Cesar Mateo, Olga Abian, Gloria Fernández-Lorente, Benevides C. C. Pessela, Valeria Grazu, Jose M. Guisan et al. Pages PDF · Glutaraldehyde in. Fernando López-Gallego, Gloria Fernandez-Lorente, Javier Rocha-Martin, Juan M. Bolivar, Cesar Mateo, Jose M. Guisan. Pages PDF. Simple protocols for the immobilization of enzymes and cells that could be very useful for nvensigtitape.ga]. Slavik, K. and.
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Immobilization of Enzymes and Cells: Third Edition, Methods in Molecular Biology, The term immobilized enzymes refers to “enzymes physically confined or. enzymes and cells have to be immobilized, via very simple and cost-effective Included format: EPUB, PDF; ebooks can be used on all reading devices. The selection of immobilization of cell or enzyme depends on so many criteria like number . nvensigtitape.ga
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.
Site-directed mutagenesis has been successfully used to produce more stable and functionally more efficient enzymes e. 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 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. 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. 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 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.
Enzyme immobilization is frequently associated with alterations in enzyme properties, particularly the kinetic properties of enzymes. There is a substantial decrease in the enzyme specificity. This may be due to conformational changes that occur when the enzyme gets immobilized.
The kinetic constants K m and V max 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. 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.
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.
The simplest form of batch reactor is the stirred tank reactor Fig. 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. 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. 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. 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. CSTR is ideal for good product formation.
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. In a continuous membrane reactor, the biocatalysts are held over membrane layers on to which substrate molecules are passed Fig. In a recycle model membrane reactor, the contents i.
The product passes out which can be recovered. 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 A selected list of important immobilized enzymes and their industrial applications is given in Table Some details on the manufacture of L-amino acids and high fructose syrup are given hereunder.
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.
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.
Some authors use the term high fructose corn syrup i. 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 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.
Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and key tips on troubleshooting and avoiding known pitfalls.
Authoritative and practical, Immobilization of Enzymes and Cells, Third Edition demonstrates simple and efficient protocols for the preparation, characterization, and utilization of immobilized enzymes and cells. Skip to main content Skip to table of contents. Advertisement Hide. Immobilization of Enzymes and Cells Third Edition.
Editors view affiliations Jose M. Front Matter Pages i-xi. New Opportunities for Immobilization of Enzymes.
Pages Immobilization of Enzymes: A Literature Survey. Glutaraldehyde-Mediated Protein Immobilization. Bolivar, Cesar Mateo, Jose M. Bolivar, Jose M. Immobilization of Enzymes by Bioaffinity Layering. Tips for the Functionalization of Nanoparticles with Antibodies.
Pablo del Pino, Scott G. Mitchell, Beatriz Pelaz.