Immobilization of Cells
Immobilized individual enzymes can be successfully used for single-step reactions. They are, however, not suitable for multi-enzyme reactions and for the reactions requiring cofactors. The whole cells or cellular organelles can be immobilized to serve as multi-enzyme systems. In addition, immobilized cells rather than enzymes are sometimes preferred even for single reactions, due to further cost factor in isolating enzymes. For the enzymes which depend on the special arrangement of the membrane, cell immobilization is preferred.
Immobilized cells have been traditionally used for the treatment of sewage. The techniques employed for immobilization of cells are almost the same as that used for immobilization of enzymes with appropriate modifications. Entrapment and surface attachment techniques are commonly used. Gels, and to some extent membranes, are also employed.
Immobilized Viable Cells:
The viability of the cells can be preserved by mild immobilization. Such immobilized cells are particularly useful for fermentations. Sometimes mammalian cell cultures are made to function as immobilized viable cells.
Immobilized Non-viable Cells:
In many instances, immobilized non-viable cells are preferred over the enzymes or even the viable cells. This is mainly because of the costly isolation and purification processes. The best example is the immobilization of cells containing glucose isomerase for the industrial production of high fructose syrup. Other important examples of microbial biocatalysts and their applications are given in Table 21.5.
Limitations of Immobilizing Eukaryotic Cells:
Prokaryotic cells (particularly bacterial) are mainly used for immobilization. It is also possible to immobilize eukaryotic plant and animal cells. Due to the presence of cellular organelles, the metabolism of eukaryotic cells is slow. Thus, for the industrial production of biochemical, prokaryotic cells are preferred. However, for the production of complex proteins (e.g. immunoglobulin’s) and for the proteins that undergo post- translational modifications, eukaryotic cells may be used.
Effect of Immobilization on Enzyme Properties:
Enzyme immobilization is frequently associated with alterations in enzyme properties, particularly the kinetic properties of enzymes.
Some of them are listed below:
1. There is a substantial decrease in the enzyme specificity. This may be due to conformational changes that occur when the enzyme gets immobilized.
2. The kinetic constants Km and Vmax of an immobilized enzyme differ from that of the native enzyme. This is because the conformational change of the enzyme will affect the affinity between enzyme and substrate.
Immobilized Enzyme Reactors
The immobilized enzymes/cells are utilized in the industrial processes in the form of enzyme reactors. They are broadly of two types — batch reactors and continuous reactors. The frequently used enzyme reactors are shown in Fig. 21.9.
Batch Reactors:
In batch reactors, the immobilized enzymes and substrates are placed, and the reaction is allowed to take place under constant stirring. As the reaction is completed, the product is separated from the enzyme (usually by denaturation).
Soluble enzymes are commonly used in batch reactors. It is rather difficult to separate the soluble enzymes from the products; hence there is a limitation of their reuse. However, special techniques have been developed for recovery of soluble enzymes, although this may result in loss of enzyme activity.
Stirred tank reactors:
The simplest form of batch reactor is the stirred tank reactor (Fig. 21.9A). It is composed of a reactor fitted with a stirrer that allows good mixing, and appropriate temperature and pH control. However, there may occur loss of some enzyme activity. A modification of stirred tank reactor is basket reactor. In this system, the enzyme is retained over the impeller blades. Both stirred tank reactor and basket reactor have a well-mixed flow pattern.
Plug flow type reactors:
These reactors are alternatives to flow pattern type of reactors. The flow rate of fluids controlled by a plug system. The plug flow type reactors may be in the form of packed bed or fluidized bed (Fig. 21.9B and 21.9C). These reactors are particularly useful when there occurs inadequate product formation in flow type reactors. Further, plug flow reactors are also useful for obtaining kinetic data on the reaction systems.
Continuous Reactors:
In continuous enzyme reactors, the substrate is added continuously while the product is removed simultaneously. Immobilized enzymes can also be used for continuous operation. Continuous reactors have certain advantages over batch reactors. These include control over the product formation, convenient operation of the system and easy automation of the entire process. There are mainly two types of continuous reactors-continuous stirred tank reactor (CSTR) and plug reactor (PR). A diagrammatic representation of CSTR is depicted in Fig. 21.9D. CSTR is ideal for good product formation.
Membrane Reactors:
Several membranes with a variety of chemical compositions can be used. The commonly used membrane materials include polysulfone, polyamide and cellulose acetate. The biocatalysts (enzymes or cells) are normally retained on the membranes of the reactor. The substrate is introduced into reactor while the product passes out. Good mixing in the reactor can be achieved by using stirrer (Fig. 21.10A). In a continuous membrane reactor, the biocatalysts are held over membrane layers on to which substrate molecules are passed (Fig. 21.10B).
In a recycle model membrane reactor, the contents (i.e. the solution containing enzymes, cofactors, and substrates along with freshly released product are recycled by using a pump (Fig. 21.10C). The product passes out which can be recovered.
Applications of Immobilized Enzymes and Cells:
Immobilized enzymes and cells are very widely used for industrial, analytical and therapeutic purpose, besides their involvement in food production and exploring the knowledge of biochemistry, microbiology and other allied specialties. A brief account of the industrial applications of immobilized cells is given in Table 21.5.
1. Manufacture of Commercial Products:
A selected list of important immobilized enzymes and their industrial applications is given in Table 21.6. Some details on the manufacture of L-amino acids and high fructose syrup are given hereunder.
2. Production of L-Amino Acids:
L-Amino acids (and not D-amino acids) are very important for use in food and feed supplements and medical purposes. The chemical methods employed for their production result in a racemic mixture of D- and L-amino acids. They can be acylated to form D, L-acyl amino acids. The immobilized enzyme aminoacylase (frequently immobilized on DEAE sephadex) can selectively hydrolyse D, L-acyl amino acids to produce L-amino acids.
The free L-amino acids can separated from the un-hydrolysed D-acyl amino acids. The latter can be recemized to D, L-acyl amino acids and recycled through the enzyme reactor containing immobilized aminoacylase. Huge quantities of L-methionine, L-phenylalanine L-tryptophan and L-valine are produced worldwide by this approach.
3. Production of High Fructose Syrup:
Fructose is the sweetest among the monosaccharide’s, and has twice the sweetening strength of sucrose. Glucose is about 75% as sweet as sucrose. Therefore, glucose (the most abundant monosaccharide) cannot be a good substitute for sucrose for sweetening. Thus, there is a great demand for fructose which is very sweet, but has the same calorific value as that of glucose or sucrose.
High fructose syrup (HFS) contains approximately equivalent amounts of glucose and fructose. HFS is almost similar to sucrose from nutritional point of view. HFS is a good substitute for sugar in the preparation of soft drinks, processed foods and baking.
High fructose syrup can be produced from glucose by employing an immobilized enzyme glucose isomerase. The starch containing raw materials (wheat, potato, corn) are subjected to hydrolysis to produce glucose. Glucose isomerase then isomerizes glucose to fructose (Fig. 21.11). The product formed is HFS containing about 50% fructose. (Note: Some authors use the term high fructose corn syrup i.e. HFCS in place of HFS).
Glucose isomerase:
This is an intracellular enzyme produced by a number of microorganisms. The species of Arthrobacter, Bacillus and Streptomyces are the preferred sources. Being an intracellular enzyme, the isolation of glucose isomerase without loss of biological activity requires special and costly techniques. Many a times, whole cells or partly broken cells are immobilized and used.
Immobilized Enzymes and Cells- Analytical Applications:
A. In Biochemical Analysis:
Immobilized enzymes (or cells) can be used for the development of precise and specific analytical techniques for the estimation of several biochemical compounds. The principle of analytical assay primarily involves the action of the immobilized enzyme on the substrate.
A decrease in the substrate concentration or an increase in the product level or an alteration in the cofactor concentration can be used for the assay. A selected list of examples of immobilized enzymes used in the assay of some substances is given in Table 21.7. Two types of detector systems are commonly employed.
- Thermistors are heat measuring devices which can record the heat generated in an enzyme catalysed reaction. Electrode devices are used for measuring potential differences in the reaction system. In the Fig. 21.12, an enzyme thermistor and an enzyme electrode, along with a specific urease electrode are depicted.
C. In Affinity Chromatography and Purification:
Immobilized enzymes can be used in affinity chromatography. Based on the property of affinity, it is possible to purify several compounds e.g. antigens, antibodies, cofactors.