immobilized plant cells

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  • 1. 5 Immobilized Plant Cells PAUL D. WILLIAMS and FERDA MAVITUNA University of Manchester Institute of Science and Technology, UK5.1 5.2INTRODUCTION SYSTEMS FOR IMMOBILIZED CULTURES 5.2.1 Immobilization Techniques 5.2.2 Bioreactor Configurations 5.3 CHARACTERISTICS OF IMMOBILIZED PLANT CELLS 5.3.1 Viability of Immobilized Plant Cells 5.3.1.1 Staining 5.3.1.2 Respiration and substrate uptake 5.3.1.3 Growth and division 5.3.1.4 NMR spectra 5.3.1.5 Plasmolysis 5.3.1.6 Scanning electron microscopy 5.3.2 Growth of Immobilized Cells 5.3.3 Biosynthetic Capacity 5.3.3.1 Bioconversions 5.3.3.2 Synthesis from precursors 5.3.3.3 De novo synthesis 5.4 MASS TRANSFER 5.4.1 Background 5.4.2 Effect on Physiology 5.4.3 Effective Diffusion Coefficients 5.4.4 Oxygen Uptake Rates 5.4.5 Light 5.4.6 Carbon Dioxide 5.5 PRODUCT RELEASE 5.5.1 Spontaneous Release 5.5.2 Permeabilization 5.6 CONCLUDING REMARKS 5.7 REFERENCES5.163 64 64 66 67 67 67 68 68 68 69 69 69 69 69 70 71 72 72 72 73 73 74 74 74 74 75 75 75INTRODUCTIONImmobilization of biocatalysts offers many potential advantages and is now a well-established technique, with the history of enzyme immobilization going back over 25 years and including many industrial applications. The immobilization of microorganisms is less well developed in terms of large scale applications, but is widely used in the laboratory. With this background it was inevitable that immobilization techniques should be applied to plant cell cultures and much work has been carried out to establish methods for plant cell immobilization and suitable bioreactors for use with the immobilized cultures. Although many of the methods used for immobilization are common to both microorganisms and plant cell cultures, it is worth noting that there are major practical differences between culturing suspensions of plant cells and microorganisms. Individual plant cells are generally larger than microbial cells and in suspension culture are usually found as aggregates, which may be up to

2. 64Systems for the Exploitation of Cell Culturesseveral millimetres in diameter. They also show greater susceptibility to damage from mechanical stresses such as sustained shear, and from environmental stresses such as changes in temperature and oxygen concentration. Any method for the immobilization of plant cells must therefore take into account these characteristics. In general the advantages of immobilizing biocatalyst may be summarized (Brodelius, 1984) as follows: (i) the biocatalyst is easily recovered and can be used over an extended period of time; (ii) the desired product is easily separated from the catalyst; (iii) the continuous operation of a process is readily achieved; and (iv) the immobilized catalyst often shows increased stability. These benefits are equally applicable to microbial systems but, more specifically, immobilization offers a solution to some of the physiological requirements and some of the process engineering problems particularly associated with plant cell cultures. Physiological requirements for plant cell cultures for the production of secondary metabolites appear to include cell to cell contact to allow transfer of materials from one cell to another. Such contact may help induce cytodifferentiation, which is related to secondary metabolism (Yeoman et al, 1982). There is also evidence that plant secondary metabolites are produced at higher concentrations in slow-growing cultures (Kurz and Constabel, 1985). In an immobilized system, growth and production phases can be decoupled and controlled by chemical and physical stress conditions. This allows cells to be retained in the bioreactor for extended periods, with alternating rejuvenation/growth and secondary metabolite production cycles. The slow growth of plant cell cultures gives a long lead time before beginning bioreactor operation and it is therefore of advantage to extend the productivity of a bioreactor as far as possible. Process engineering problems may develop from the tendency of plant cells to aggregate, which can lead to blockages in pipes and openings and to the culture rapidly sedimenting, if it is not continually agitated. However, the shear sensitivity of the cultures means that mechanical agitation may be detrimental to cells and that cultures cannot be transported using conventional pumps, without significant loss of viability. Again, immobilization may be a solution to these problems and may offer a microenvironment protected from sustained shear. The main disadvantage of immobilization is that it is only of use with cell lines which excrete the product of interest into the culture medium. Attempts to induce the release of products which are normally retained within the cell, by such techniques as permeabilization, have generally decreased cell viability to an undesirable extent, although Brodelius and Nilsson (1983) have produced encouraging results with reversible permeabilization of Catharanthus roseus using dimethyl sulfoxide (DMSO). Systems for inducing product release may eventually further increase the applicability of immobilization, but even without such methods the range of secondary metabolites produced by immobilized plant cell cultures is extensive. Brodelius (1985a) suggests that the potential role of immobilized plant cells for the large scale production of secondary metabolites cannot be fully evaluated, until such biological problems as low productivities and genetic instability have been addressed. However, although improvements in these areas are of key importance to the commercialization of plant tissue culture products, immobilization offers sufficient advantages to suggest that it will have important applications in future developments in this technology.5.2 5.2.1SYSTEMS FOR IMMOBILIZED CULTURES Immobilization TechniquesAny immobilization method selected for plant cells should be harmless to the cells, easy to carry out under aseptic conditions, capable of operating for long periods and, particularly for large scale applications, low in cost (Mavituna et al., 1987). In practice this has meant the use of some sort of entrapment immobilization in almost all cases. Table 1 gives a number of examples of the systems of immobilization which have been used with plant cells, together with the associated plant species and their products. This list is not exhaustive, but aims to indicate the variety of cultures which have been immobilized and the popularity of the various methods of immobilization. Entrapment methods which have been used with plant cell cultures can be categorized after Novais (1988) into: (i) gel entrapment by ionic network formation; (ii) gel entrapment by precipitation; (iii) gel entrapment by polymerization; and (iv) entrapment in preformed structures. The most widely used form of immobilization with plant cell cultures is entrapment by ionic network formation, especially in the form of alginate beads. Alginate is a polysaccharide which forms a 3. Immobilized Plant Cells65Table 1 Some Examples of Immobilized Plant Cell Systems Substrate/ precursorProductImmobilization methodByconversions Catharanthus roseus Digitalis lanata Daucus carota Daucus carotaCathenamine Digitoxin Digitoxigenin GitoxigeninAgarose Alginate Alginate AlginateFelix etal, 1981 Brodelius et ai, 1981 Jones and Veliky, 1981 Veliky and Jones, 1981Mentha spicata Mucuna pruriens Papaver somniferum( )-Menthone L-Tyrosine CodeinoneAjmalicine Digoxin Periplogenin 5-Hydroxygitoxigenin ( + )-NeomentholPolyacrylamide Alginate AlginatePapaver somniferum Nicotiana tabacumCodeinone Acetoacetic esters Keto estersPlant speciesNicotiana tabacumSynthesis from precursors Tryptamine, Catharanthus roseus Secologanin Isocapric acid, Capsicum frutescens Vanillylamine, Valine, ferulic acid Datura innoxia Ornithine Phenylalanine Nicotiana tabacumRef.Codeine 3-Hydroxybutanoates Hydroxy estersPolyurethane foam AlginateGalun etal., 1983 Wichers et al, 1983 Furuya et , 1984 Furusaki et al, 1988 Corchete and Yeoman, 1989 Naoshima et ai, 1989AlginateNaoshima and Akakabe, 1989AjmalicineAlginate, agarose, Agar, carrageenan Polyurethane foamL-DOPACodeineScopolamine CafTeoylputriscine CaffeineAlginate AlginateBrodelius et al., 1979 Brodelius and Nilsson, 1980 Brodelius and Nilsson, 1983 Lindsey et al, 1983 Lindsey and Yeoman, 1984 Mavituna et al, 1987 Lindsey, 1982 Berlin etal, 1989MembraneLang et a/., 1990Anthraquinones Ajmalicine, SerpentineAlginate Alginate, agarose, Agar, carrageenan Polyester fibresGlycine max Catharanthus roseusStrictosidine lactam, Ajmalicine, Epivindolinine, Tabasonine, Catharanthine Steroid glycosides Phenolics EnzymesBrodelius etal, 1980 Brodelius and Nilsson, 1980 Brodelius etal, 1981 Lambe and Rosevear, 1983 Rho et al, 1990Vicia faba Nicotiana tabacum Lavandula veraEthane Epiandrosterone PigmentsPolyphenylene oxide Hollow fibres Agarose, Polyurethane Alginate Alginate Polyurethane gelCapsicum frutescensCapsaicinPolyurethane foamApium graveolens Salvia miltiorrhizaPhthalides Cryptotanshinone Methylxanthine Berberine Diosgenin GinkgolidesAlginate AlginateSchnabl etal, 1983 Tsuchiya, 1983 Tanaka et al, 1984 Nakajima et al, 1986 Lindsey and Yeoman, 1984 Wilkinson et al, 1988 Watts and Collin, 1985 Miyasaka et al, 1986Alginate Alginate Polyurethane foam Polyester fibreHaldimann and Brodelius, 1987 Kobayashi et al, 1987 Ishida, 1988 Carrier et al, 1990Coffea arabica De novo synthesis Morinda citrifolia Catharanthus roseus Catharanthus roseusSolanum aviculareCoffea arabica Thalictrum minus Dioscorea deltoidea Ginkgo bilobaTheobromineCapsaicinJirku etal, 1981 Macek etal, 1981 Shuler, 1981 Felix and Mosbach, 1982reasonably stable gel in the presence of multivalent cations, with calcium being commonly used. Beads of alginate-containing cells are formed by dripping a cell/sodium alginate solution into a calcium chloride solution. This method of immobilization has the advantage of being easily reversible, by the addition of a cal

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