes. US Pat. No. 6,858,718 discloses a gene encoding a hydrogenase and a method of using the gene product for the microbial production of molecular hydrogen. More specifically, the invention discloses an isolated nucleic acid sequence encoding a stable hydrogenase enzyme (HydA) that catalyzes the reduction of protons to form molecular hydrogen.

  U.S. Pat. No. 4,532,210 discloses the biological production of hydrogen in algal culture using alternating light-dark cycles. The process comprises culturing algae in water under aerobic conditions in the presence of light to accumulate photosynthetic products (starch) in the algae, and aerobic microbial conditions in the dark to generate hydrogen. Below, it comprises alternating steps of culturing algae in water and degrading the accumulated substances by photosynthesis. This method uses a nitrogen gas purge technique to remove oxygen from the culture.

U.S. Pat. No. 4,442,211 cultivates algae decolorized during the first stage of irradiation in a culture medium under an aerobic atmosphere until the color is restored, and then hydrogen is added at an increased rate. It is disclosed that subjecting the algae to the generated second stage irradiation increases the efficiency of the process of generating hydrogen by subjecting the algae in the aqueous phase to light irradiation. A reaction cell is used that irradiates the culture with light in an environment substantially free of CO ₂ and atmospheric O ₂ . This environment is maintained by passing an inert gas (eg, helium) through the cell to remove all of the hydrogen and oxygen generated by the splitting of water molecules in the aqueous medium. H 2 _- Continuous purging with an inert gas product culture has allowed sustained production of H _2, such purging is expensive, impractical for large mass culture of algae.

Accordingly, the present invention is enzymatic H ₂ formation, photocatalytic nanomaterials, and electronic / optical - felt over the years that a commercial scale was achieved by combining knowledge from different fields of chrome polymer gel technology to produce hydrogen It satisfies the need that has been made.

The present invention relates to composite materials and methods for generating hydrogen from renewable sources, such as simple raw materials. In one aspect, the present invention relates to a photocatalytic H ₂ generating composite material comprising 1) a polymer gel, 2) a photocatalyst, and 3) a protein-based H ₂ catalyst. The H ₂ catalyst includes, but is not limited to, Clostridium pasteurianum, Lamprobacter modegalophilus, various microorganisms such as Thiocapsa rosepericina, Thiocapsosain, It may be an enzyme such as a hydrogenase enzyme that may be derived from an organism. The composite material can further include a redox mediator such as polyviologen, and an oxygen scavenger such as Cu (0). The photocatalyst can be formulated as nanoparticles and encapsulated within a protein cage structure.

The H ₂ catalyst can 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 can include a protein where the protein is a 24 subunit protein such as a small heat shock protein (HSp). The core of the protein cage can include a metal selected from the group consisting of platinum, nickel, iron, and cobalt.

The present invention also relates to a method for producing H ₂ using the composite material of the present invention. For example, the present invention, the electron donating, 1) Polymer gel, 2) a photocatalyst, and 3) relates to a method for generating and H ₂ comprising reacting a composite material comprising a protein-based H 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.

DETAILED DESCRIPTION OF THE INVENTION All publications and patent applications in this specification are incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually incorporated by reference. Yes.

  The following description includes information that may be useful in understanding the present invention. None of the information provided herein is admitted to be prior art or related to the invention claimed by the present invention, either specifically or implicitly cited publications. Is not an admission that it is prior art.

  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.

The present invention, enzymatic H ₂ formation, photocatalytic nanomaterials, and electronic / optical - about chrome polymer gel technology. The present invention provides a system that substantially increases efficiency, where H ₂ can be generated from very simple raw materials, ie, visible light and a simple organic acid (acetate-vinegar). In one aspect, the present inventors have found that a simple organic acid, using a visible light, and the photocatalyst, elicited hydrogenase enzyme activity was demonstrated to be able to produce enough reducing equivalents to generate the H _2. To optimize these results, to immobilize the hydrogenase enzyme, encapsulate it in a polymer gel matrix, and generate H ₂ , the resulting enzyme gel pellet is transferred to an electron donor such as dithionite and an electron transfer such as methyl viologen. Incubate with vehicle. This approach takes advantage of the efficiency and specificity of the hydrogenase enzyme and the synthetic regulation that is made into polymer formulation to build a robust composite that meets many targets and objectives of economical hydrogen gas production.

  In another aspect, the present invention relates to the use of novel protein cages or nanoparticles as hydrogenase mimics to generate hydrogen gas. Nanoparticles comprising both a protein “shell” and a “core” can be mixed together to form a novel composition of a complete nanoparticle or core mixture. Furthermore, the shell can be loaded to form full nanoparticles with any number of different materials, including organic, inorganic and metal organic materials, mixtures thereof. Particularly preferred embodiments utilize a metal catalyst such as platinum to allow efficient reduction of protons. Furthermore, because the shells are proteinaceous, they can be altered to vary any number of physical or chemical properties by various methods, including but not limited to covalent and non-covalent derivatization and recombinant methods. Can be changed.

  Usually, one of the metal catalysts used for chemical reactions is platinum. However, platinum is known for its high cost and limited supply. In order to maximize the utilization of this catalyst, it is necessary to explore a method based on maximizing the catalytic efficiency of Pt per atom in order to develop an economically viable catalyst ( Spiegel et al., 2004). In particle-based approaches aimed at developing Pt catalysts, it is necessary to minimize particle diameter and thus increase the surface area (ie, the number of exposed Pt atoms per particle). A number of different synthetic approaches have been used to synthesize platinum nanoparticles with various passivating layers (Brugger et al., 1981; Chen et al., 2000; Chen). 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). Passivation layers generally interfere with exposed Pt atoms and reduce efficiency (Brugger et al., 1981). In the present invention, unlike the passivating layer, we do not coat the entire surface of the nanoparticles, but we still have a protein cage as a synthetic platform that isolates particles in solution and prevents aggregation. used.

  Protein cage structures have been used as biotemplates to create an interface between protein and metal (Allen et al., 2003; Flenniken et al., 2003). Cage-like structures have previously been shown to act as molecular containers for the encapsulation of both organic and inorganic materials (Flenniken et al., 2003; McMillan et al., 2002). The protein cage structure is self-assembled from a limited number of protein subunits (Douglas and Young) to create well-defined container-like forms that are chemically distinct on the inner and outer surfaces. 1998; Flenniken et al., 2003). Molecular access to the interior can also be controlled by pores at the subunit interface (Kim et al., 1998). We have previously shown that a proton cage structure can be used as a reaction environment that is constrained in size and shape for nanomaterial 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 a defined size and crystalline form. In addition, genetic and chemical modifications can create active sites at the exact location within the cage structure to create dramatically new functionality (Klem et al., 2005).

Using internal cage for spatially selective mineralization, the inventors of the present application successfully protein cage structure that mimics the controlled molecular access to the indicated active sites in H ₂ ase It was constructed. Briefly, xerogels and hydrogels containing hydrogenases were prepared using tetramethoxysilane (TMOS) as the starting material. Tetrahydroxysilane is produced by sonication of TMOS, water and HCl. The addition of a buffered (pH 8.0) protein solution (1: 1 v: v) initiates condensation to form a Si—O—Si network. The sol-gel pellets were cast from this mix in Teflon wells or directly into reaction vials. The gel was rinsed repeatedly with anaerobic buffer to remove unencapsulated protein. All H ₂ ase: sol-gel samples were prepared under anaerobic conditions. Hydrogen production was initiated by adding methyl viologen and dithionite to a gel suspended in a buffer contained in a sealed anaerobic vial as previously described. Samples were taken from the headspace of these reaction vials and analyzed by gas chromatography for quantification of the hydrogen produced. Various aspects of the invention are further provided below.

Material durability Purified hydrogenase from Clostridium pasteurianum is a very efficient catalyst for the reduction of H ⁺ to form H ₂ . The long-term stability of this enzyme is significantly enhanced by the addition of a thin polymer coating to the outer surface of the protein or by modification of the protein by encapsulation in a polyelectrolyte multilayer. In addition, the enzyme is immobilized within a 3-D cross-linked polyviologen gel matrix to increase electron transfer efficiency.

Materials engineering The ability to adjust the composition and morphology of nanoparticles, and thus the photocatalytic properties of nanomaterials, was developed with the goal of optimizing nanoparticles for efficient electron transfer to the hydrogenase system. . Furthermore, this synthesis control utilizes readily available electron donors such as cheap and simple organic acids (acetates, tartrate, citrate). Furthermore, due to expertise in the formulation of electroactive polyviologen gels, the efficiency of electron transfer between the electron donor and the active hydrogenase catalyst is enhanced by adjusting the stoichiometric and spatial interrelationship between these two components. Optimized to allow the inventors to incorporate the nanoparticle photocatalyst and hydrogenase into the solid matrix.

Efficiency Efficient H ₂ production from sunlight is achieved by adjusting light intensity and by directly analyzing the amount of H ₂ produced as a function of hydrogenase, photocatalysts, redox mediators, and electron donors. . A Xe arc lamp that mimics the solar spectrum was used as the light source for these tests.

H ₂ Production Rate The hydrogen production rate can be measured directly in an enzymatic assay, as previously described. The rate of H ₂ is measured under conditions that minimize the mass transfer of the redox partner in the composite formulation described in detail in the Examples.

Hydrogenase and O ₂ inhibition Incorporation of hydrogenase into an electroactive polymer gel allows the creation of materials with a core-shell structure. The outer layer can incorporate photocatalysts, redox mediators (viologens) and O ₂ reactive Cu colloids generated by photolysis of the catalyst. Cu is commonly used as a de-O ₂ agent and the high surface area of the shell becomes very attractive for this purpose. Thus, the outer layer can act as an O ₂ cleaning layer to protect the hydrogenase present in the inner layer. The inner layer can include a photocatalyst, hydrogenase, redox mediator, and electron donor. This procedure can significantly enhance the overall stability of the hydrogenase against O ₂ .

  It has been previously demonstrated that various enzymes can be immobilized on oxidized silica gel matrix (sol gel) and remain fully active. Indeed, in many cases, enzyme stability and the long-term stability or half-life of certain enzymes are increased. Enzyme encapsulation capabilities are very promising in both basic scientific and biotechnological applications. In some cases, immobilization of the enzyme can extend the stabilization of the intermediate, which has a very short lifetime in solution. In biotechnology, encapsulation can result in the production of durable heterogeneous catalysts for potential industrial applications.

  Encapsulation of purified active hydrogenase in tetramethylorthosilicate-derived sol-gel was demonstrated. The inventors have shown that when these enzymes are embedded in these porous oxidized silica polymer gels, a high percentage of both hydrogen oxidation and proton reduction overall hydrogenase activity is retained. Encapsulated in a reaction in the hydrogenated reaction from Clostridium pasteurianum, Lamprobacter modegalophilus, and Thiocapsa rosesepersicina hydrogenase in the reaction of thiocapsa rosesepersicina It can be immobilized with an apparent activity of at least 65-70% of the enzyme activity. Encapsulated hydrogenase exhibits some stability enhancement under storage and elevated temperatures. L. Immobilized NiFe hydrogenase from Modetogalophilus retains 85% of its hydrogen production activity over 10 days when stored at room temperature under a nitrogen atmosphere. However, the result that the hydrogenase enzyme can be immobilized in an active form is a major step in addressing the utility of utilizing hydrogenases in solid phase hydrogen generating materials with heterogeneous catalysis.

H ₂ Immobilization of hydrogen producing enzyme in stability electroactive polymer gel matrix generating material hydrogenase present invention provides an immobilized hydrogenase recombinase system consists of using a variety of synthetic polymers to encapsulate the hydrogenase molecules. Various methods of encapsulation result in increased enzyme stability. Other factors related to the optimization of the polymer matrix in which the hydrogenase enzyme is embedded are 1) the ability to dope polymers with various electron transfer agents / mediators, and 2) the permeability of the polymer to substrates and reaction products. . The following hydrogen producing enzymes were successfully encapsulated: 1) Fe-only hydrogenase, 2) NiFe bidirectional hydrogenase, and 3) Alkaline phosphatase (phosphate oxidation linked to hydrogen production). Immobilization within these porous polymers allows for high throughput heterogeneous catalysts.

  An immobilized catalyst system provides the possibility of reduction obtained by chemical or electrical means. By doping the three-dimensional catalyst with an electron transfer agent / mediator (from a synthetic chemical source or biological source), an external source of reducing power can be applied to the surface of the three-dimensional immobilized catalyst system.

Heterologous expression of regulatory hydrogenases The present invention provides methods for regulatable expression of heterologous hydrogenases in a host cell. Regulated heterologous expression of Shewanella oneldensis was achieved in the host E. coli. This was achieved by co-expression of a hydrogenase structural gene and a putative accessory gene product involved in hydrogenase maturation. Maximum hydrogenase expression can be achieved by optimization of the current system and by replacement of the hydrogenase gene from various sources. Regulated expression allows for genetic manipulation of the hydrogenase enzyme for enhanced stability, catalytic activity, and derivatization for composite construction.

Photocatalytic H ₂ Production The present invention further includes a method of utilizing a photocatalyst in the process of generating hydrogen gas. The addition of a photocatalyst adds additional regulatory elements to the system, giving the possibility to use lower potential electron transfer agents / mediators as a source of reducing equivalents. The catalyst system is operated by applying an electrical or chemical oxidation / reduction potential through the catalyst itself. Important components of photocatalysts and their specific synthesis include:
1) Synthesis of size-controlled catalyst nanoparticles and compositions 2) Electron transfer from photoactivated nanoparticles to electron transfer mediators 3) Electron transfer from photoactivated light harvesting molecules to protein cage encapsulated catalyst nanoparticles ,
Is mentioned.

Synthesis with Size-Adjusted Nanoparticles and Compositions The present invention further provides methods of synthesizing size-adjusted nanoparticles and compositions. A biomimetic scientific approach was adopted to systematically change the composition of metal oxide-based nanomaterials with the aim of adjusting the efficiency of the photoredox process. A range of protein-encapsulated nanomaterials was created to evaluate the effectiveness of the MV generation effectiveness composition. These materials include, but are not limited to, Fe ₂ O ₃ , Fe ₃ O ₄ , Mn ₂ O ₃ , Mn ₃ O ₄ , Co ₂ O ₃ , Co ₃ O ₄ , TiO ₂ .nH ₂ O and various Fe and Co based materials doped with amounts of Ni (II), ZnSe, CdSe, CdS, ZnS, and MoS ₂ . These materials are all synthesized and structurally characterized.

De-O _{2 In} one embodiment, the present invention uses a de-O ₂ agent in the construction of nanoparticles. For example, Cu (0), which is highly O ₂ reactive and can act as a de-O ₂ agent, can be encapsulated within a protein cage structure to protect the activity of hydrogenase (or other) redox active enzymes. Thus, hydrogenase protein cages containing copper oxide can have several advantages. First, Cu acts as an in situ de-O ₂ system, and the large surface area of the nanoparticles becomes very attractive for this purpose. Secondly, the reducing equivalents generated by photoreduction of MV ⁺ can be used to drive the turnover of the hydrogenase system. Since reduced MV ⁺ cannot reduce Cu (II) to Cu (0), these two products of photoreduction are naturally independent of each other.

Photocatalyst and light harvesting chromophore In another embodiment, the present invention provides a method of using a protein case structure as a platform for light harvesting molecules. For example, CCMV (and other virus) protein cage structures, ferritin (and ferritin-like proteins), small heat shock proteins, Dps proteins can be used as multivalent templates for binding light harvesting molecules, and electron transfer mediators ( It can be used to drive photochemical reduction of methylviologen-like). These include molecules such as Ru (II) bipyridine and Ru (II) phenanthroline that can bind to the cage in a site-specific manner. Reduced methyl viologen can be generated from oxidized methyl viologen (MV) through photochemical oxidation of organic species (eg, EDTA) having Ru (bpy) ₃ ²⁺ as a catalyst. Furthermore, the light harvesting chromophore can be directly coupled to a redox-active enzyme such as hydrogenase, potentially having the limitations of mass transport and eliminating the need for electron transfer mediators such as methyl viologen.

Protein Cage Encapsulated Catalyst Nanoparticles The present invention further provides a composition comprising a protein cage in which the catalyst nanoparticles are encapsulated. We have demonstrated that Pt nanoparticles can be efficiently encapsulated within protein cage structures (CCMV, Ferrin, Hsp, Dps). These particles are constrained in size and shape by the protein cage and increase the Pt colloid with a very high specific surface area, resulting in high H ₂ formation due to reduction of H ⁺ . The reducing equivalent required for this reaction can be supplied by reduced methyl viologen (MV). Alternatively, reduced viologen can be chemically generated by Zn (Hg / Zn amalgam) oxidation in the presence of EDTA. Using this binding system, Pt particle size dependence, nature of the protein cage structure (ie, diffusion access of reduced viologen to Pt nanoparticles), prevention of catalytic poisoning with respect to useful life, nanoparticle catalysts (eg, Pd, CoPt the composition of FePt), and the investigation of the nature of the photocatalyst couple, can be optimized with _{H 2} generation.

The success of the inventor can be used to incorporate small peptides (derived from phage display) into protein cage structures in the synthesis of nanoparticles of various compositions, particularly alloy particles. These peptides have been shown to be specific for nucleation and particle growth of certain inorganic solids and also for polymorph selection. Therefore, the synthesis of multiple inorganic phases can be directed to screen the optimal balance between long-term catalyst stability and activity upon reduction of H ⁺ to form H ₂ .

  The Pt particles encapsulated within the protein cage disclosed herein serve as a synthetic hydrogenase mimic. Such a system allows the synthetic material to incorporate the best-characteristic colloidal catalyst with a biological catalyst. Both colloidal catalysts and biological catalysts have their own limitations (sensitivity to oxygen, poisoning, cost, reaction conditions and lifetime), but combining these two types of catalysts can limit these limitations. Skillfully avoid.

Reductants The present invention further includes reductants for reduced methyl viologen (and other) mediator formation. Examples of organic substances include ethylenediaminetetraacetic 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 ₃ ²⁻ ) is that it is a petroleum by-product contaminated byproduct (SO ₂ + H ₂ O → HSO ₃ ⁻ ). Sulfite oxidation leads to sulfate (SO ₄ ²⁻ ) formation. Thus, by utilizing sulfite as a reductant, the problem of CO ₂ production caused by oxidation of organic species is overcome.

Composite Material The present invention further provides a method of promoting electron transfer from a redox protein to an electrode. The nano-range containment of redox-active proteins into silica-derived sol-gel materials requires a mediator such as methyl viologen and facilitates electron transfer by the protein. The porosity of these gels provides access to the encapsulated protein. Materials that promote direct electron transfer between the electrode surface and the redox centers of hydrogenases and other redox-active proteins eliminate the catalytic dependence on chemically reduced forms. In the case of hydrogenases, these novel materials facilitate the flow of electrons from the encapsulated enzyme to the electrode during enzymatic association catalyst generation and oxidation of H-2 (g). The material is derived from an electroactive matrix. Various bioelectronic glasses have been reported such as Sn-doped silica [Sn / SiO ₂ ], V ₂ O ₅ , MoO ₃ , and MnO ₂ .

Combined enzyme system for hydrogen generation from sulfite Hydrogenase is either 1) in a freely diffusing solution-based system, 2) by covalent bonding (cross-linking of sulfite oxidase and hydrogenase) or 3) electroactive porosity Incorporation of both components into the gel allows binding to the enzyme sulfite oxidase. In this system, reducing equivalents derived from enzymatic oxidation of sulfite to sulfate can be directed to proton reduction by hydrogenase. As mentioned above, the advantage of using sulfite (SO ₃ ²⁻ ) is that it is a petroleum by-product contaminated byproduct (SO ₂ + H ₂ O → HSO ₃ ⁻ ). Sulfite oxidation leads to sulfate (SO ₄ ²⁻ ) formation. Thus, the use of sulfite as a reductant also overcomes the problem of CO ₂ production caused by oxidation of organic species.

Binding photocatalysts to hydrogenases Hydrogenase enzymes can be photocatalyzed (such as nanoparticle photocatalysts) in the development of heterogeneous catalysts that can utilize light energy to generate hydrogen from abundant electron donor sources such as sulfites, organic acids, or perhaps ethanol. To join. The coupled system can operate in aqueous solution or immobilized gel. In order to produce a heterogeneous catalyst such that the substrate and product can enter the liquid phase, the components of the composite material can be immobilized in an electroactive gel (silica oxide or other polymers such as polyviologen). The addition of oxygen consuming catalyst nanoparticles such as Cu (0) serves to protect the oxygen sensitive hydrogenase from oxygen inactivation.

  Without further explanation, using the foregoing description and the following illustrative examples, one of ordinary skill in the art would be able to make and utilize the compounds of the present invention to perform the claimed methods. Accordingly, the following working examples specifically point out preferred embodiments of the invention and should not be construed as limiting the remainder of the disclosure in any way.

Example 1: Encapsulation embodiment hydrogenase such in the polymer gel, specifically H ₂ ase: focus on hydrogen production activity of the sol-gel material. Hydrogenases are also bidirectional enzymes that also catalyze the oxidation of hydrogen. H ₂ ase: sol-gel pellets, under the head pressure of hydrogen, by placing in buffered (pH 8.0) solution, the pellets were assayed for hydrogen oxidation activity. Electron flow was monitored by reduction of methyl viologen. H ₂ ase of NiFe (run Pro Arthrobacter Modesto Gallo stearothermophilus (Laprobacter modestogalophilus) (Lm) and Chiokapusa-Roseoperushichina (Thiocapsa roseopersicina) (Tr)) or Fe only in the form of (Clostridium Pasutsurianamu (Clostridium pasteurianum) (CpI)) with H ₂ ase: sol-gel pellets, hold approximately 60% to 70% of the specific activity of the hydrogen generated observed in solution (Table 1) (isolation of hydrogenase class:. (a) L Modesto Gallo stearothermophilus ( L. modestogalophilus): Zadoborny, OA (Zadvorny, OA); Zolin, NA (Zorin, NA); ogotov, I.N.); Gorlenko, VM (Gorlenko, VM) Biochemistry (Mosc) 2004, 69, 164-169, (b) T. roseopersicina: Sherman, M.B. (Sherman, M.B.); Orlova, E.V. (Orlova, E.V.); Smirnova, E.A. (Smirnova, E.A.); Zolin, NA (Zorin, NA) J Bacteriol, 1991, 173, 2576-2580. (C) C. pasteurianum: Chen, J. S. Chen, JS); Mortenson, LE (Mortenson, LE) B ochim.Biophys.Acta 1974 years, pp. 371,283-298).

  Blank gels assayed under the same conditions showed no hydrogen production. C. Partially purified formulation consisting of heat-treated crude extract formulation of Fe-only hydrogenase CpI from C. pasteurianum (processed crude extract was prepared by cell lysis with pressure-treated cells and then at 55 ° C. for 10 minutes Incubation of the cells and removal of cell particles and precipitated protein by overnight precipitation at 4 ° C. and centrifugation at 20000 g) resulted in similar activity levels after encapsulation when compared to more highly purified formulations. Retained (70% and 60% respectively). This suggests that the co-encapsulation of additional protein macromolecular components of the extract does not significantly affect the activity. Encapsulation of the crude extract yields a less well-defined material, but is nearly equal because the heterogeneous expression of hydrogenase is not easy, is difficult to purify, and the preparation of bulk purified material is very expensive The ability to encapsulate and immobilize a crude enzyme preparation while retaining activity levels is important in practice. To date, CpI has been grown in C. aeruginosa grown under anaerobic conditions. Purified directly from cells of C. pasteurianum, the enzyme is purified in the absence of oxygen and in the presence of a reducing agent.

  Long-term stability and temperature compatibility are essential features in potential high-throughput hydrogen generation biocatalysts. Activated hydrogenase enzymes are sensitive to oxygen and typically diluted enzyme preparations may have a limited shelf life at room temperature. These properties are barriers that need to be overcome for the perceived utility of these biocatalysts. Detailed tests on shelf life (FIG. 2) and thermal stability (FIG. 3) were performed using purified Lm NiFe hydrogenase and CpI heat treated extract. The gel-encapsulated enzyme can be stored at room temperature under anaerobic buffer while maintaining about 80% activity of the starting material for 4 weeks (FIG. 2). Enhancing the stability of the encapsulated hydrogenase is largely determined over a longer period. The temperature test shows that the encapsulated enzyme functions as well as that in solution (FIG. 3). Future studies will monitor longer shelf life, but will support the results presented herein, considering that no attempt has been made to optimize the conditions for encapsulation of these enzymes.

At this point, what factors, H ₂ ase: or contribute to the time-dependent decline in the sol-gel activity is unclear. The reduction mediator appears to bind to the sol-gel material after prolonged exposure. This is not surprising since the Si-O network and unreacted Si-OH portion of the TMOS-derived sol-gel has a strong ion-pair potential in the sol-gel material. This time H ₂ ase: may contribute to slow decrease of the sol-gel activity.

Hydrogen solutions and sol-gel encapsulated samples CpI H ₂ ase, was treated with a protease (Samples of C. Pasutsurianamu (C.Pasteurianum) extract was treated for 25 minutes with a protease cocktail before activity assay), observed It was confirmed that the production activity was derived from enzymes embedded in the porous structure of the sol-gel material (FIG. 4) (Tr and Lm hydrogenases are not sensitive to proteolytic digestion). CpI _{H 2} ase activity of the solution is reduced substantially to zero after protease exposure 25 minutes. CpI H ₂ ase: sol-gel activity is reduced to less than 7% after the same protease treatment. This small reduction may result from an accessible surface bound or unencapsulated H ₂ ase protease digestion. These results indicate that most of the active H ₂ ase, embedded in the gel clearly shows that it is protected against proteolytic degradation. Furthermore, these results indicate that there is a high encapsulation efficiency and that the observed decrease in hydrogenase activity is not due to partial encapsulation of the protein. Thus, the loss of activity may be due to enzyme inactivation as a result of the encapsulation procedure, or an overall decrease in the rate of mass transfer in the matrix.

The nanoscope containment to H ₂ ase porous sol-gel inside, resulting in the synthesis of a functional hydrogen generation biomaterial. As heterogeneous catalysts, these new materials have enormous potential and utility.

Example 2: Synthesis of Pt nanoparticles encapsulated in an Hsp cage This example describes the synthesis of Pt nanoparticles encapsulated in an Hsp cage. A small heat shock protein (Hsp) self-assembled cage-like structure from Methanococcus jannaschii has been used to encapsulate metal clusters with defined spatial alignments (Flenniken) Et al., 2003). Hsp is built from 24 subunits in a 12 nm cage that defines a 6.5 nm lumen with a pore through the cage structure, so that molecules can be transported back and forth between the internal and external environments ( FIG. 5) (Kim et al., 1998).

Briefly, purified Hsp was incubated with PtCl4 ^2- at 65 ° C. for 15 minutes. Protein cages were incubated at 150, 250, or 1000 Pt per protein cage. Subsequent reduction with dimethylamine borane complex ((Ch ₃ ) ₂ NBH ₃ ) formed a brown solution. Characterization of the reaction product by gel filtration chromatography revealed the same retention capacity as untreated Hsp, indicating co-elution of protein (280 nm) and Pt (350 nm) components (FIG. 6). The dynamic light scattering method showed no change in the particle diameter after reaction. Visualization of Pt-treated Hsp (Pt-Hsp) with a transmission electron microscope (TEM) reveals an electron density core identified as Pt metal by electron diffraction (inset of FIG. 7A) and no staining when negatively stained. The treated 12 nm protein cage (Figure 7B) was revealed. In this stained sample, the Pt particles are clearly visible on the background stain and are localized within the cage structure. For an average loading of 1000 Pt / cage, 2.2 metal particles (0.7 nm was observed) (FIG. 7C). At a theoretical loading of 250 Pt / cage, one particle (0.2 nm was observed) (FIG. 8), while at 150 Pt / cage loading, particles could not be identified due to electron microscope limitations. The Hsp free control reaction formed aggregated Pt colloids that rapidly precipitated from solution. A control reaction with bovine serum albumin (BSA) at the same total protein concentration also resulted in bulk precipitation, and only the Pt fraction was in solution with a broad distribution of particle sizes (3-120 nm) as observed by TEM. The remaining.

Pt-Hsp protein cage complexes are highly active artificial catalysts capable of reducing H ⁺ to form of H ₂ in a very efficient hydrogenase enzyme a rate equivalent. In a typical hydrogenase assay, reduced methyl viologen (MV ⁺ ) was used as a source of reducing equivalents to drive the reaction. Unlike most in vitro hydrogenase kinase assay using dithionite to generate a MV ^+, visible and cocatalyst (Ru ^{_(bpy) 3 2+)} is, in order to generate a MV ⁺ by oxidation of simple organic substances such as EDTA (Brugger et al., 1981; Jiang et al., 2004) (FIG. 9). In this modification 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). Pt-Hsp (0.51 μM) was illuminated at pH 5.0 in the presence of MV ²⁺ (0.5 mM), Ru (bpy) ₃ ²⁺ (0.2 mM), and EDTA (200 mM) and the resulting H ₂ Was quantified by gas chromatography. When calculated on a per cage basis, the onset rate of H ₂ formation is 4.47 × 10 ³ H ₂ / sec (394H ₂ / sec) and 250 loading factors for a loading factor of 1000 Pt per Hsp. against 7.63 × ¹⁰ _{2 H} 2 / sec was (405H ₂ / sec) (Fig. 6). These rates are comparable to those reported for hydrogenase enzymes (4 × 10 ³ to 9 × 10 ³ H ₂ / sec per protein molecule) (Adams et al., 1990).

_{H 2} generating activity for at least Pt loading (150Pt / Hsp) was detected. This is consistent with previous reports of Pt size-dependent activity (Greenbaum et al., 1988). It is also consistent with our inability to detect individual Pt particles by TEM in these samples, meaning that the synthesized Pt particles are below some threshold threshold required for activity.

If H ₂ production rate is calculated by the reference per 1pt, they, are highly preferred to compare with other reported Pt nanoparticles. The onset rate of Pt-Hsp with 1000 Pt / cage is 268 H ₂ / Pt / min, which is a reported literature value (20 H ₂ / Pt / min (Brugger et al., 1981) that can be compared. Year), 16H ₂ / Pt / min (Keller et al., 1980), and 6.5 H ₂ / Pt / min (Song et al., 2004). starting H ₂ production rate of -Hsp long-term stability of the bond photochemical reaction to produce a .H ₂ also about 20 times greater than that obtained for Pt particles produced in protein-free control reactions is not optimized, significant slowing of the reaction is observed after the first 20 minutes (Figure 10). attenuation of the _{H 2} generation is mainly photocatalyst Ru ^{_(bpy) 3 2+,} It is subjected to hydrogenation Pt catalyst and is believed to be due to the degradation of the electron mediator (MV ^2+).

Unlike the hydrogenase enzyme, the artificial Pt-Hsp system is not sensitive to O ₂ and does not show significant inhibition of H ₂ production by CO, but is poisoned by thiols. The Pt-Hsp catalytic reaction was driven by the presence of reduced viologen (MV ⁺ ). MV ⁺ is produced by using Jones Reductor (Harris et al., 1999) (Zn amalgam) which produced a H ₂ production rate that was about 40% slower than the photoreduction or combined photoreduction reactions described above. did it. Pt-Hsp can also catalyze the reverse reaction (H ₂ → 2H ⁺ + 2e ⁻ ) monitored by in situ reduction and decolorization of methylene blue (Seefeldt et al., 1989). Importantly for this artificial system, the Pt-Hsp construct is remarkably stable and can be heated to 85 ° C. without precipitation of the complex or without loss of catalytic activity.

Well-defined thermostable protein cage structures have been used to produce artificial hydrogenases that have many features common to these biological catalysts. In a spatially selective manner, small metal clusters are introduced inside the cage-like structure of Hsp that acts as an active site involved in the reduction of H ⁺ to form H ₂ . The specific activities of these artificial enzymes are comparable to known hydrogenase enzymes and are significantly better than the previously described Pt nanoparticles. The protein cage structure of Hsp acts to maintain the integrity of small clusters, prevents aggregation and regulates access to these “active sites”. The Pt-Hsp complex is stable up to 85 ° C., demonstrating the utility of using protein structures for the design and implementation of functional nanoparticles.

  The foregoing detailed description has been set forth for clarity of understanding only, and no unnecessary limitations should be considered therefrom, and variations will become apparent to those skilled in the art.

  Although the present invention has been described with reference to specific embodiments thereof, it will be appreciated that further modifications are possible and that this application will generally be subject to any modification, use of the present invention consistent with the principles of the invention. Or are intended to encompass adaptations, are within the scope of known or customary practice in the technical field to which this invention pertains, and are applicable to the essential forms described herein above, and are It is intended to include such deviations from the present disclosure in accordance with the claims.

  All publications cited in this specification disclose specific embodiments of the invention in which the publications are cited and are incorporated herein by reference for the purpose of illustration.

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For a more complete understanding of the present invention and further benefits thereof, reference is made to the following drawings:
It is drawing which shows the composite material used for a hydrogen production | generation device. C. encapsulated in solution and sol-gel. Pasteurianum (CpI) and L. FIG. 2 is a graph showing hydrogen generation activity of L. modetogalophilus (Lm) hydrogenase: ◆, CpI in solution; ●, CpI in sol gel; ▲, Lm in solution; ■, Lm in sol gel . C. encapsulated in solution and sol-gel. Pasteurianum (CpI) and L. It is a graph which shows the temperature dependence of the hydrogen-producing activity of L. modetogalophilus (Lm) hydrogenase: ◆, CpI in solution; ●, CpI in sol-gel; ▲, Lm in solution; Lm in sol-gel. B. presence of protease (open) and absence (shade), C.I. 2 is a graph showing the hydrogen generation activity of a solution and sol-gel encapsulated hydrogenase from C. pasteurianum. (A) Space-filled image of a small heat shock protein (Hsp) cage derived from Methanococcus jannaschii (pdb: 1shs) and (B) a cut-away view of Hsp showing the lumen of the cage. FIG. 1 shows gel filtration chromatography of Hsp: (A) non-mineralized Hsp; (B) Hsp mineralized with 250 Pt / Hsp showing co-elution of protein (280 nm) and mineral (350 nm); (C) protein Hsp mineralized with 1000 Pt / Hsp, showing co-elution of (280 nm) and mineral (350 nm). (A) Image showing TEM of unstained Hsp1000Pt, inset shows electron diffraction of Pt ⁰ from Hsp1000Pt, (B) TEM of Hsp1000Pt stained with 2% uranyl acetate, (C) Hsp1000Pt A columnar diagram of the Pt particle diameter in the middle is shown, with an average of 2.2 (0.7 nm scale bar) 20 nm. (A) Image showing a TEM of Hsp250Pt stained with 2% uranyl acetate, (B) Columnar view of Pt particle diameter in Hsp250Pt, scale bar) 20 nm. FIG. 6 is a schematic diagram showing photo-mediated H ₂ production from Pt—HsP, where methylviologen (MV ²⁺ ) is used as an electron transfer mediator between Ru (bpy) ₃ ²⁺ photocatalyst and Pt-Hsp responsible for H ₂ production. It is done. FIG. 5 is a graph showing H ₂ production from Pt-Hsp in 0.2 mM Ru (bpy) ₃ ²⁺ , 0.5 mM methylviologen, 200 mM EDTA, and 500 mM acetate pH 5.0: ▲, 1000 Pt / Hsp5 1 × 10 ⁻¹⁰ mol Pt; ■, 250 Pt / Hsp 8.2 × 10 ⁻¹⁰ mol Pt.

Claims (41)

1) a polymer gel;
2) a photocatalyst;
3) a protein-based H ₂ catalyst;
A composite material for producing photocatalyst H ₂ . The composite material according to claim 1, wherein the H ₂ catalyst is a hydrogenase.   The composite material according to claim 2, wherein the hydrogenase is encapsulated in the polymer gel.   The composite material according to claim 3, wherein the gel is porous.   The composite material according to claim 4, wherein the gel is a sol-gel. The composite material of claim 1, wherein the H ₂ catalyst is a hydrogenase mimic.   The composite material according to claim 6, wherein the hydrogenase mimic is a nanoparticle.   The composite material according to claim 7, wherein the nanoparticles are in the form of a protein cage comprising a shell and a core.   The composite material according to claim 8, wherein the shell contains a protein.   The composite material according to claim 9, wherein the protein is a 24 subunit protein.   The composite material according to claim 10, wherein the protein is a small heat shock protein (HSp).   The composite material according to claim 8, wherein the core includes a metal.   The composite material according to claim 12, wherein the metal is selected from the group consisting of platinum, nickel, iron, and cobalt.   The composite material according to claim 13, wherein the metal is platinum.   The composite material of claim 1 further comprising a redox mediator.   The composite material of claim 15, wherein the redox mediator comprises a polyviologen.   The composite material of claim 16 further comprising an oxygen scavenger.   The composite material according to claim 17, wherein the oxygen scavenger comprises Cu (0).   The composite material according to claim 1, wherein the photocatalyst is formulated as nanoparticles.   20. A composite material according to claim 1 or 19, wherein the photocatalyst is encapsulated within a protein cage structure.   The hydrogenase enzyme is derived from Clostridium pasteurianum, Lamprobacter modernogalophilus, Thiocapsa roseopericina, Thiocapsa rosesein, or a combination of these materials. The material is
i) an inner layer further comprising the photocatalyst and the hydrogenase enzyme;
ii) an outer layer further comprising the photocatalyst, the redox mediator, and the oxygen scavenger;
The composite material according to claim 17, comprising: Electron donor, 1) Polymer gel, 2) a photocatalyst, and 3) a method of generating and H ₂ which comprises reacting a composite material containing H ₂ catalysts based on protein. The H ₂ catalyst The method of claim 23 which is a hydrogenase.   25. The hydrogenase enzyme is derived from Clostridium pasteurianum, Lamprobacter modegalophilus, Thiocapsa roseopericina (Thiocapsa rosesein), or thiocapsa rosesein 24.   25. The method of claim 24, wherein the hydrogenase is encapsulated within a polymer gel.   27. The method of claim 26, wherein the gel is porous.   28. The method of claim 27, wherein the gel is a sol-gel. The H ₂ catalyst The method of claim 23 which is a hydrogenase mimics.   30. The method of claim 29, wherein the hydrogenase mimic is a nanoparticle.   31. The method of claim 30, wherein the nanoparticles are in the form of a protein cage that includes a shell and a core.   32. The method of claim 31, wherein the shell comprises a protein.   35. The method of claim 32, wherein the protein is a 24 subunit protein.   34. The method of claim 33, wherein the protein is a small heat shock protein (HSp).   32. 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.   The method of claim 36, wherein the metal is platinum.   24. The method of claim 23, wherein the photocatalyst is formulated as nanoparticles.   24. The method of claim 23, wherein the photocatalyst is encapsulated within a protein cage structure.   24. 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.   24. The method of claim 23, wherein the electron donor is one of the group consisting of sulfite, thiosulfate, and dithionite.

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