Cultured plant cells, immobilization

In recent years, extensive attention has been focused on finding cultured plant cells that can be used as catalysts for organic functional group transformations. A number of transformations employing freely suspended or immobilized plant cell cultures have been reported.24 For example, Akakabe et al.25 report that immobilized cells of Daucus carota from carrot can be used to reduce prochiral carbonyl substrates such as keto esters, aromatic ketones, and heterocyclic ketones to the corresponding secondary alcohols in ( -configuration with enantiomeric excess of 52-99% and chemical yields of 30 63%). 

To date, progress achieved clearly demonstrates the potential of cultured plant cells for secondary metabolite production. Use of concurrent immobilization/permeabilization procedures, as well as precursor and elicitor treatments, may open new avenues of increasing product yields and will consequently affect the economic aspects of plant cell culture in a positive manner. However, our understanding of the many biosynthetic pathways of desired secondary metabolites is incomplete and successful industrial scale plant cell culture processes are still limited. Results of research in the area of plant cell culture will increase our understanding of the biosynthesis of plant metabolites, enhance our knowledge of plant-microorganism or plant-plant interactions and can lead to entirely new products or product lines of desirable compounds currently not available to use. Such work can also lead to development of industrial scale production processes for products now produced and recovered by conventional methods. Also, the genetic variety of the 250,000 to 750,000 plant species available remains to be explored. Presently only 5 to 15% of these species have been subject to even... 

Immobilization of cultured plant cells by entrapment in a gel matrix appears to provide conditions that are optimum to cell differentiation resulting in higher yields of secondary products. Compact or organized and slow-growing cultures often synthesize higher amounts of secondary products than growing cultures. 

The alkaloid productivity and the storage capacity in cultured plant cells can be influenced by the pH gradient between the medium and the accumulation sites inside the plant cell (vacuoles). A shift in the medium pH from low to high value was used to release the intracellularly stored alkaloids into the culture medium in cell suspension culture of C. roseus [42]. Similarly, transient modifications in the medium pH value led to the release of indole alkaloids in culture medium during immobilized cell cultivation of C. roseus [43]. 

J.M. (2001) The effect of immobilization on recombinant protein production in plant cell culture. Plant Cell Rep., 20, 562-566. 

Other kinds of plant cell cultures such as immobilized tobacco cells have also been studied for the analogous transformation. The results show that plant cell cultures provide an accessible way of converting several prochiral ketones into the corresponding chiral secondary alcohols with reasonable chemical yield and high enantioselectivity. 

Lindsey, K., M. M. Yeoman, G. M. Black, and F. Mavituna, "A novel method for the immobilization and culture of plant cells," FEBS Letters 155 (1983) 143-149. 

Both cell culture with a lipophilic extraction phase and with a polar extraction phase have been reported to be helpful for the accumulation and detection of secondary substances [7,8]. Plant cell cultures release lipophilic and volatile substances such as ethylene, ethanol, and acetaldehyde. The addition of a lipophilic phase to the culture medium can be used as a means of accumulating and detecting these substances. Maisch et al. [8] found that the addition of XAD-4 resin to Nicotiana tabacum cultures enhanced the production of phenolic secondary metabolites several times compared to the adsorbent-free control. Kim and Chang [9] reported in situ extraction for enhanced shikonin production by Lithospermum erythrorhizon. When n-hexadecane was added to the cultivation, higher specific shikonin productivity was obtained than that from the cultures of free cells without extraction. They also suggested that n-hexadecane addition at an early stage in calcium alginate immobilized cell cultures was effective for shikonin production. Most of the produced shikonin was dissolved in n-hexadecane, so it would reduce the costs for shikonin separation. 

Various adsorbents have been examined for their potential to increase in situ product separation in plant cell culture. Suspended solid adsorbents were popular, and the use of immobilized adsorbent has been investigated recently [17-20]. The advantages of immobilized adsorbent are that it is easy to use in a bioreactor operation and that it allows adsorbents to be easily separated from culture broth for the repeated use of cells and adsorbents [21, 22]. The design and optimization of in situ separation process for phytochemicals using immobilized adsorbent required a detailed mathematical model. It was difficult to achieve an optimal design based on purely empirical correlations, because the effects of various design parameters and process variables were coupled. 

In this review the use of adsorbent for the in situ separation of product in plant cell culture is discussed, and a mathematical model which describes immobilized adsorbent coupled with selective separation is presented. Several examples of the application of the technique are also provided. 

The integration of elicitation, in situ product adsorption with XAD-7, and the immobilization of Catharanthus roseus cells lead to an increase in productivity and a significant increase in extracellular ajmalicine production [5]. The integration of in situ product separation by two-phase culture and immobilized plant cells could be feasible for continuous production in immobilized plant cell bioreactors requiring the repeated use of cells. 

Integrated bioprocesses can be used to enhance the production of valuable metabolites from plant cell cultures. The in situ removal of product during cell cultivation facilitates the rapid recovery of volatile and unstable phytochemicals, avoids problems of cell toxicity and end-product inhibition, and enhances product secretion. In situ extraction, in situ adsorption, the utilization of cyclodextrin, and the application of aqueous two-phase systems have been proposed for the integration of cell growth and product recovery in a bioreactor. The simultaneous combination of elicitation, immobilization, permeabilization, and in situ recovery can promote this method of plant cell culture as a feasible method to produce various natural products including proteins. 

Embryogenic rice calli tend to form larger clumps during cultivation. Therefore, immobilization of the calli has hardly been carried out until now. Porous supports such as polyurethane foam have often been used for the immobilization of mycelial cells [64, 65] and plant cells [66-68]. In almost all cases, effective production of biological materials by the immobilized cells has been reported. To avoid the damage due to the hydrodynamic stress, we proposed the immobilization culture of rice callus using a macroporous urethane foam support. A turbine-blade reactor (TBR), which has been developed for hairy root culture, was also used in the culture. In the culture space, polyurethane foam was added as an immobilization support. 

Cells are grown either in suspension in a free or immobilized form 102), or by adherence to a solid surface 100). Materials used for promoting surface-dependent cell growth are glasses, metals, plastics, carbohydrate polymers etc. the media used contain substances such as blood plasma, amniotic fluids, tissue extracts, etc.103). Recent developments in animal cell culture are aimed at the improvement of strains and culture techniques, medium optimization, and scale-up. In contrast to plant cell culture, animal cell culture has already found its technical application. Large-scale... 

Plant cell cultures represent a potentially rich source of secondary metabolites of commercial importance and have been shown to produce them in higher concentrations than the related intact plants. However, plant cell cultures often produce metabolites in lower concentrations than desired and commonly store them intracellularly. These limitations can be overcome by product yield enhancement procedures, including immobilization of cultured cells, and permeabilization, or ideally using a combined immobilization/ permeabilization process with retained plant cell viability. Complex coacervate capsules consisting of chitosan and alginate or carrageenan proved to be effective biomaterials for entrapment, controlled permeabilization of cells and to allow control of capsule membrane diffusivity. 

Plant cell and organ cultures can produce higher metabolite concentrations than found in the corresponding intact plant organs (6, 9). However, plant cells grown in culture may also produce lower quantities of the desired secondary metabolites which are commonly stored intracellularly. The challenges to increase product yield and to enhance the release of secondary metabolites can be met in various ways (7). These include immobilization (9), permeabilization (12, ), the use of precursors (12,13), and the induction of secondary metabolite production via elicitors (14). 

Other experiments on the chitinolytic activity of immobilized cells and culture medium of carrot cells (Table V) indicate that chitosan, in concentrations available to plant cells during the preparation of kappa-carrageenan-chitosan coacervate capsules, is detrimental to chitinase activity in plant cells and culture medium. This is also of interest in light of the data presented on the elicitor effect of chitosan on chitinase production at minute chitosan concentrations (see Table IX). 

Products synthesized by the parent plant, in a variety of cell types and throughout the development of the plant, are made in culture under a range of conditions and considerable scope exists for improving the productivity of such cultures. Development of stable plant cell lines of sufficiently high productive capacity on which to base commercial processes remains an important problem. Immobilization of plant cells within a support matrix appears to offer both bioengineering and biochemical advantages compared with free cells. These include ease of use in a continuous process with retention of biomass reuse of biocatalyst (cells), cofactors or precursors protection of cells from mechanical stresses and superior productivity and longevity of cells. 

Petersen, M., Seitz, H., Alfermann, A. and Reinhard, E. (1987) Immobilization of digitoxin 12p-hydroxylase, a cytochrome P450-dependent enzyme, from cell cultures of Digitalis lanata EHRH. Plant Cell Rep., 6, 200-3. 

Gas Permeable Membrane Aerator Bioreactor. This type of bioreactor has not yet been fully developed. Nevertheless, some information is available. For example, one bioreactor is equipped with an aerator composed of fine tubes made of polycarbonate, polypropylene, silicone gum, etc. This type of bioreactor should be valuable for immobilized plant cell cultures. 

Immobilized Culture. Immobilization of plant cells was first reported by Brodelius et al. in 1979,1 1 and since then many reports have been published. Unfortunately, an immobilized cell culture technique has not yet been established as an industrial process for secondary metabolite production. However, this technique has many excellent features and should be the subject of future development research. 

Figure 30. Immobilized plant cell cultures. (Prenosil and Pederson 1983).

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