Mesoporous materials enzymes

The large size of the pores of MCM-41 has also allowed the entrapment of enzymes, such as cytochrome c, papain and trypsin [193]. Enzyme entrapment has been extensively performed with sol-gel materials. The types of applications of redox catalysis using enzyme-mesoporous materials is expected to parallel the sol-gel materials, which is discussed in the last section of this chapter. 

Since Diaz and Balkus first attempted to immobilize enzymes on mesoporous MCM-41 [101], several research groups have investigated the influence of various physical factors such as pore size, ambient pH, and ionic strength, on the adsorption efficiency of proteins [102-118]. This research revealed the general tendencies of protein adsorption behavior and outlines for successful immobilization of proteins onto mesoporous materials. As one of the representative examples, systematic... 

For application of protein-immobilized porous materials to sensor fields, use of an electroactive substance as the framework material is important. DeLouise and Miller demonstrated the immobilization of glutathione-S-transferase in electrochemically etched porous silicon films [134], which are attractive materials for the construction of biosensors and may also have utility for the production of immobilized enzyme bioreactors. Not limited to this case, practical applications of nanohybrids from biomolecules and mesoporous materials have been paid much attention. Examples of the application of such hybrids are summarized in a later section of this chapter. 

One of the most promising applications of enzyme-immobilized mesoporous materials is as microscopic reactors. Galameau et al. investigated the effect of mesoporous silica structures and their surface natures on the activity of immobilized lipases [199]. Too hydrophilic (pure silica) or too hydrophobic (butyl-grafted silica) supports are not appropriate for the development of high activity for lipases. An adequate hydrophobic/hydrophilic balance of the support, such as a supported-micelle, provides the best route to enhance lipase activity. They also encapsulated the lipases in sponge mesoporous silicates, a new procedure based on the addition of a mixture of lecithin and amines to a sol-gel synthesis to provide pore-size control. 

Addition of third components to nanohybrids of proteins and mesoporous materials sometimes brings advantages in their functions. Kim, Hyeon, and coworkers immobilized enzyme molecules together with magnetite (Fe304) nanoparticles in hierarchically ordered, mesocellular, mesoporous silica (HMMS) (Figure 4.25)... 

Kumar, C.V. and Chaudhari A. (2003) Unusual thermal stabilities of some proteins and enzymes bound in the galleries of layered alpha-Zr(IV) phosphate/phosphonates. Microporous and Mesoporous Materials, 57,181-190. 

Figure 1). Finally, high ordered pure silica mesoporous material, containing lipase enzyme by entrapping procedure, has been obtained by sol-gel method (XRD results not showed).

The last two examples have demonstrated in particular how subtle the effects can be and that much work is required to fine-tune the properties of a mesoporous material as a suitable host for active enzymes. Besides the stability of the enzyme itself, important factors include the pore size, the robustness of the host, and the distribution of functional groups within the material. 

Immobilized CPO on SB A-16 mesoporous materials The Cs+-doped material incremented the CPO load and its catalytic activity. The Cs+-doped and CPO covalent bonded materials showed a higher enzyme activity compared to physical random immobilization [7]... 

Depending on the conditions and chemical precursors used for mesoporous materials synthesis, different morphologies, such as hexagonal, cubic, or lamellar, and different pore sizes can be obtained (Fig. 9.2). Additionally, these materials can easily be functionalized with organic groups to produce a variety of hybrid inorganic-organic materials with new properties, which directly affect the functionality of the enzyme [91, 92]. 

In the first report about immobilization of peroxidases on mesoporous materials, Takahashi and coworkers shed light on different parameters that affect the process. Using horseradish peroxidase (HRP) as a model, the authors reported that higher stability to temperature and organic solvent, important variables on industrial processes, were obtained when the size of the pore match the size of the enzyme, in such a way that the encapsulated enzyme was located in a restricted space that slowed down its free movement, preventing its denaturation [4],... 

Fig. 9.3 Schematization for chemical (a) and physical (b) protein adsorption in mesoporous material. The pore size for high enzyme load should be at least three times the enzyme size...

Several studies have demonstrated the improved stability of peroxidases when they were subjected to immobilization. Akhtar and Husain observed that bitter gourd peroxidase (BGP) was able to remove higher percentage of phenols over a wider range of pH when immobilized on a bioaffinity support [37]. Sasaki et al. highlighted an improvement of thermal stability of MnP immobilized on FSM-16 mesoporous material [59]. Furthermore, some other studies demonstrated a protective effect of peroxidase immobilization against inactivation by H202 [7, 20]. The different behavior of immobilized peroxidases with respect to soluble ones points out the necessity of an optimization of the process conditions when immobilized enzyme is used. Nevertheless, the possible improvement in stability should balance the usual decrease in kinetic rates, due to substrate transfer limitations to reach the enzyme inside the support. 

Since the discovery of ordered mesoporous materials, researchers have explored many possible applications that can take advantage of the unique compositional or structural features of mesoporous materials. In addition to apphcations in traditional areas such as catalysis, separation, and ion exchange, new applications that might involve mesoporous materials include stationary phases in HPLC, bio and macromolecular separations, low dielectric constant materials, enzyme immobilization, optical host materials, templates for fabrication of porous carbons, and reactions in confined enviromnents. 

We have discussed in some detail ET reactions occurring in zeolites, mesoporous materials and sol-gels. The unifying theme across these three different types of materials is their porosity. ET reactions within the pores with molecules as small as propylene to complex enzymes such as glucose oxidase have been discussed. However, what is also apparent from the discussions are that each host matrix imposes its own unique characteristics on the reactions within its pores. We conclude by highlighting the uniqueness of each matrix. 

Summarizing the field of ET using porous materials, it appears that zeolites act as active hosts and have advantages of manipulating reaction rates and product yields involving small molecules due to the close fits of the size of the molecule and cages. Mesoporous materials make it possible to examine the redox chemistry of larger molecules as compared with zeolites, while sol-gel materials form the primary hosts for enzyme-based redox reactions. 

This area of research is also denoted as nanobiocatalysis. A range of possible ways to stabilize enzymes in these nanostructures has been developed. Figure 2.23 reports, as an example, a cartoon of the encapsulation of an enzyme inside a silica shell (single enzyme nanoparticles, SENs) [185], and its support over (i) conductive materials (carbon or oxide semiconductor nanofibers or nanotubes) to realize biosensors or electrodes for biofuel cells and (ii) mesoporous materials (e.g., SBA-15, or MCM-41) to develop robust biocatalysts for bioremediation or chemical applications. 

Figure 2.23 Encapsulation of an enzyme inside a silica shell (single enzyme nanoparticles, SENs), and the support of these SENs over conductive supports or silica mesoporous material. Source adapted from Kim et al. [178].

Many target organic molecules are too bulky to enter or leave zeolites, l- or this category the ordered mesoporous materials, like MCM-41, MCM-48 and SBA-15, became available. The latter material can even accommodate enzymes. Quite recently the use of these ordered mesoporous materials in catalysis has been reviewed [14]. The review covers no less than 447 references. Another recent review [15] deals with alkylation, hydrogenation and oxidation by mesoporous materials. 

The use of microporous solid catalysts such as zeolites and related molecular sieves has an additional benefit in organic synthesis. The highly precise organization and discrimination between molecules by molecular sieves endows them with shape-selective properties [12] reminiscent of enzyme catalysis. The scope of molecular sieve catalysis has been considerably extended by the discovery of ordered mesoporous materials of the M41S type by Mobil scientists [13,14]. Furthermore, the incorporation of transition metal ions and complexes into molecular sieves extends their catalytic scope to redox reactions and a variety of other transition metal-catalyzed processes [15,16]. 

A new type of materials has been developed by delaminating the lamellar precursors of some zeolites. These materials show external surface areas > 600 m. g from where active sites can be accessible to very large molecules. If on one hand delamination eliminates geometrical shape selective properties of zeolites, it allows on the other hand to dispose of catalysts with the good reactant accessibility of mesoporous materials, but with the stability and active sites characteristics of zeolites. The very large and well structured external surface area can be specially suited for supporting different catalytic functions, which include, among others, metals, transition metal complexes and enzymes. 

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