Methods of Cell Immobilization

Introductionintact or disintegrated dead cells that contain active enzymesresting or growing cells (this technique is used especially with eukaryotic cells where the whole metabolic machinery is often required for their specific application)

Methods of Cell Immobilization

The reactions catalyzed by immobilized whole-cell biocatalysts can be classified as follows: 1. Reactions involving single enzymes (bioconversions)2. Reactions involving multienzyme systems with or without cofactors3. Reactions involving a complete metabolic pathway yielding primary or secondary metabolites

Methods of Cell ImmobilizationThe use of immobilized microbial cells is advantageous in the following areas

1.when the desired enzymes are intracellular and the extracted, purified enzymes become unstable after immobilization

2. when the microorganism does not contain interfering enzymes or when such enzymes can be inactivated without loss of desired catalytic activity

3. when substrates and products do not have a high molecular mass and can diffuse through the cell membrane

Methods of Cell Immobilization1.The material should be available in sufficient quantities and at low price.2. The material should have a large surface area accessible to cells and reactants3. The material must be mechanically, chemically, and thermally stable under process and storage conditions.4. The matrix should contain a sufficient number of functional groups to bind the cells.5. The material should not reduce cell activity or initiate cell lysis.6. The material should be easy to handle in the immobilization procedure.7. The material should be capable of recycling or safe disposal.8. In the case of viable growing cells, the matrix should have asufficiently large void volume or be elastic enough to accommodate new cells.

Methods of Cell ImmobilizationImmobilization Techniques

1. Carrier-free immobilization2. Immobilization of a given biomass onto a

preformed carrier surface3. Immobilization of a given biomass during

the course of carrier formation (e.g., by polymerization)

4. Immobilization by controlled growth of an inoculum or by germination of immobilized spores

Classification of cell immobilization methods

Cell Immobilization without a Support

intrinsic tendency to aggregate or flocculate at high cell densities. Yeast, mold, and plant cells aggregand are relatively stable to shear fields in fluidized-bed reactors Flocculation can also be induced by polyelectrolytes such as chitosan Cell aggregation can be induced by low molecular mass bi- or multifunctional reagents such as glutaraldehyde, diazotized diamines, or toluene diisocyanate

Binding of Cells to a Carrier Physical Adsorption.

• Additional chemicals are usually unnecessary, and fixation is carried out under growth conditions; viable cell preparations can, therefore, be obtained. mammalian cells bound to preformed surfaces to produce therapeutic biochemicals. The cells are immobilized on microcarriers (small-diameter beads, 100 – 200 µm) manufactured from different synthetic polymers (e.g., polystyrene, gelatin, dextran, polyacrylamide, or glass) that offer a large specific surface area for cell growth monolithic ceramic matrix has been developed for large-scale cell

cultures. For adherent cell growth, the scalability is almost linear and depends on the available surface area.

• Ionic Binding. Ionic binding is a special case of physical adsorption where charged microbial cells can electrostatically interact with the ions on a carrier surface to form stable complexes. Synthetic ion-exchange resins, modified cellulose derivatives, or inorganic materials can be used as carriers. Cell adsorption is mainly affected by factors such as pH, ionic strength, surface charge, cell age, or composition of the carrier surface.

Covalent Binding

Cells can also bind to the functional groups of the carrier surface by covalent bonds. This technique is frequently used for enzyme immobilization but has only limited use in whole-cell immobilization because the toxicity of the coupling agents often results in loss of cell viability or enzyme activity

Immobilization of Cells by Entrapment

Lattice Entrapment.retention of cells within the network of a polymer matrix.

forms pellets high viable cell density (e.g., polyacrylamide gel, alginate gel, k-carrageenan, and photo-cross-linkable resins)

Polyacrylamide Gel.

free-radical polymerization in an aqueous acrylamide monomer solution containing the cells at low temperature. The degree of cross-linking determines the porosity and mechanical properties of the pellets. The major disadvantage of the method is the toxicity of the acrylamide monomer, the cross-linking agent (e.g., methylenebisacrylamide [110-26-9]), and the polymerization initiator (e.g.,tetramethylethylenediamine [110-18-9]) which can decrease cell viability and enzyme activity], Hydroxyethyl methacrylate [868-77-9] .....less toxic alternative to acrylamide With acrylamide monomers, optimal results are achieved in isotonic buffered solution at low temperature (4 – 12 °C) by keeping exposure time to a minimum (3 – 4 min).

Alginate [9005-38-3]

is extracted from seaweed and is a linear copolymer of b-D-mannuronic acid and a-L-guluronic acid linked by 1,4-glycosidic bonds. It forms a gel in the presence of multivalent ions, usually calcium or aluminum. The controlled entrapment of cells is simple and generally nontoxic. Various cell types can be immobilized with negligible loss of viability.

κ-Carrageenan [11114-20-8]sulfonated polysaccharide extracted from seaweed. It consists of b-D-galactose 4-sulfate and 3,6-anhydro-D-galactose units. Only the sodium salt of k-carrageenan is soluble in cold water. Induction of gelation with potassium ions can be performed under very mild conditions without the use of chemicals that decrease enzyme activity. Another advantage of this technique is that the immobilized cell biocatalysts can be tailor-made into pellets, sheets, beads, etc.

one-step procedure

Photo-cross-linkable resins

Active groups (e.g., vinyl groups) are coupled to polyethylene or poly-(propylene glycol) oligomers of controlled chain length to prepare prepolymers. The prepolymers are then mixed with the cell culture, and cross--linking is initiated with UV radiation

Immobilization of Cells by Entrapment

The advantages of this method are: 1. entrapment is simple and proceeds under very

mild conditions2. prepolymers do not contain toxic monomers3. the network structure of the gels can be adapted

as required4. optimal physicochemical gel properties can be

achieved by selecting suitable prepolymers

Immobilization of Cells by Entrapment

Membrane Entrapment

The supply of oxygen and nutrients to the cells and the removal of carbon dioxide from the cells occur by diffusion and may be insufficient.

Microencapsulation

entrapped in hollow spheres where they can be cultured. Different capsule diameters (from 20 nm to 2 mm) and controlled membrane porosity can be achieved In larger microcapsules, however, radial concentration gradients of nutrients or oxygen can result in a necrotic core

Methods of Organelle Immobilization

mitochondria, chloroplasts, peroxisomes (microbodies), vacuoles, and nuclei.

chloroplasts as potential solar energy photoconverters Immobilization of rat liver mitochondria on a solid support consisting of alkysilanized glass beads Rat liver mitochondria have also been entrapped in polyacrylamide gelyeast mitochondria have been entrapped in a polyurethane matrix

Coimmobilization of Biocatalysts

• galactosidase and Saccharomyces cerevisiaefor the conversion of cellobiose to ethanol

• Yeast cells or dead mycelia have been employed for direct coupling of enzymes to cell walls in food-processing applications

• bioproduction of hydrogen gas with hydrogenase and chloroplasts Coimmobilization results in a fivefold increase in hydrogen production compared with the system employing two separately immobilized catalytic species.

Economic Aspects25 % of the world pharmaceutical market is already covered by biotechnologically manufactured products. In the chemical industry, this fraction is approaching 10 %. According to market growth projections, U.S. biotechnology industry sales will increase from about $ 400×106

in 1987 to $ 2.5×109 by 1990, and more than $ 25×109 by 2000 . The world biotechnology market in the year 2004 is predicted to be $ 75×109

Immobilized Enzymes.The total market for industrial enzymes is about $ 400×106 and is growing by about 15 % each year. Enzyme immobilization has not initiated the expected revolution in the enzyme industry Soluble use-and-discard enzymes have, by far, the most dominant market share. Only one immobilized enzyme product, immobilized glucose isomerase, is used in amounts > 50 t a year; 1500 – 1750 t are used worldwide annually. The three other major immobilized enzymes are aminoacylase (about 5 t/a), lactase (< 5 t/a), and penicillin G acylase (3 – 4 t/a) .Other immobilized enzymes are also available but are used in even smaller amounts: glucoamylase, hydantoinase, invertase, nitrilase, RNAse, and penicillin V acylase.

Immobilized microorganismsmultiple enzyme reactions especially where cofactor regeneration is essential.

Recombinant Escherichia coli can be used for the production of relatively simple peptide hormones with pharmaceutical applications such as human insulin, human growth factor, or human interferons An immobilized biocatalyst is reportedly used for the conversion . A hepatitis B surface antigen vaccine prepared in recombinant yeast was approved for marketing, and many other products from geneticallyengineered microorganisms and yeast are in the clinical test stage (interferons, lymphokines, hormones, enzymes, and vaccines.

Immobilized Mammalian Cells.Many complex proteins of pharmacological interest have precise folding and glycosylation requirements that cannot be fulfilled in simple microorganisms. By using hybridoma or recombinant DNA techniques, mammalian cells can often be persuaded to produce and secrete useful quantities of these compounds

Tissue plasminogen activator (TPA) recombinant E. coli or yeast or in glycosylated form in mammalian cells. The first three months' sales in the United States were almost $ 60×106. Worldwide sales are projected to reach about $ 500×106 in the next few years..

Monoclonal antibodies 100 monoclonal antibodies are offered, with annual sales of nearly $ 500×106.

Generally, the economics of microbial systems are more favorable than those of mammalian cell culture systems for recombinant proteins. When the use of recombinant microbial systems is impossible or impractical, cell culture systems will gain a market position if the products are high-value biologicals (> $ 100/g).

Immobilized Plant Cells.Plant cell cultures can also be used for the production of metabolites such as pharmaceuticals, chemicals, flavors, and fragrances. The first product obtained from mass plant cell cultures was shikonin [517-89-5], a red pigment composed of eight naphthoquinone molecules. Shikonin is produced by a two-stage fermentation process and is a high-value chemical ($ 4000/kg) with a limited annual market capacity of ca. 15 kg.

Immobilized plant cell systems will be used mainly for products of cells in the stationary growth phase. The release of intracellularly stored products by intermittent permeabilization of immobilized cells can be a great economic advantage, allowing reutilization of the biomass. The continuous immobilized plant cell process in combination with strain selection and improved product leakage allows production of plant-derived chemicals in the range of $ 20 – 25/kg. However, in the present industrial state of technology for plant cell cultures, a relatively small number of products have both high value per weight and sufficient market size.

Safety, Environmental, and Legal Aspects

• Immobilized Enzymes1. catalytic activity of the pure enzyme,2. allergenic reactions produced by the

enzyme itself or by other proteins in the preparation,

3. presence of toxic metabolites such as mycotoxins or antibiotics.

Safety, Environmental, and Legal Aspects

Joint FAO/WHO Expert Committee on Food Additives (JECFA)

The committee laid down criteria for evaluating enzymes according to the source material . Enzymes obtained from animal, plant, or microbial sources commonly used as food, or used in the preparation of food are accepted as foods if satisfactory chemical and microbiological specifications can be established. No toxicological studies are required for such enzymes. For enzymes obtained from nonpathogenic microorganisms commonly found as contaminants in food, JECFA recommended short-term toxicity experiments. More extensive toxicological studies, as well as chemical and microbiological specifications, are required for enzymes obtained from less well-known microorganisms.

Safety, Environmental, and Legal Aspects