Immobilization of plant cells

Immobilization of plant cells in hybrid sol-gel materials. Journal of Sol-Gel Science and Technology, 7, 87-97. 

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. 

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. 

Fixation of plant cells in a matrix of, for example, polyurethane foam or entrapment of the cells in calcium alginate beads provides an artificial surrounding for the cells, which protects them from hydrodynamic stress, high cell densities inside the matrix also allow cell to cell contact and communication. Inside the immobilized matrix nutrient and product gradients may occur. Furthermore, immobilized biomass is easily separated from the medium, which is useful in production and biotransformation systems. Immobilization of plant cells has been reviewed 103,104). 

Alkaloid production in cell culture can be more effective with the immobilization of plant cells and enzymes and generally by using large-scale technological systems.Alkaloid produced in cell cultures can be isolated directly from this culture or sometimes from young plants grown from this culture. More than 300 alkaloids are reported to be successfully produced by cell-culture techniques. Only a limited number of species have been researched in this respect. The species studied are known to produce alkaloids with special use in applications. The most researched alkaloids produced by cell cultures are mentioned in Table 6.3. 

In principle, precursor feeding is another approach to increase the titer of final products however, since the expression of the involved genes is subjected to strict regulation, increasing precursor concentration does not usually increase the metabolic fluxes proportionally. In a final approach, immobilization of plant cells is a method that enhances cell-to-cell contact. The associated changes in cellular physiology can bring up the levels of certain secondary metabolites [19]. 

Relatively less work has been done on immobilization of plant and animal cells and spores of microbes in silica matrixes. The main drawback is less viability of the cells in sol-gel matrices. Thus more refined methods are required to utilize harness of the whole cells entrapped in sol-gel matrices and biosensing applications. At the same time studies such as interactions between sol-gel matrices and whole cells and metabolic changes during immobilization have to be closely monitored for the exploration of new matrices and methods. 

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. 

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. 

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. 

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... 

Abstract The use of various immobilized biocatalysts in industrial research and production will be introduced. The applied catalysts span the range from isolated enzymes to microbial whole cells, and even examples of the use of plant cells and mammalian cells could be found. Approximately 65 processes have been reviewed in this article, roughly 50% of which are actual production processes in the chemical industry. The remaining 50% refer to biocatalytic transformations which were carried out at laboratory scale up to pilot scale. In this review special attention was drawn to the range of transformable substrates and the variety of different supports. 

An improved technique for entrapment of plant cells has been recently developed with Capsiaum frutesaene cell suspensions. Cells were shown to passively invade the pores of a reticulated polyurethane matrix, inside which viability and metabolic activity were better preserved than in other immobilizing matrices (23). This sytem was used in a deteuled study of the synthesis of capsaicin (an ester of vanillylamine), which is the pungent principle of chili pepper fruits. In the absence of specific precursors to capsaicin, the entrapped cells produced 2-3 orders of magnitude higher yields than c ll suspensions over a 5-10 day culture period (4-5 mg capsaicin g dry weight 1 versus 30 yg... 

Bioconversion rates can be optimized by using immobilized cells, cell-free preparations, or immobilized (purified) enzymes. Furuya et al. lOI) reported the reduction of codeinone to be more efficient with immobilized P. somniferum cells than with suspended cells. Strictosidine could be produced in large quantities using immobilized strictosidine synthase 102). The potential for this use of plant cell cultures is enormous however, the number of successful applications is still limited. 

Serum albumin Cysteine Water + urea -20 to -25 -20 -3 to -78 for the immobilization of thylakoids Preparation of macroporous carrier for the immobilization of bacterial cells. Preparation of macroporous carrier for the immobilization of submembrane fraction of plant cells Preparation of macroporous albumin [179, 180] [181] [31]... 

Goedhart, J, Hink, M. A, Visser, A. J, Bisseling, T. and Gadella, T. W, Jr. (2000). In vivo fluorescence correlation microscopy (FCM) reveals accumulation and immobilization of Nod factors in root hair cell walls. Plant J. 21, 109-19. 

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