Biomolecular Catalysis - American Chemical Society

Chapter 4

Microperoxidase-11 Immobilized in a Metal Organic Framework

Downloaded by STANFORD UNIV GREEN LIBR on April 14, 2013 | http://pubs.acs.org Publication Date: June 23, 2008 | doi: 10.1021/bk-2008-0986.ch004

Kenneth J . Balkus, J r . , Thomas J . Pisklak, and Rita H u a n g

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Department of Chemistry and the NanoTech Institute, University of Texas at Dallas, Richardson, TX 75083-0688

Ultra large pore molecular sieves have proven to be viable hosts for biomolecule adsorption and separation. Microperoxidase-11 has been immobilized for the first time in a nano-crystalline metal organic framework (MOF). Microperoxidase-11 was physically absorbed from solution into the 3-dimensional [Cu(OOC-C H -C H -COO) /2 C 6 H 1 2 N 2]n MOF. The activity of the MOF immobilized peroxidase for the oxidation of methylene blue and amethylstyrene in organic solvents was determined. .1

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Enzymes are generally more active and show higher selectivities than synthetic, biomimetic catalysts (1). While biocatalysts are generally more active, enzymes have some inherent drawbacks as well. These problems include low thermal stability, low stability in organic solvents, and difficulty in recycling (1-4). Biocatalyst lifetime can be significantly improved by supporting the biomolecules on polymers and metal oxides. However, the non-uniform porosity of these support materials may limit access to the enzymes or allow leaching. Attempts to overcome these obstacles have resulted in the immobilization of enzymes and proteins in mesoporous materials (1-47). Mesoporous molecular sieves, comprised of one- to three-dimensional channel systems, have well defined pores ranging from 2-50 nm. The uniform pores of molecular sieve materials make them excellent materials for the size selective absorption of

© 2008 American Chemical Society

In Biomolecular Catalysis; Kim, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2008.


77 various biocatalysts (1-4). Mesoporous materials can also be compositionally tailored to provide an environment which is favorable for the adsorption of biocatalysts, such as changing the hydrophobicity of the material or adding surface binding sites (1-4). Mesoporous materials are often chemically and thermally stable; these attributes provide a protective environment for the biocatalyst after immobilization. We first demonstrated that mesoporous molecular sieves such as MCM-41, MCM-48, SBA-15, and DAM-1 are capable of immobilizing enzymes (37). Subsequently, many different mesoporous hosts and enzymes have been studied (1-47). The pore size of mesoporous silicates must be chosen which closely match the size of the enzyme or protein which is to be immobilized (23). If the pores are too small, the biocatalyst will not be absorbed and if the pores are too large, the biocatalyst can leach out. To reduce leaching, the hydrophilic interiors of mesoporous silicates have been fiinctionalized with organic groups to enhance hydrophobicity and help retain the biocatalyst in the pores (42-44). In an effort to provide more hydrophobic environments for inclusion of biocatalysts in mesoporous materials, we and others have immobilized biocatalysts in periodic mesoporous organosilicas (PMO) (11, 45-48), such as mesoporous benzene silica (MBS) and ethane bridged organosilica (MSE). Even though the hydrophobicity is greater than mesoporous silicas, leaching in PMO's is still highly dependant upon pore size (45). Recently, Hudson and coworkers have shown that electrostatic interactions also play an important role in biocatalyst retention (11). A material which exhibits all of these properties, such as: hydrophobicity, tunable pore size, and electrostatic interaction with the biocatalyst would provide an ideal environment for the biocatalyst and prevent leaching. Inorganic/organic hybrid framework materials may satisfy these requirements. Metal organic frameworks (MOF) are a family of materials which exhibit these properties and should be excellent hosts for biocatalysts Metal organic frameworks (MOF) comprise a relatively new class of porous materials (49-54). The novelty of these materials arises from the ability to selectively coordinate variable organic linkers to metals or inorganic clusters to produce porous inorganic-organic hybrid networks. When metals are used to connect the frameworks they contain one (55-60) or more vacant or labile sites to which the organic linkers can attach. Typically, the organic linkers contain functionalities such as carboxylate, amine, or pyridine groups through which they bind to the open sites on the metal centers (49, 54, 61-63). The metals or inorganic clusters act as anchoring points to which the organic linkers are bound. The organic ligands are subsequently connected in such a fashion that voids are formed. The pores are defined by the organic linkers which can impart properties to these materials which are not easily attained with porous metal oxides, such as zeolites. In fact, by tailoring the length of the linkers, MOFs have been synthesized with pore sizes and pore volumes greater than zeolites (49, 64-67). Catalytic properties (61, 68-69) as well as luminescence (70-75),


78 chirality (64, 69\ adsorption (64-65, 67, 69, 74-87) and framework polarity (49, 54, 77-78, 82) can also be manipulated by varying the type of organic linker used to form the framework. This nanoporous family of metal organic frameworks exhibits the largest surface areas of any known materials (67, 88). With pore sizes ranging as high 28.8 A (65) MOFs show a great potential for adsorption of guest molecules (64-65, 67, 74-87) and inclusion chemistry (69). Recently, Chael et al. reported the inclusion of polycyclic molecules such as C o, Astrazon orange R, Nile red, and Reichardt's dye in a metal organic framework (67). With pore sizes in the mesoporous range MOF's should also be capable of adsorbing small proteins and enzymes. Additionally, MOF pores are generally hydrophobic which could result in an affinity for and promote immobilization of proteins and enzymes. Also redox active MOF's could be synthesized that might operate in concert with proteins and enzymes in catalyzed reactions. In order to test the immobilization of a biomolecule in a MOF the proteolytic degradation product of cytochrome c, Microperoxidase-11 (MP-11) was selected (Figure 1) (38). The shortened peptide chain (11 amino acids) prevents denaturing of MP-11 by organic solvents (89). Figure 1 (random coil configuration) shows that MP-11 is composed of a heme group attached to an 11 amino acid peptide chain through thioether linkages of two Cysteines (Cys 14 and Cys 17) as well as to the imidazole group from histadine which is coordinated to the iron center as an axial ligand. The second axial position is coordinated with a molecule of water. Although there are no crystal structures of MP-11 available, the figure is drawn in the lowest energy configuration similar previously performed molecular dynamics calculations for MP-11 in methanol solution (90). MP-ll's usefulness arises from its ability to reduce hydrogen peroxide to water while oxidizing a substrate. However, free MP-11 has the tendency to aggregate in solution due to both intermolecular attractions and ligation through the metal center (38). When MP-11 oligimerizes through coordination to the metal center, the heme becomes less accessible and the activity of the enzyme is adversely affected. Immobilization in a suitable host material prevents aggregation and renders the heme more accessible to substrate molecules. The small size of MP-11 relative to complete enzymes, as well as the peroxidase activity of MP-11 makes it a suitable biocatalyst for immobilization in materials with pore sizes around 20 A. MP-11 has been shown to degrade organic soluble azo dyes such as Solvent Yellow, Solvent Blue 11, Solvent Green 3, and Solvent Orange 7 (89, 91) while entrapped in reversed micelles. Therefore, by immobilizing MP-11 in a hydrophobic matrix such as the MOF [Cu(OOC-C H -C H -COO) !/2C6H N2]„, (Figure 2) the microperoxidase can be dispersed in organic media and the hydrophobic interior of the MOF will allow the substrate access to the active site of the microperoxidase. Synthetic dyes are a large part of the waste stream from industrial manufacture of textiles and various consumer products. The removal of the dyes from the environment is a pressing concern. Although, peroxidases have been shown to degrade dyes via


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oxidation they have been limited to water soluble dyes. Many of the dyes used are water insoluble and a method of degrading these dyes is needed.

Figure L Structure of MP-11 calculated using Materials Studio

Recently, Kadnikova et al. immobilized microperoxidase-11 (MP-11) in solgel silica glass (38) and reported that the a-helical structure is promoted by a hydrophobic environment. MP-11 has also been immobilized on surfaces such as gold, in hybrid polymers, and mesoporous materials (98-101). Immobilization on surfaces and in polymers may not sufficiently sequester the microperoxidase which would allow it to dimerize and lose activity. Also, because MP-11 is relatively small, when immobilized in mesoporous materials there is a high propensity for leaching. The metal organic framework [Cu(OOCC H4-C H4-COO) /2C6Hi2N ]n (Figure 2) is composed of 2-dimensional sheets of copper dimers connected via biphenyl dicarboxylates. These 2-dimensional sheets are connected together by l,4-diazabicyclo[2.2.2]octane to form a 3J

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dimensional framework. Consequently, the interior of this MOF is composed of phenyl rings which form a hydrophobic environment in the pores. Immobilization of the microperoxidase MP-11 in the pores of this MOF would provide a hydrophobic environment around the MP-11 and may promote the

Biomolecular Catalysis - American Chemical Society

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