JP3476933B2 - Method for producing polysaccharide-producing cell and cell for producing polysaccharide - Google Patents

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for producing a high-polysaccharide-producing cell and a polysaccharide-producing cell. More specifically, the present invention provides an improved method for producing a polysaccharide-producing cell by subculturing callus of a plant cell secreting extracellular polysaccharide, and a polysaccharide-producing cell obtained by the improved method. Regarding

[0002]

2. Description of the Related Art Many types of plants that produce polysaccharides are known. In order to obtain a polysaccharide from such a plant, conventionally, a method by extraction or tapping from a seed of natural or cultivated plant, fruit, stem, stem, leaf, root, tuber, or tuberous root, etc. It was used. However, these natural plants or cultivated plants have a drawback that they are easily affected by the natural environment and the weather, and thus the production amount and the price are not stable. Therefore, a method has been proposed in which cells or tissues of these plants are cultured in a liquid medium to grow the cells or tissues and simultaneously produce a polysaccharide, which is then discharged into the medium.

The above-mentioned method for producing a polysaccharide by culturing cells in a tissue is not affected by the natural environment or the weather and can be stably produced, which is an advantageous method. However, it has been known so far. However, the method has a big problem that the yield of polysaccharide produced from cultured cells is low.
For this reason, studies have been conducted to improve the production amount of polysaccharides by changing the culture conditions.

[0004] For example, in the invention described in JP-A-5-207888, when a plant cell such as callus derived from tuberose of the genus Polyantes is cultured in a liquid medium, a certain range of amount of the medium is added to the liquid medium. The presence of the metal salt suppresses the increase in the viscosity of the liquid medium,
This aims to improve the production amount of polysaccharides. That is, a polysaccharide-producing cell is generally obtained by inducing callus from a target plant and then proliferating by subculture of this callus to obtain a large number of cells, and a large amount of this expanded polysaccharide-producing cell. It is used in a method of culturing in a liquid medium to produce a polysaccharide, secreting it extracellularly, and finally separating the extracellularly secreted polysaccharide from the cell to obtain the desired polysaccharide. However, in the invention described in this publication, a method is used in which a metal salt is present in a polysaccharide production medium to suppress an increase in viscosity of the liquid medium. Then, as a result of suppressing the increase in the viscosity of the liquid medium for the polysaccharide production of the polysaccharide-producing cells in this way, the diffusion of the culture solution, the mixing is improved, and the productivity of the polysaccharide is improved. It is said that the separation of callus or cells from the supernatant containing the target substance is facilitated and the productivity is improved.

[0005]

The present invention further improves the known method for producing polysaccharide-producing cells by subculturing callus of plant cells secreting extracellular polysaccharides to produce polysaccharide-producing cells. It is an object of the present invention to provide a method for obtaining a polysaccharide-producing cell that produces a saccharide with high productivity.

[0006]

The present invention provides a method for producing a polysaccharide-producing cell by subculturing callus of a plant cell secreting extracellular polysaccharides in a liquid medium. On the way, carbon source component , nitrogen source component in the medium component composition,
A method for producing a high-polysaccharide-producing cell, which comprises an operation of culturing in a liquid medium changed so as to increase the concentration of at least one of metal salts and plant hormones by 20% or more. is there. It should be noted that changes in the composition of the components in the liquid medium during the subculture may be due to
Concentration increase der Rukoto is desirable.

The callus of the present invention is a plant of the genus Polyantes
(In particular, Poriantesu genus Chu bellows plants) is preferably one derived from cells.

That is, according to the research conducted by the inventor of the present invention,
When callus of plant cells secreting extracellular polysaccharides is subcultured in a liquid medium to obtain polysaccharide-producing cells, the concentration of any component of the liquid medium during the subculture It was found that a polysaccharide-producing cell having a markedly improved polysaccharide productivity can be obtained by changing (changing) the value. The present invention has been completed based on this novel finding by the present inventor.

The plant cells used in the method for producing a high-polysaccharide-producing cell of the present invention are not particularly limited as long as they are of a type capable of producing a polysaccharide. For example, okra of a mallow family plant. It is possible to use various plants such as carrots of the Apiaceae plant and mint of the Lamiaceae plant. However, in the present invention, in particular, as a cell, a plant cell of the genus Polyanthes (Polianthes L.), particularly tuberose (Polyanthes) of the genus Polyanthes, is used.
es tuberosa L. Since the present invention can be advantageously used when using a callus derived from a plant, the present invention will be described in the following with reference to the callus of cells of a tuberose plant of the genus Polyantes.

Callus obtained from tuberose plants of the genus Polyantes is known to produce acidic heteropolysaccharides whose constituents are arabinose, mannose, galactose, xylose and glucuronic acid. Then, the subculture is carried out by using a known basic medium, for example, Murashige-Skoog medium, Linsmeier-Skoog medium.
Skoog's medium, Gamborg's medium, White medium, Niche-Nitach medium, Trekke (T
ulecke) medium or the like. Among them, it is preferable to use Murashige-Skoog's medium or Rinsmeier-Skoog's medium. These media usually contain small amounts of metal salts.

Various well-known additives such as plant hormones and carbon sources may be added to these subculture media. The type and concentration of the added plant hormone affect the productivity of polysaccharides. For example, 2,4-dichlorophenoxyacetic acid (2,4-D), α-naphthalene acetic acid (NA
A), indole acetic acid (IAA), indole butyric acid (I
Auxins such as BA); cytokines such as kinetin, benzyladenine (BA), and dimethylaminopurine (2IP); gibberellins such as gibberellin A ₃ (GA ₃ ) and the like can be used. In this
2,4-D or NAA alone or NAA and B
It is preferable to use A or kinetin in combination. In those cases, the concentration of 2,4-D or NAA alone is in the range of 5 × 10 ⁻⁴ M to 1 × 10 ⁻⁷ M, particularly 5 × 10 ⁻⁵ M to 5 × 10 5. ^When the concentration of NAA and BA or the combination of NAA and kinetin is used in the range of ⁻⁶ M, the concentration of NAA is 1 × 10 ⁻⁴ M.
To 1 × 10 ⁻⁷ M, particularly 1 × 10 ⁻⁴ M to 5 × 10 ⁻⁶ M, and BA or kinetin concentration of 5 × 10 ⁻⁵ M to 1 × 10 ^−9. M, especially 1 × 10
A concentration in the range of ^-6 M to 1 x 10 ^-7 M is preferred.

As the carbon source, glucose, fructose, mannose, xylose, sucrose, rhamnose, fucose, starch and the like are used. The production amount of polysaccharides is not significantly affected by the type of carbon source to be added, and normally oligosaccharides such as sucrose are used as a carbon source. In general, the amount of carbon source does not significantly affect the production amount of polysaccharides, but it is usually used at a concentration in the range of 1 to 6% by weight.

In the present invention, the liquid medium used for the above subculture contains 10 to 180 mM (particularly 20 to 100 mM).
Metal salt in a concentration within the range of 30 to 200 mM (particularly 40 to 150 mM) in the liquid medium.
It is preferred to use it as mM). Addition of this metal salt further increases the ability of cells obtained from subcultured callus to produce polysaccharides.

Examples of the metal salt include alkali metals such as sodium and potassium, alkaline earth metals such as calcium, magnesium and barium, and chlorides of 1 to 3 valent metals such as metals such as iron, zinc and aluminum, and sulfuric acid. Salts, carbonates, phosphates, nitrates are used. Examples of specific metal salts include sodium chloride, potassium chloride,
Calcium chloride, barium chloride, magnesium chloride, iron chloride, zinc chloride, aluminum chloride, sodium sulfate,
Potassium sulfate, calcium sulfate, magnesium sulfate, barium sulfate, zinc sulfate, ferrous sulfate, ferric sulfate, aluminum sulfate, sodium carbonate, potassium carbonate, calcium carbonate, barium carbonate, magnesium carbonate, sodium phosphate, potassium phosphate , Calcium phosphate, magnesium phosphate, barium phosphate, sodium nitrate, potassium nitrate, calcium nitrate, magnesium nitrate, barium nitrate and the like.

When a metal salt is added during subculture, it is not always necessary to add it to the subculture liquid medium each time, but in order to significantly improve the polysaccharide-producing ability, many times of subculture are performed. It is preferable to allow a metal salt to be present in the subculture liquid medium. Further, varying the metal salt concentration in the subculture medium at each time is also effective for significantly improving the polysaccharide-producing ability.

The subculture of the callus of the present invention is usually 20
(There is no particular limit to the number of times, usually more than three times, more than 10 times) several times on the order of 15 to 30 days of culture (preferably shaking culture <br/> nutrient is) in the range of to 30 ° C. embodiment be Performed by. And, in the production of cells having high polysaccharide production ability of the present invention, the concentration of a liquid medium component such as a sugar component (carbon source component), a nitrogen source component, a metal salt, and a plant hormone in the course of the subculture. Is increased by 20% or more . The cells produced by the change in the component composition of the subculture liquid medium as described above show a high polysaccharide-producing ability in the polysaccharide-producing medium.

There are no particular restrictions on the culture device, culture tank, etc. used in the callus (cell) subculture method of the present invention. As a culture tank, vertical drum, horizontal drum,
Various types such as a rotating drum can be used. However, it is preferable to use a shaken culture tank. A stirring device is usually attached to the culture device and used, but the installation and use of the stirring device are not always necessary as long as the liquid medium is circulated and moved inside the culture tank.

The cells with improved polysaccharide-producing ability obtained by the subculture of the present invention are directly used as polysaccharide-producing cells under known culture conditions for industrial or laboratory production of polysaccharides. Available. As the liquid medium at that time, it is possible to use the same composition as the liquid medium used for the above subculture, or a liquid medium particularly suitable for the production of polysaccharides (particularly, a liquid medium containing a metal salt as well). preferable.

[0019]

【Example】

[Example 1] (1) Induction of callus: Tubulose buds 2 to 7 days before flowering were cut out, sterilized with 70% ethanol solution for 1 minute, and further sterilized with 1% sodium hypochlorite solution for 10 minutes, and then sterilized. It was washed with water. The sterilized explant was cut into an appropriate size and inoculated into a callus induction medium. As a callus induction medium, a Rinsmeier-Skoog medium (LS medium) containing 0.8% agar was used as a basic medium. As a plant hormone, 10 ^-5 M as an auxin
NAA and 10 ⁻⁶ M BA as cytokinin were added. 3% sucrose was added as a carbon source. This medium was adjusted to pH 5.7 with 0.1N-KOH and then autoclaved at 120 ° C. and 1.2 atm for 1 hour.
Sterilized for 5 minutes. The culture was performed at 25 ± 1 ° C. under illumination.
After culturing for 30 to 60 days, it was confirmed that callus was induced from each explant.

(2) Callus subculture 1st stage: 20 subculture unit steps in which the callus induced in the above step is cultured for 30 days under the same conditions using the same medium as the induction medium.
Times (callus is transferred to fresh medium each time; 20 subcultures).

(3) Second stage of subculture of callus: In a 500 ml Erlenmeyer flask, a basic medium (LS medium, 4 w / v% sucrose and 10 ⁻⁵ M 2,4-D was added). 100 mL of the added) was added, and callus was inoculated into this medium at an inoculation rate of 20 w / v%, and the mixture was cultured with shaking at 26 ° C. and 90 rpm for 4 days to examine the amount of polysaccharide produced in the medium. Next, the cells produced by this culture were transplanted to a liquid medium containing increased sugar components in which 8 w / v% sucrose and 10 ⁻⁵ M 2,4-D were added to the L-S medium and shaken for 2 hours. . Then, the cells were separated from the liquid medium and transplanted to the basal medium again. Then, in order to examine the polysaccharide production ability of the cells transplanted to the basic medium after the above-mentioned transplantation of the sugar component-increasing liquid medium, the polysaccharide production amount in the medium after 6 days, 7 days, and 10 days was examined. Further, using only the basic medium as a control, cells were subcultured in the same manner to obtain control cells, and the production amount of polysaccharide was similarly examined during the process.

The results are shown as a graph in FIG. This graph shows the relative amount of the polysaccharide concentration in the medium at each measurement, with the polysaccharide concentration in the medium in the first 4 days of shaking culture as a reference (1).

[Example 2] In the second stage of subculture of callus in (3) of Example 1, 5 w was added to LS medium as a basic medium.
/ v% sucrose, 10 ^-5 M 2,4-D and 50 mM potassium chloride were added, and cells in the middle were transplanted to a liquid medium with an increased sugar component, and the sucrose concentration was 12 w / v.
Subculture was carried out in the same manner except that it was immersed in an aqueous sucrose solution of 2% for 2 hours. In order to examine the polysaccharide production ability of cells transplanted to the basic medium after the transplantation of the liquid medium with increased sugar component, the production amount of polysaccharides in the medium 14 days after the transplantation was examined. At the same time, the cell growth rate was also examined. Further, using only the basic medium as a control, cells were subcultured in the same manner to obtain control cells, and the production amount of polysaccharide was similarly examined during the process. At the same time, the cell growth rate was also examined.

The results are shown as a bar graph in FIG.
This graph is a bar graph with reference to the production amount of polysaccharide by the control using only the basal medium (100%). Figure 2
From the results, it was confirmed that callus (cells) temporarily cultured in a medium having a changed sugar concentration in the course of subculturing according to the present invention showed a high polysaccharide-producing ability without cell proliferation. Obviously it will change.

[Example 3] In Example 2, as the basal medium, L-S medium supplemented with 4 w / v% sucrose and 10 ^-5 M 2,4-D was used. Subculture was carried out in the same manner except that the transplantation into the liquid medium with increased sugar component was carried out by immersing in a sucrose aqueous solution having a sucrose concentration of 8 w / v% for 2 hours. Then, the polysaccharide-producing ability of the cells subjected to the above-mentioned sugar concentration varying treatment was similarly examined. In order to investigate the effect of the repeated treatment with the medium having an increased sugar concentration, after transplanting the cells to the basic medium, the cells were further subjected to the same sugar component fluctuation treatment on the second and fourth days. Then, the polysaccharide-producing ability of the cells subjected to the above-mentioned sugar concentration varying treatment was similarly examined. Further, using only the basic medium as a control, cells were subcultured in the same manner to obtain control cells, and the production amount of polysaccharide was similarly examined during the process. At the same time, the cell growth rate was also examined.

The results are shown as a bar graph in FIG.
This graph is a bar graph with reference to the production amount of polysaccharide by the control using only the basal medium (100%). Figure 3
From the results, it can be seen that callus (cells) repeatedly cultured in a medium having a changed sugar concentration during the subculture changes so as to show a high polysaccharide-producing ability.

[Example 4] Callus obtained in the first stage of subculture of callus of (3) of Example 1 was mixed with a basic medium (LS medium containing 4 w / v% sucrose, 10 ^-5 M 2,4-D and 5
200 mL with 0 mM potassium chloride added)
Inoculate at 10 w / v% and incubate at 26 ° C and 90 rpm for 4
After shaking culture for one day, the production amount of polysaccharide in the medium was examined.

Next, the cells produced by this culture were divided into three parts, and each was immersed in the following component varying medium for 2 hours. (A) Only high sucrose concentration in the medium components (1.
5 times). (B) Nitrogen source (KNO ₃ , NH ₄ NO ₃ ) in medium components
Only high concentration (3 times). (C) A high concentration (2.0 times) of the concentration of all medium components. After the above treatment, the cells were transferred to a basal medium and subcultured for 2 weeks under the same conditions as above. Then, the treatment with the above-mentioned component varying medium was performed again. Then, by performing such a component varying medium treatment a total of 5 times, the variation in the polysaccharide production ability of the cells due to the treatment was examined by the same method as described above. In addition, as a control, only the basal medium was used, and subculture was performed in the same manner to obtain control cells, and in the middle of the process, the polysaccharide production amount was similarly examined.

The results of the above (a), (b) and (c) are shown as bar graphs in FIGS. 4, 5 and 6, respectively.
These graphs show the relative amount of polysaccharide concentration in the medium at each measurement, with the polysaccharide production ability of the cells obtained by subculturing only the basal medium as 100%. From the results of Fig. 4, Fig. 5, and Fig. 6, the callus (cells) that had been repeatedly cultivated in the medium in which the sugar concentration, the nitrogen source concentration, or the concentration of all components changed during the subculture had a high polysaccharide-producing ability. You can see that it becomes as shown.

[0030]

INDUSTRIAL APPLICABILITY According to the present invention, cells treated with a medium in which sugar concentration, nitrogen source concentration, total component concentration, etc. were changed during the subculture, cells subcultured without changing the composition of the conventional medium. It has a higher polysaccharide-producing ability than that of Therefore, the polysaccharide-producing cell produced by using the production method of the present invention can be particularly advantageously used when industrially producing a polysaccharide.

[Brief description of drawings]

FIG. 1 shows an example of transition of polysaccharide productivity in cells subcultured with treatment with a medium in which the sugar concentration in the medium is changed according to the present invention and cells subcultured by a conventional method. It is a graph.

FIG. 2 shows other changes in the polysaccharide productivity of cells subcultured with treatment with a medium having a different sugar concentration in the medium according to the present invention and cells subcultured by a conventional method. It is an example graph.

FIG. 3 shows changes in the polysaccharide productivity of cells subcultured by repeated treatment with a medium in which the sugar concentration in the medium is changed according to the present invention and cells subcultured by a conventional method. It is a graph of the example of.

FIG. 4 shows changes in the polysaccharide productivity of cells subcultured by repeated treatment with a medium in which the sugar concentration in the medium is changed according to the present invention and cells subcultured by a conventional method. It is a graph of the example of.

FIG. 5: Example of variation in polysaccharide productivity by cells subcultured with treatment with a medium having a different nitrogen component concentration in the medium according to the present invention and cells subcultured by a conventional method It is a graph shown.

FIG. 6 shows an example of variation in polysaccharide productivity by cells subcultured with treatment with a medium in which the concentrations of all components in the medium are changed according to the present invention and cells subcultured by a conventional method. It is a graph shown.

─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Yutaka Iga 1-8762-23 Doihonhonmachi, Hasaki-cho, Kashima-gun, Ibaraki Prefecture (56) Reference JP-A-4-53495 (JP, A) JP-A-5-207888 (JP, A) (58) Fields investigated (Int.Cl. ⁷ , DB name) C12N 5/04 BIOSIS / WPI (DIALOG)

Claims (5)

(57) [Claims] 1. A method for the callus of the plant cells that secrete polysaccharides extracellularly producing polysaccharide producing cells subcultured in liquid media, media component composition in the middle of該継subculture
Of a high-polysaccharide-producing cell, characterized in that it comprises an operation of culturing in a liquid medium in which the concentration of the carbon source component, the nitrogen source component, the metal salt, or the phytohormones is increased by 20% or more. Production method. 2. A metal salt of 30 to 200 mM in a liquid medium.
The method for producing a high-polysaccharide-producing cell according to claim 1, which comprises: Wherein a concentration increase is claim plural kinds of component change in the chemical composition is in a liquid medium in the middle of subculture 1 if
A method for producing a high-polysaccharide-producing cell according to item 2 . 4. Callus derived from cells of a Polyanthes plant
4. Any one of claims 1 to 3, which is induced .
The method for producing a high-polysaccharide-producing cell according to the item. 5. A tubero of the genus Polyanthes with callus.
The method for producing a high-polysaccharide-producing cell according to any one of claims 1 to 4, which is derived from a plant .