Engineering and Manufacturing for Biotechnology - Marcel Hofman & Philippe Thonart

A new polysaccharide derived from plant rhizosphere

Tests for food formulation indicated that the EPS has a good stability in a broad range of pH (figure 4), show a very important cohesiveness and very good healing after the applications of important strains (figure 5). During storage, the gelling properties are reinforced, indicating a renaturation phenomenon ; no syneresis is observed.

4. Conclusion

Original properties of the EPS in terms of gelling effect have been underlined. Numerous applications are to be considered in food and non-food areas. First sampling

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and evaluation have been made towards cosmetic, pharmaceutical and food industries. Collaborations in these domains are in progress with industrial partners. The behaviour of "Soligel" joined to a good yield of conversion and large availability of raw materials authorise optimistic views for this new polysaccharide.

Acknowledgements

The authors are grateful to Pr M. Rinaudo (CERMAV-CNRS), Dr T. Heulin (LEMIRCNRS), Pr G. Cuvelier (ENSIA) and Pr F. Duchiron (Europol'Agro) for their contribution to this work. This research was in part supported by the ADEME and a doctoral fellowship (Europol’ Agro, Reims).

References

Alami, Y. (1997) Role of an exopolysaccharide-producing bacterium in the aggregation of rhizospheric soil of sunflower: consequence of inoculation on the structuration of the soil and on the mineral nutrition of the plant, PhD thesis, Université H. Poincaré, Nancy, 139 pp.

Alami, Y., Heulin, T., Milas, M., de Baynast, R., Heyraud, A.and Villain, A. (1998) Polysaccharide, microorganism and method for obtaining same, composition containing it and application, Patent WO 985993.

Amanullah, A., Satti, S. and Nienow, A.W. (1998) Enhancing xanthan fermentations by different modes of glucose feeding, Biotechnol. Prog. 4, 265-269.

Crompin, J.M. (2000) Industrial scaling up of the production and purification of a new bacterial polysaccharide derived from plant rhizosphere, PhD thesis, Université Reims Champagne Ardenne, 240 pp.

De Baynast, R., Crompin, J.M., Gamier, T., Wintrebert, A., Heulin, T., Alami, Y., Achouak, W., Rinaudo, M., Milas, M. and Villain, A. (1998) Characterisation of new microbial polysaccharides isolated from plant rhizosphere, internal report.

Hebbar, K.P., Gueniot, B., Heyraud, A., Colin-Morel, P., Heulin, T., Balandreau, J. and Rinaudo, M. (1992) Characterisation of exopolysaccharides produced by Rhizobacteria, Appl. Microbiol. Biotechnol. 38, 248-253.

Paul, F., Morin, A. and Monsan, P. (1986) Microbial polysaccharides with actual potential industrial applications, Biotech. Adv. 4, 245-259.

Sutherland, I.W. (1998) Novel and established applications of microbial polysaccharides, TIBTECH 16, 4146.

Villain-Simonnet, A. (1999) New polysaccharides of bacterial origin : structure and properties, PhD thesis,

Université J. Fourier, Grenoble , 242 pp.

Villain-Simonnet, A., Milas, M. and Rinaudo, M. (2000) A new bacterial exopolysaccharide (YAS 34). II. Influence of thermal treatments on conformation and structure. Relation with gelation ability, Int. J.

Biol. Macromol 27, 77-87.

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healthcare (Akerele, 1993). A list of medicinal plants used worldwide contains 21 000 species (Penso, 1983). Some believe that this is a conservative estimate and the number of plant species used for medicinal purposes is between 35 000 and 70 000 (Farnsworth and Soejarto, 1991).

Some examples of prescribed drugs that are obtained from plants include steroids, cardiotonic glycosides (Digitalis glycosides), antimalarials (Cinchona alkaloids, artemisinin-based drugs from Artemisia annua), analgesics and antitussives (opium alkaloids), anaesthetic (cocaine), anticholinergics (belladonna-type tropane alkaloids), antihypertensive (reserpine), cholinergics (physostigmine, pilocarpine), antigout (colchicine), skeletal muscle relaxant (tubocurarine), antiviral drugs (michellamine B from Ancistrocladus abbreviates) and anticancer drugs (Balandrin et al., 1993; Cardellina et al., 1993; Wright, 1995; Pezzuto, 1997; Phillipson et al., 1997).

Anticancer drugs from plants include paclitaxel (Taxol®) from Taxus brevifolia L., vincristine (Oncovin®) from Catharanthus roseus, podophyllotoxin from Podophyllum peltatum L. and camptothecin from Camptotheca acuminata Decne. In addition to these natural compounds extracted from plants, scientists have also produced semisynthetic compounds based on these with improved properties, such as water-solubility and different or enhanced pharmacokinetic profiles. Examples of these are vinorelbine (Navelbine®) which is closely related to vincristine, teniposide (Vumon®) which is a podophyllotoxin analog, topotecan hydrochloride (Hycamtin®) which is an analog of camptothecin, artemether which is based on artemisinin, and taxotere which is a semisynthetic derivative of taxol (Pezzuto, 1996; Phillipson, 1997).

There are approximately 250 000 species of higher plants in the world and the majority of these have not been examined yet for their pharmocological properties. The ethnomedical information and epidemiological data often play important roles in the screening of plants for pharmaceuticals. The rational plant selection also benefits from using a literature-based correlative approach such as the NAPRALERT database (Loub et al., 1985) coupled with advanced analytical techniques (Constant and Beecher, 1995) and bioassays. Although this approach does not lead to the identification of structurally novel compounds with pharmacological properties, it nevertheless enhances our understanding of molecular recognition sites and structure-activity relationships as well as leading to new uses of known compounds. The approach for novel drug discovery is usually based on bioassay-directed fractionation (Cordell et al., 1991; Suffness and Pezzuto, 1991). There are exciting developments in computational chemistry, molecular recognition, combinatorial chemistry and high throughput screening that will have significant effects on the drug discovery process.

With advances in extraction, purification and isolation techniques, it has become possible to produce single active ingredients in standardised tablet or capsule form instead of the “old” medicinal plant extracts. These advances have also led to the chemical synthesis of the active compounds or the moiety of these natural plant compounds. Although the total chemical synthesis of most of these compounds has been generally successful (Nicolaou et al., 1994a; Nicolaou and Sorensen, 1996), it has proved to be uneconomic on an industrial scale in most cases compared to extraction from plant material. The current production based on extraction from plant material however, has its own long-term problems in the case of compounds extracted from wild

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plants. The international trade in the medicinal plants in Europe is reported to involve mainly wild-collection species (Cunningham and Schipman, 1995). There is a serious threat of the loss of medicinal plant species through over-harvesting and habitat destruction. Considering the estimated 25% extinction of higher plants, corresponding to about 60 000 species, by the year 2050 (Akerele et al., 1991), the increasing demand for medicinal plants should be met either from cultivated species or other methods of production should be found. It is in this respect that plant biotechnology can offer a potentially attractive alternative.

1.2. PLANT BIOTECHNOLOGY

The technical aspects of plant cell, tissue and organ culture and experimental protocols can be found in a number of publications (Street, 1977; Pierik, 1987; Fowler and

Warren, 1992; Dixon and Gonzales, 1994). Although the initial expectation from the application of plant biotechnology was in the in vitro production of fine chemicals commercially using plant cell, tissue or organ cultures in the bioreactors (Bajaj, 1999), this has not materialised except for a few cases. Instead, the main application of plant biotechnology has been in the area of horticulture, agriculture and forestry (Lindsey and Jones, 1989; Vasil, 1994; Davey et al., 1998).

The main reasons for the failure to produce fine chemicals by plant cell cultures commercially were our initial lack of understanding of the complex metabolism of plants, especially the biochemical routes for the synthesis of these compounds and the mechanisms for their regulation and control, and of plant molecular biology and genetic engineering. Further complications arose from the instability of plant cell lines, cell line storage problems and extreme susceptibility of plant cell and tissue cultures to microbial contamination. Although there is still a great deal to discover and elucidate in plant metabolism and genetics, with our current knowledge and technology in these fields, and the numerous strategies developed over the years for the manipulation of plant cell, tissue and organ cultures, the exciting scope for the production of fine chemicals in vitro by genetically engineered plant cell, tissue and organ cultures is still viable (Goodwin and Mercer, 1983; Herbert, 1989; Burbridge, 1993; Dennis et al., 1997).

1.3. ANTITUMOR COMPOUNDS FROM TAXUS SPP.

Paclitaxel (Taxol) is a relatively new anticancer drug which was first isolated from T. brevifolia. Paclitaxel and the related taxane compounds mainly occur in the plants of Taxus spp. (Strobel et al., 1993). The bark of T. brevifolia was reported to have the highest content of Taxol (Vidensek et al., 1990; Witherup et al., 1990; Fang et al., 1993), but the concentration was very low, only about 0.007-0.04% of the dry weight (Vidensek et al., 1990; Wheeler et al., 1992; Elsohly et al., 1994). Paclitaxel and related taxane compounds act on cells by stabilising microtubules, preventing their depolymerisation and therefore blocking mitosis at the transition between metaphase and anaphase. The inability of the cell to depolymerise microtubules induces cell death. The structure and biological activities of Taxol were first published in 1971 by Wani et al. Since then the total synthesis of Taxol has been achieved but on an industrial scale it has been considered both unfeasible and uneconomic (Holton et al., 1994; Nicolaou et

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al., 1994b). The current method of commecial production involves the extraction of intermediary compounds from the needles and then the chemical transformation of these compounds (Nicolaou et al., 1994a; Hezari and Croteau, 1997). Extraction from the bark of the old trees is not a viable option because of the obvious environmental concerns. Therefore, plant biotechnology can provide an alternative source for this drug.

Several research groups have reported success in inducing suspension cultures of T.

(Christen et al., 1989; Jaziri et al., 1991; Wickremesinhe and Arteca, 1991, 1993; Fett-

Neto et al., 1992, 1993, 1995; Durzan and Ventimiglia, 1994; Chee, 1995; Kim et al., 1995) and production of taxane compounds (Christen et al., 1991; Fert-Neto et al.,

1992, 1994a, 1994b; Zhiri et al., 1994; Mirjalili and Linden, 1995). However, there are not many reports on the immobilised cultures of Taxus spp (Seki and Furusaki, 1996; Seki et al., 1997).

Although secondary metabolites are mainly produced by slow growing or nongrowing cultures, it is a good process strategy to grow cultures fast to reach the desired biomass concentration in the growth stage and then switch to a production strategy in the second stage. When considering such a production system with two stages, cell immobilisation is one of the most effective ways to maintain higher productivity of target metabolites. Therefore, this work provides data for initiating and growing cultures rapidly for the first stage and then immobilisation for the second stage in a production process.

In the immobilised plant cell cultures, the actual productivity often changes due to the effects of intraparticle mass transfer (Furusaki et al., 1988), redifferentiation and physicochemical interaction between the immobilising materials and cells (Haldimann and Brodelius, 1987). Immobilisation of plant cells in reticulated polyurethane foam matrices in situ in the bioreactors is easy, fast and economical (Lindsey et al., 1983; Mavituna and Park, 1985; Mavituna et al., 1987; Williams and Mavituna, 1992). This paper describes successful initiation, growth and immobilisation of Taxus cultures for paclitaxel production. The immobilised cultures showed improvement of cell growth and paclitaxel production compared to the suspension cultures.

2.Materials and methods

2.1.PLANT MATERIAL AND CHEMICALS

Seedlings, stems and needles of media cv. Hicksii and T. cuspidata var. nana were used as explants. These plants were obtained from a commercial nursery and grown in our department.

All reagents were purchased from Sigma Chemical Company (Poole, UK), except for the basal media which were purchased in a pre-made form excluding the plant growth regulators and sucrose from Imperial Laboratories Ltd. (Salisbury, UK). Vitamins used were in a pre-mixed form, while coconut water was prepared in the laboratory by

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draining the juice from a large number of coconuts, deproteinising by boiling for 10 minutes and then filtering before storing it in small batches in the freezer until required.

2.2. CULTURE INITIATION AND MAINTENANCE

2.2.1. Callus initiation

2.2.1.1Explant Preparation. Seedlings, stems and needles of media and T. cuspidata were used as explants in this study. Seedlings were established using the same protocol of Zhiri et al. (1994) for the in vitro germination of T. baccata seeds. Stems and needles from pot-grown plants were surface sterilised before placing on the media. For surface sterilisation, plant materials were washed and immersed in 70% ethanol for

2 min. They were then immersed in 2% sodium hypochlorite solution for 20 min. and rinsed with sterile water. Finally, the surface sterilised explants were aseptically cut into pieces of ca. 10 mm in length, placed on solid media, and incubated at either in the dark or using a 16 h light / 8 h dark photo-regime. The light intensity was 20-30

2.2.1.2Media Composition. Different basal media, MS (Murashige and Skoog, 1962),

Gamborg's B5 (Gamborg, 1968) and woody plant (WP) (Lloyd and McCown, 1980) supplemented with different plant growth regulators (PGR), all at 1 mg/1, were used for

the initiation of callus from the needles of media (Table 1). In addition to the growth regulators, these basal media were supplemented with 20g/l sucrose and 3.5g/l

After the first set of experiments on callus initiation and growth using different basal media and PGR, B5 (Gamborg et al., 1968) and NAA were chosen to investigate further the effect of the plant species, explant type and coconut water on callus initiation.

For the maintenance of the calli, seven pieces of callus were placed on either CWT, a new medium formulated in this research (Table 2), or TM5 medium (Ketchum et al., 1995) which was modified (Zalat & Mavituna, unpublished) by solidifying with 0.3g/l gelrite instead of agar and adding ascorbic acid (100 mg/1) aseptically by filtering after autoclaving and cooling.

and seedlings and T. cuspidata seedling to Gamborg’s B5 medium supplemented with 20g/l sucrose and lmg/1 NAA and 0.0025 mg/1 BA. 50mg/l ascorbic acid and 292 mg/1 glutamine were filtered sterilised through 0.2 µm cellulose acetate filter (Gelman, Northampton, UK) and added to the medium after autoclaving. The suspension cultures were subcultured every two weeks by transferring about 4g cells (fresh weight) into 100ml of fresh medium in a 250ml Erlenmeyer flasks.

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2.3. CELL IMMOBILISATION

Cells from suspension cultures were immobilised in reticulated polyurethane foam

(Declon, UK) particles in shake flasks and sheets in the bioreactors. The pore size of this foam matrix was 45 ppi (pores per inch). Five empty foam particles were threaded onto an L shaped, stiff, stainless steel wire, which was held stationary and submerged in the liquid medium in the 250 ml Erlenmeyer flask. After inoculation the plant cells and cell aggregates were self-immobilised in these empty foam particles and

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grew within the foam matrix. The details of immobilisation in shake flasks and bioreactors can be found in Mavituna et al. (1987).

2.4. BIOREACTORS

Four different types of reactors were used in this study; airlift, bubble, stirred tank and immobilised cell reactor. One 4L reactor, with 3.5L working volume and dimensions of 24cm height x 15cm diameter, was modified to work as airlift, stirred tank and immobilised cell reactor. Air was introduced to the bioreactor through an L-shaped sparger and the airflow was maintained during the whole experiment at 0.25 v.v.m. In order to use it as an airlift bioreactor, an additional stainless steel tube draft with dimensions of 17cm length x 13cm diameter was placed in the centre of the bioreactor. In the stirred tank operation, two impellers of 9cm diameter and 12cm apart, rotating at

90 rpm were used. In order to use it as an immobilised cell reactor, 8 sheets of 3cm x 15cm X 0.5cm reticulated polyurethane foam were arranged vertically like baffles around the impeller shaft in the stirred tank bioreactor (Figure 7). Only one impeller was used with 90 rpm in the initial stage of immobilisation what took about 7 to 10 days. After this, the bulk medium was mixed by sparged air only.

A glass jar of 2L capacity with 1.5L working volume and dimensions of 18cm height x 12cm diameter was used as the bubble reactor. A sintered glass sparger placed in the bottom centre of the jar was used for the aeration and mixing. In all the bioreactor experiments, an inoculum size of 10% v/v was used in CWT liquid medium.

2.5. ANALYTICAL MEASUREMENTS

2.5.1. Growth

0.8g Callus was aseptically transferred to 50ml capped jars (Fisher Scientific) containing 10ml of either CWT or modified TM5 medium. On TM5, callus growth was determined by both the fresh and dry weight measurements at close intervals. On CWT, callus growth was monitored by measuring the increase in fresh weight which was then used to calculate the growth index as [(final wt - initial wt)/initial wt]. All samples were in triplicates.

In order to study the growth of suspension cultures, one gram fresh weight of cell suspension was inoculated into 50 ml medium in 125-ml Erlenmeyer flasks that were placed on shakers with 100 rpm. Three flasks were harvested at each point for the determination of fresh and dry weight, pH of the medium, viability of cells as well as sucrose, glucose and fructose concentration in the medium.

2.5.2. Viability

Cell viability of the various cultures was determined by fluorescein diacetate (FDA) staining according to the method of Widholm (1972).

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2.5.3. Sugar analysis

The residual sugars in the suspension medium were analysed by HPLC using a

Spherisorb5 NH2 column (Phenomenex, Macclesfield,UK). Acetonitrile:water ratio of 80:20 v/v was used as the mobile phase at a flow rate of 1ml/min. Sugars were detected by a differential refractometer (Waters 110). The concentration of glucose, fructose and sucrose were calculated using a standard curve for each sugar.

2.5.4. Taxane analysis

Paclitaxel and related taxane compounds were analysed by extracting the cell free medium with equal volume of dichloromethane. The organic layer was evaporated completely, and the residue was resuspended in HPLC grade methanol and filtered through 0.45µm filter for HPLC analysis (Waters). Analyses were performed using Curosil G column (Phenomenex, Macclesfield, UK), 250mm x 4.6mm, and acetonitrile:water ratio of 45:55 v/v as the mobile phase at a flow rate of 0.8ml/min. Taxol was detected using photo-diode array detector and identified by its retention time and UV spectrum.

3.Results and discussion

3.1.CALLUS INITIATION

3.1.1 Effect of media and plant growth regulators

Table 3 shows the result of callus initiation from the needles of media. It was observed that callus initiation started from the cut surface of the needles and anywhere else on the leaf surface where the epidermal layer was stripped off. The initiated callus was fine, friable and light yellow.

Although there was not a significant difference between the different types of basal salt media in terms of their effect on callus initiation, in general, MS and B5 gave better results than WP as the callus yields were higher.

Hormone regimes played a key factor in callus formation. There was no significant difference between 2,4-D and NAA in terms of their effect on callus initiation. When the media contained both 2,4-D and NAA, this would slightly increase the percentage of callus formation. The media containing 1mg/L kinetin did not produce any callus or produced significantly smaller amounts of callus compared to the other treatments. This was because of the detrimental effect of kinetin on the explants (the explants turned brown). Kinetin also gave the lowest percentage of callus initiation in all groups. If either 2,4-D or NAA was added to kinetin in the medium, the percentage of callus initiation would increase significantly compared to the case when only kinetin was present.

The effect of increasing the NAA concentration on callus initiation was also evaluated. It was found that increasing the concentration of NAA to 5mg/l was better

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