Immobilization of Enzymes and Cells: Methods, Effects and Applications
Traditionally, enzymes in free solutions (i.e. in soluble or free form) react with substrates to result in products. Such use of enzymes is wasteful, particularly for industrial purposes, since enzymes are not stable, and they cannot be recovered for reuse.
Immobilization of enzymes (or cells) refers to the technique of confining/anchoring the enzymes (or cells) in or on an inert support for their stability and functional reuse. By employing this technique, enzymes are made more efficient and cost-effective for their industrial use. Some workers regard immobilization as a goose with a golden egg in enzyme technology. Immobilized enzymes retain their structural conformation necessary for catalysis.
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There are several advantages of immobilized enzymes:
a. Stable and more efficient in function.
b. Can be reused again and again.
c. Products are enzyme-free.
d. Ideal for multi-enzyme reaction systems.
e. Control of enzyme function is easy.
f. Suitable for industrial and medical use.
g. Minimize effluent disposal problems.
There are however, certain disadvantages also associated with immobilization.
a. The possibility of loss of biological activity of an enzyme during immobilization or while it is in use.
b. Immobilization is an expensive affair often requiring sophisticated equipment.
Immobilized enzymes are generally preferred over immobilized cells due to specificity to yield the products in pure form. However, there are several advantages of using immobilized multi-enzyme systems such as organelles and whole cells over immobilized enzymes. The immobilized cells possess the natural environment with cofactor availability (and also its regeneration capability) and are particularly suitable for multiple enzymatic reactions.
Methods of Immobilization:
The commonly employed techniques for immobilization of enzymes are—
- adsorption
- entrapment
- covalent binding
- cross-linking
Adsorption:
Adsorption involves the physical binding of enzymes (or cells) on the surface of an inert support. The support materials may be inorganic (e.g. alumina, silica gel, calcium phosphate gel, glass) or organic (starch, carboxymethyl cellulose, DEAE-cellulose (Diethylaminoethyl cellulose (DEAE-C) is a positively charged resin used in ion-exchange chromatography, a type of column chromatography, for the separation and purification of proteins and nucleic acids), DEAE-sephadex (DEAE–Sephadex is a positively-charged slurry that will have electrostatic interactions with the negatively charged atoms, making them elute later than the positively-charged molecules in the interested sample. This is a separation technique used widely to discover specific proteins, or enzymes in the body)).
Adsorption of enzyme molecules (on the inert support) involves weak forces such as van der Waals forces and hydrogen bonds (Fig. 21.3). Therefore, the adsorbed enzymes can be easily removed by minor changes in pH, ionic strength or temperature. This is a disadvantage for industrial use of enzymes.
Entrapment:
Enzymes can be immobilized by physical entrapment inside a polymer or a gel matrix. The size of the matrix pores is such that the enzyme is retained while the substrate and product molecules pass through. In this technique, commonly referred to as lattice entrapment, the enzyme (or cell) is not subjected to strong binding forces and structural distortions.
Some deactivation may however, occur during immobilization process due to changes in pH or temperature or addition of solvents. The matrices used for entrapping of enzymes include polyacrylamide gel, collagen, gelatin, starch, cellulose, silicone and rubber. Enzymes can be entrapped by several ways.
1. Enzyme inclusion in gels:
This is an entrapment of enzymes inside the gels (Fig. 21.4A).
2. Enzyme inclusion in fibres:
The enzymes are trapped in a fibre format of the matrix (Fig. 21.4B).
3. Enzyme inclusion in microcapsules:
In this case, the enzymes are trapped inside a microcapsule matrix (Fig. 21.4C). The hydrophobic and hydrophilic forms of the matrix polymerise to form a microcapsule containing enzyme molecules inside. The major limitation for entrapment of enzymes is their leakage from the matrix. Most workers prefer to use the technique of entrapment for immobilization of whole cells. Entrapped cells are in use for industrial production of amino acids (L-isoleucine, L-aspartic acid), L-malic acid and hydroquinone.
Microencapsulation:
Microencapsulation is a type of entrapment. It refers to the process of spherical particle formation wherein a liquid or suspension is enclosed in a semipermeable membrane. The membrane may be polymeric, lipoidal, lipoprotein-based or non-ionic in nature. There are three distinct ways of microencapsulation.
1. Building of special membrane reactors.
2. Formation of emulsions.
3. Stabilization of emulsions to form microcapsules.
Microencapsulation is recently being used for immobilization of enzymes and mammalian cells. For instance, pancreatic cells grown in cultures can be immobilized by microencapsulation. Hybridoma cells have also been immobilized successfully by this technique.
Covalent Binding:
Immobilization of the enzymes can be achieved by creation of covalent bonds between the chemical groups of enzymes and the chemical groups of the support (Fig. 21.5). This technique is widely used. However, covalent binding is often associated with loss of some enzyme activity. The inert support usually requires pretreatment (to form pre-activated support) before it binds to enzyme. The following are the common methods of covalent binding.
1. Cyanogen bromide activation:
The inert support materials (cellulose, sepharose, sephadex) containing glycol groups are activated by CNBr, which then bind to enzymes and immobilize them (Fig. 21.6A).
2. Diazotation:
Some of the support materials (amino benzyl cellulose, amino derivatives of polystyrene, aminosilanized porous glass) are subjected to diazotation on treatment with NaNO2 and HCI. They, in turn, bind covalently to tyrosyl or histidyl groups of enzymes (Fig. 21.6B).
3. Peptide bond formation:
Enzyme immobilization can also be achieved by the formation of peptide bonds between the amino (or carboxyl) groups of the support and the carboxyl (or amino) groups of enzymes (Fig. 21.6C). The support material is first chemically treated to form active functional groups.
4. Activation by bi- or poly-functional reagents:
Some of the reagents such as glutaraldehyde can be used to create bonds between amino groups of enzymes and amino groups of support (e.g. aminoethylcellulose, albumin, amino alkylated porous glass). This is depicted in Fig. 21.6D.
Cross-Linking:
The absence of a solid support is a characteristic feature of immobilization of enzymes by cross- linking. The enzyme molecules are immobilized by creating cross-links between them, through the involvement of poly-functional reagents. These reagents in fact react with the enzyme molecules and create bridges which form the backbone to hold enzyme molecules (Fig. 21.7). There are several reagents in use for cross-linking. These include glutaraldehyde, diazobenzidine, hexamethylene diisocyanate and toluene di- isothiocyanate.
Glutaraldehyde is the most extensively used cross-linking reagent. It reacts with lysyl residues of the enzymes and forms a Schiff’s base. The cross links formed between the enzyme and glutaraldehyde are irreversible and can withstand extreme pH and temperature. Glutaraldehyde cross- linking has been successfully used to immobilize several industrial enzymes e.g. glucose isomerase, penicillin amidase. The technique of cross-linking is quite simple and cost-effective. But the disadvantage is that it involves the risk of denaturation of the enzyme by the poly-functional reagent.
Choice of Immobilization Technique:
The selection of a particular method for immobilization of enzymes is based on a trial and error approach to choose the ideal one. Among the factors that decide a technique, the enzyme catalytic activity, stability, regenerability and cost factor are important.
Immobilization of L-amino acid acylase:
L-Amino acid acylase was the first enzyme to be immobilized by a group of Japanese workers (Chibata and Tosa, 1969). More than 40 different immobilization methods were attempted by this group. Only three of them were found be useful. They were covalent binding to iodoacetyl cellulose, ionic binding to DEAE-Sephadex and entrapment within polyacrylamide.
Stabilization of Soluble Enzymes:
Some of the enzymes cannot be immobilized and they have to be used in soluble form e.g. enzymes used in liquid detergents, some diagnostic reagents and food additives. Such enzymes can be stabilized by using certain additives or by chemical modifications. The stabilized enzymes have longer half-lives, although they cannot be recycled. Some important methods of enzyme stabilization are briefly described.
1. Solvent Stabilization:
Certain solvents at low concentrations stabilize the enzymes, while at high concentrations the enzymes get denatured e.g. acetone (5%) and ethanol (5%) can stabilize benzyl alcohol dehydrogenase.
2. Substrate Stabilization:
The active site of an enzyme can be stabilized by adding substrates but the required condition for optimum reaction not created e.g. starch stabilizes a-amylase; glucose stabilizes glucose isomerase.
3. Stabilization by Polymers:
Enzymes can be stabilized, particularly against increased temperature, by addition of polymers such as gelatin, albumin and polyethylene glycol.
4. Stabilization by Salts:
Stability of metalloenzymes can be achieved by adding salts such as Ca, Fe, Mn, Cu and Zn e.g. proteases can be stabilized by adding calcium.
5. Stabilization by Chemical Modifications:
Enzymes can be stabilized by suitable chemical modifications without loss of biological activity. There are several types of chemical modifications.
a. Addition of poly-amino side chains e.g. polytyrosine, polyglycine.
b. Acylation of enzymes by adding groups such as acetyl, propionyl and succinyl.
6. Stabilization by Rebuilding:
Theoretically, the stability of the enzymes is due to hydrophobic interactions in the core of the enzyme. It is therefore, proposed that enzymes can be stabilized by enhancing hydrophobic interactions. For this purpose, the enzyme is first unfold and then rebuilt in one of the following ways (Fig. 21.8).
1. The enzyme can be chemically treated (e.g. urea and a disulfide) and then refolded.
2. The refolding can be done in the presence of low molecular weight ligands.
3. For certain enzymes, refolding at higher temperatures (around 50°C) stabilize them.
7. Stabilization by Site-Directed Mutagenesis:
Site-directed mutagenesis has been successfully used to produce more stable and functionally more efficient enzymes e.g. subtilisin E.