Enzyme encapsulation procedure

FTIR analysis was also used to study the efficiency of the sol-gel enzyme encapsulation method that uses TEOS as precursor. The silica matrix gelation procedure produces a tridimensional reticulate formed by interacting polymeric inorganic chains that form a holding net around the enzyme, and FTIR can be used to follow this encapsulating process. The FTIR spectrograms obtained for the pure silica matrix and the encapsulated CGTase are shown at Fig. 6. 

Fig. 7 Schematic representation of the procedure for encapsulating enzyme in polyelectrolyte microcapsules using MS spheres as templates (I) enzyme immobilization in MS spheres (II) LbL assembly of oppositely charged polyelectrolytes (PE) (III) MS sphere template dissolution using buffered hydrofluoric acid (IV) enzyme encapsulation in a polyelectrolyte microcapsule and (V) enzyme release via altering the shell permeability by pH or salt changes. Reprinted with permission from Advanced Materials [76]...

Similarly to the above-mentioned entrapment of proteins by biomimetic routes, the sol-gel procedure is a useful method for the encapsulation of enzymes and other biological material due to the mild conditions required for the preparation of the silica networks [54,55]. The confinement of the enzyme in the pores of the silica matrix preserves its catalytic activity, since it prevents irreversible structural deformations in the biomolecule. The silica matrix may exert a protective effect against enzyme denaturation even under harsh conditions, as recently reported by Frenkel-Mullerad and Avnir [56] for physically trapped phosphatase enzymes within silica matrices (Figure 1.3). A wide number of organoalkoxy- and alkoxy-silanes have been employed for this purpose, as extensively reviewed by Gill and Ballesteros [57], and the resulting materials have been applied in the construction of optical and electrochemical biosensor devices. Optimization of the sol-gel process is required to prevent denaturation of encapsulated enzymes. Alcohol released during the... 

The first belief in the possibility of enzyme stabilization on a silica matrix was stated by Dickey in 1955, but he did not give experimental evidence, only mentioning that his experiments were unsuccessful [65]. A sol-gel procedure for enzyme immobilization in silica was first developed by Johnson and Whateley in 1971 [66]. The entrapped trypsin retained about 34 % of its tryptic activity observed in solution before the encapsulation. Furthermore, the enzyme was not released from the silica matrix by washing, demonstrating the increased stability and working pH range. Unfortunately, the article did not attract attention, although their method contained all the details that may be found in the present-day common approach. This was probably due to its publication in a colloid journal that was not read by biochemists. 

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. 

Fig. 7.2 Schematic representation of the procedure for the encapsulation of enzyme in PE microcapsules (I) and preparing nanoporous protein particles (II) using MS spheres as templates.

Liposome conjugates may be used in various immunoassay procedures. The lipid vesicle can provide a multivalent surface to accommodate numerous antigen-antibody interactions and thus increase the sensitivity of an assay. At the same time, it can function as a vessel to carry encapsulated detection components needed for the assay system. This type of enzyme-linked immunosorbent assay (ELISA) is called a liposome immunosorbent assay or LISA. One method of using liposomes in an immunoassay is to modify the surface so that it can interact to form biotin-avidin or biotin-streptavidin complexes. The avidin-biotin interaction can be used to increase detectability or sensitivity in immunoassay tests (Chapter 23) (Savage et al., 1992). 

The sol-gel procedure enables encapsulation of enzymes in optically transparent, porous silicate matrices, under mild room-temperature conditions. The small pores prevent microbial degradation and, due to the biomolecule size, they will not diffuse out of the polymer network. The physical encapsulation avoids self-aggregation effects as well as protein unfolding and denaturalization. At the same time, the catalytic activity is maintained as the enzymes are able to react with small substrates that can transfer across or within the support, assuring continuous transformations [75]. 

Enzyme micro-encapsulation is another alternative for sensor development, although in most cases preparation of the microcapsules may require extremely well-controlled conditions. Two procedures have usually been applied to microcapsule preparation, namely interfacial polymerization and liquid drying [80]. Polyamide, collodion (cellulose nitrate), ethylcellulose, cellulose acetate butyrate or silicone polymers have been employed for preparation of permanent micro capsules. One advantage of this method is the double specificity attributed to the presence of both the enzyme and the semipermeable membrane. It also allows the simultaneous immobilization of many enzymes in a single step, and the contact area between the substrate and the catalyst is large. However, the need for high protein concentration and the restriction to low molecular weight substrates are the important limitations to this approach. 

For the preparation of lipase-encapsulated derivatives, a procedure was adopted similar to that described for Method 1, except, in this case, the lipase solution (2.70 g of enzyme diluted in 15 mL of ultrapure water) was added simultaneously with the hydrolysis solution (NH4OH). The encapsulation of CRL in the hydrophobic silica gels was performed in the absence and presence of PEG-1450, resulting in the EN1 and EN2 derivatives, respectively. 

The support obtained by the sol-gel technique was used to immobilize commercial CRL following three procedures. In the first, the lipase was immobilized on PS by ADS in the second, the enzyme was covalently bonded on the support previously silanized and activated with glutaralde-hyde (SPS) in the absence (CB1) and presence of an additive (CB2) and, in the third, the enzyme was encapsulated in the absence (EN1) and presence of an additive (EN2). 

DNA trapped between the layers. Upon rehydration, vesicles reform that contain highly concentrated DNA, a process that can be visualized by staining with a fluorescent dye (Fig. 6). Several enzymes have also been encapsulated using similar procedures [55]. 

An enzyme can be dissolved in an aqueous phase and retained on a column of a hydrophilic solid such as cellulose. Substrate in the solvent phase diffuses into the aqueous phase where reaction with the enzyme occurs. Products diffuse back into the mobile phase and pass out of the column. Using invertase, such a system retained nearly complete activity for a number of weeks. This system resembles the micro-encapsulated enzyme procedure, except that a solvent phase substitutes for a semipermeable membrane. 

Compared to the evaporation procedure, extraction of organic solvent occurs relatively more rapidly, therefore, microspheres produced by solvent extraction are more porous usually resulting in faster release of drugs. Hence, for sustained release purposes, solvent evaporation is preferred. Solvent extraction could be more appropriate and effective compared to evaporation method for encapsulation of delicate and sensitive drugs such as proteins and peptides, enzymes, hormones, and antigens that are susceptible to thermal degradation at temperatures above room temperature (i.e.,... 

With the encapsulation process, the enzyme is dissolved in a solution of monomer, cross-linking agent, and initiator, and the solution then polymerized. Acrylamide or 2-hydroxyethyl methacrylate are the monomers most often used. The simplest procedure embeds the enzyme in the polymer matrix. But enzyme accessibility is reduced because of more difficult diffusion... 

---