Porous biocatalyst

This example involves the same diffusion-reaction situation as in the previous example, ENZSPLIT, except that here a dynamic solution is obtained, using the method of finite differencing. The substrate concentration profile in the porous biocatalyst is shown in Fig. 5.252. 

Because the monoliths allow total convection of the mobile phase through their pores, the overall mass transfer is dramatically accelerated compared to conventional porous structures. Based on the morphology and porous properties of the molded monoliths, which allow fast flow of substrate solutions, it can be safely anticipated that they would also provide outstanding supports for immobilization of biocatalysts, thus extending the original concept of monolithic materials to the area of catalysis. 

The main idea demonstrated by Willner and coworkers [20] is the ability to construct multilayered nanoparticle electrodes, which are porous. In a related study Patolsky et al. extended this idea further using biocatalysts to detect H2O2 [18]. In this example, the construction of the electrode is similar to the one described above but the redox-active bridging molecule was replaced with microperoxidase-11 (MP-11). 

When a biocatalyst is immobilized on or within a solid matrix, mass transfer effects may exist because the substrate must diffuse from the bulk solution to the immobilized biocatalyst. If the biocatalyst is attached to non-porous supports there are only external mass transfer effects on the catalytically active outer surface in the reaction solution, the supports are surrounded by a stagnant film and substrate and product are transported across this Nemst layer by diffusion. The driving force for this diffusion is the concentration difference between the surface and the bulk concentration of substrate and product. 

The internal mass-transfer effects can be reduced, however, by decreasing the particle dimensions of the porous support containing the biocatalyst. Particle-diameter decrease results in a reduction of the distance from the outer support surface that the substrate must cross and, consequently, also results in a decrease of the substrate concentration gradient. 

Internal diffusion in porous catalysts, if dominant, also reduces the observed activity of the biocatalyst. The decisive coefficient for mass transfer is the effective diffusion coefficient De((, which is defined in Eq. (5.56), where D0is the diffusion coefficient in solution, e the porosity of the carrier, and t the tortuosity factor. 

Natural supports (agarose, dextran, cellulose, porous glass, silica, the optical fiber itself or alumina) and synthetic resins (acrylamide-based polymers, methacrylic acid-based polymers, maleic anhydride-based polymers, styrene-based polymers or nylon, to name a few) have been applied for covalent attachment of enzymes. These materials must display a high biocatalyst binding capacity (as the linearity and the limit of detection of the sensing layers will be influenced by this value), good mechanical and chemical stability, low cost, and ease of preparation. 

A composite biomaterial formed by Pd metal, carbon-ceramic mixture and oxidoreductase enz3ones constitutes a new t3rpe of renewable smface biosensor with a controllable size reaction layer [198]. The carbon provides the electrical conductivity, the enzymes are used for biocatalyst process, metallic palladimn is used for electrocatalysis of biochemical reaction product and the porous silica provides a rigid skeleton. The hydrophobicity of this composite material allows only a limited section of the electrode to be wetted by the aqueous analyte, thus providing a controlled thickness reactive layer. Another biocomposite material containing enzyme-modifled boron-doped diamond was used in the development of biosensors for the determination of phenol derivatives [199], alcohol [200] and glucose [201]. 

Many immobilization techniques provide biocatalysts in which the enzyme is immobilized in the porous structure of the biocatalyst particle. In such cases, the... 

Chitosan is of importance because of its primary amino groups that are susceptible for coupling reactions. Furthermore, porous spherical chitosan particles are commercially available (Chitopearl, Fuji Spinning) allowing noncovalent or covalent attachment of enzymes [55]. This support matrix can be easily prepared [56] and activation methods have been summarized [57]. Treatment with polyethyleneimine or with hexamethylenediamine and glut-ardialdehyde can improve the mechanical characteristics [53,58] of the biocatalyst, which is poor otherwise. However, this is often accompanied by some activity loss or increase of diffusional limitations. 

The adsorption of biomolecules onto carriers that are insoluble in water is the simplest method of immobilization. An aqueous solution of the biomolecules is contacted with the active carrier material for a defined period of time. Thereafter the molecules that are not adsorbed are removed by washing. Anionic and cationic ion exchange resins, active charcoal, silica gel, clay, aluminum oxide, porous glass, and ceramics are being currently used as active material. The carrier should exhibit high affinity and capacity for the biomolecule and the latter must remain active in the adsorbed state. The carrier should adsorb neither reaction products nor inhibitors of the biocatalyst. 

Whole-cell, hollow-fiber MBR are still under development. Despite their significant potential they have, so far, found only limited application for biochemicals production. One of the reasons is that cleaning of the hollow-fiber membranes is difficult, especially when whole-cell biocatalysts are immobilized in the small fibers. The mass transfer between the nutrients and cells has also to be taken into consideration and enhanced. Immobilizing the biocatalysts in porous beads, instead of directly on the membrane, may tend to avoid some of these problems, and to simplify membrane cleaning. The concept of using MBR as bioartificial organs is technically very attractive the various MBR under development, however, must still be validated with clinical results. One can expect, however, that their development will follow the success of artificial kidneys, which are currently employed worldwide. 

MBR are finding application for many important enzymatic reactions. In MBR enzymes are, often, immobilized onto the membrane surface or in its porous structure. Free enzymes and also enzymes grafted onto a soluble polymer (to increase their molecular size) are utilized. In the latter case the membrane may simply act as a barrier to retain the enzyme, while allowing the removal of products and/or the addition of reactants. A key advantage of immobilization is that it permits easier enzyme recovery in addition, grafting the biocatalyst does, on occasion, increase its thermal stability and its resistance towards organic solvents. 

The morphologic characterization of the immobilized enzyme is important to correlate the biocatalyst performance with porous structure parameters. BET analysis, which is usually based on N2 isothermal adsorption at 77 K, allows determining the solid-specific surface area, total pore volume, pore size distribution, and mean pore diameter. It is not recommended for solids with a low specific surface area (<5 m g ). Table 2 shows the specific smface area, mean pore diameter, and total pore volume determined by BET for the pure sol-gel silica matrix having TEOS as the precursor and the same matrix with the encapsulated CGTase. 

---