JP3219317B2 - Method for producing brassinosteroid-like substance - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for producing a brassinosteroid-like substance by culturing photosynthetic bacteria.

[0002]

2. Description of the Related Art Brassinosteroids are plant growth regulators having a steroid skeleton, and include various plant bodies, such as rapeseed pollen, chestnut worms, tea leaves, hornworms, immature seeds of Fuji Bean , Green kidney beans, rice stover, corn pollen and the like. Brassinosteroid is a general term for brassinolide and its homologues, and 22 homologues have been identified so far. The term "brassinosteroid-like substance" as used herein is meant to include not only brassinosteroids and their homologues, but also analogs thereof.

[0003] Brassinosteroids are widely produced by higher plants and are contained in various plants as described above. The content in the plants is 1 µg to 100 µg / k.
g Live weight and extremely small. This brassinosteroid exhibits physiological activity on plants at a concentration of 0.0001 ppm in the rice lamina joint test, and also has an elongation promoting effect on pea epicotyls, cucumber hypocotyls and the like.

Heretofore, various homologs of brassinosteroids have been chemically synthesized, and their physiological effects on agricultural crops have been extensively investigated. The following effects have been confirmed to date. In other words, rice, corn, cucumber, increased rice, rice,
In addition to enhancing cold resistance against cucumber and eggplant, enhancing disease resistance against Chinese cabbage, it has been found to have useful effects such as enhancement of drug resistance and salt resistance [“Chemistry and Biology”, 23, 717 (1985) )].

[0005] Although brassinosteroids are being demonstrated to be useful as agricultural chemicals as described above, their production methods are currently limited to chemical synthesis.
The chemical synthesis of brassinosteroids is composed of a complex combination of chemical reactions, and requires advanced techniques such as separation of by-product isomers. Although sample preparation for testing is possible, chemical synthesis is said to have limitations as a method for producing agricultural chemicals. Therefore, preparation by biological production has been attempted, and, for example, search for microorganisms that produce brassinosteroids has been performed, but has not been successful yet.

The present inventors have previously infected plant cells with bacteria of the genus Agrobacterium, introduced a part of the plasmid gene carried by the bacteria into the plant cells, and transformed them into tumor cells. Plant cells (crown gall cells)
Found that it produced brassinolide. After extracting and purifying the cultured cells, the presence of brassinosteroid and castosterone was confirmed using a mass spectrometer. Based on this finding, a method for producing brassinosteroids by culturing crown gall cells was proposed (Japanese Unexamined Patent Publication No.
No. 269500). However, the production of brassinosteroids from crown gall cells is as low as 1 to 100 μg / kg fresh weight, and it is necessary to further improve the productivity in order to produce an economical method for producing brassinosteroids.

The present inventors have studied various conditions for culturing crown gall cells in order to solve the above-mentioned problems. I found some. Further, it has been found that when culturing crown gall cells, irradiation of red light or yellow light improves the production of brassinosteroid. Then, based on these findings, the culture medium for culturing the crown gall cells,
Brassinosteroid by adding at least one culture aid selected from auxins, sterols, squalene and casamino acids, and irradiating a medium for culturing the crown gall cells with red light or yellow light A method for producing brassinosteroids by culturing productive crown gall cells has been proposed (JP-A-3-191.
No. 793).

[0008] Despite the various production methods proposed above, brassinosteroids are still very expensive drugs and their supply is limited. Therefore, a method for efficiently producing brassinosteroid in high yield has been desired.

[0009]

SUMMARY OF THE INVENTION An object of the present invention is to provide a method for producing brassinosteroids efficiently and in high yield, which can be substituted for the conventional method.

[0010]

Means for Solving the Problems As a result of searching for various organisms, the present inventors have found that Rhodo-pseudodes, a kind of photosynthetic bacterium.
monas speroides) IFO-12203
They found that the strain produced brassinosteroid-like substances efficiently and in high yield, and completed the present invention.

[0011] Since the present bacterium has an overwhelmingly high growth rate due to its basic property of being a bacterium as compared with a plant cultured bacterium, the amount of brassinosteroid activity produced per unit time is increased. Dramatically improved.

[0012] The basic structure of the present invention is to grow the cells by cultivating Rhodopseudomonas spheroides IFO-12203 strain, and then recover the brassinosteroid-like substance from the obtained cells and / or culture solution. Is to do.

[0013] In the production method according to the present invention, the L. pseudomonas spheroides IFO-12203 strain is a L. pseudomonas sp.
Culture can be performed by a conventional method using a medium usually used for culturing the 2203 strain. Furthermore, the brassinosteroid-like substance can be recovered from the cells and / or the culture solution grown by the culture according to a conventional method.

[0014]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be described below in detail with reference to embodiments.

Embodiment 1 Road Pseudomonas speroides IFO-122
The 03 strains were cultured in the following culture solution. Polypeptone 3.0 g Yeast extract 0.6 g MgSO ₄ .7H ₂ O 0.3 g Lever powder 1.0 g Potato starch 1.0 g pH 7.0 The mixture was made up to 300 ml with water.

One inoculation of a loopful of a seed strain of Rhodopseudomonas spheroides IFO-12203 was inoculated into a culture solution having the above composition, and cultured at 32 ° C. for 24 hours under light irradiation conditions. The obtained culture solution was centrifuged, and the cells 0.5
g was obtained.

1 liter of methanol was added to 0.5 g of the obtained cells, and the cells were disrupted using a homogenizer.
After dispersing, extraction was performed by allowing to stand at 5 ° C. for 24 hours. This methanol extract was centrifuged to remove cell debris, and then concentrated under reduced pressure. Distilled water was added to the obtained concentrate to obtain 300 ml of a water-soluble concentrate, which was extracted three times with an equal amount of chloroform. The chloroform extract was collected, chloroform was distilled off under reduced pressure, and the resulting residue was dissolved in 50 ml of hexane. The hexane solution was extracted three times with 50 ml of 85% methanol, and the 85% methanol extracts were collected and concentrated under reduced pressure. This concentrate was dissolved in 50 ml of ethyl acetate and 20 ml of 0.2 MK was added.
_The extract was washed three times with ₂ HPO ₄ , dried over anhydrous sodium sulfate, and concentrated under reduced pressure. As a result, 35.3 mg of a brassinosteroid (BS) fraction was recovered.
This BS fraction showed an activity equivalent to 0.002 ppm of brassinolide in the rice lamina joint test as shown in FIG.

Comparative Example 1 According to the method described in JP-A-1-269500,
1 gallium periwinkle crown gall cell line
After culturing for 2 days, 7 g of the obtained cells were fractionated in the same manner as in Example 1 and the activity was confirmed.
A pm brassinolide equivalent was confirmed. In this comparative example, 1059 g of cells were actually obtained. Accordingly, the production efficiency of the brassinosteroid in this comparative example can be expressed as 0.076 ppm equivalent amount / 1059 g cells / 12 days.

On the other hand, the production efficiency of the brassinosteroid-like substance in the above embodiment can be expressed as 0.002 ppm equivalent / 0.5 g cell / day.
For comparison, the production amount per 10 g of fresh cell weight for 10 days of culture is shown in Table 1 below. As a result, 0.00059p
The production efficiency corresponding to pm has been improved to the equivalent amount of 0.4 ppm, which means that a production efficiency of about 678 times has been achieved.

[0020]

[Table 1] Fracinosteroid per unit time and unit weight {Active substance production amount}生産 Producing strain fracinosterite ゛ Active substance production / 10 days culture 10 g live cell weight ━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━ ━ V208 0.00059ppm equivalent R.speroides 0.4ppm equivalent R.speroides / V208 677.96 ━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━ ━━

Example 2 A method similar to that of Example 1 was used to load a road pseudomonas spero.
Culturing Ides IFO-12203 strain and removing the cells
A fraction of the brassinosteroid-like substance was obtained from the culture broth.
A medium having the same composition as in Example 1 under the same conditions
Road Pseudomonas Speroides IFO-122
Culturing the 03 strains and separating the strains at 7000 rpm,
Centrifugation was performed for 20 minutes. After that, add more cells
Sterile filtration with a pore size of 0.2 microns for complete removal
Filtrate all 300 ml of culture using an over-filter
did. The obtained culture solution was diluted with 300 ml of chloroform.
Extracted twice and concentrated under reduced pressure. Collect chloroform extract
The residue obtained by distilling off chloroform under reduced pressure
Dissolved in 0 ml hexane. This hexane solution is added to 50
3 times with 85 ml of methanol and 85% methanol
The extract was collected and concentrated under reduced pressure. 50% of this concentrate
dissolved in 20 ml of ethyl acetate and 20 ml of 0.2 MK _TwoH
PO_Four3 times, and then dehydrated with anhydrous sodium sulfate
After that, the mixture was concentrated under reduced pressure. As a result,
8.8 mg was collected as the id (BS) fraction. this
The BS fraction was used for the rice lamina joint test described below.
And as shown in Fig. 1, a brush of 0.001 ppm or more
It showed activity equivalent to nolide. Therefore, it is used in the present invention.
Efficiency from only the culture solution excluding the bacterial cells used
That brassinosteroid-like substances can be produced
confirmed.

Rice Lamina Joint Test (Rice Leaf Bending Test) The rice lamina joint test is performed by the method of Maeda (E.M.
aeda: Physiol. See Plant, vol. 1
8, p. 813 (1965)) as follows. (1) Rice (Koshihikari) seeds at 28 to 30 ° C for 2 days
Soak in water at temperatures up to and germinate. (2) The germinated seeds are sown in a mesh vat, immersed in water, and cultivated hydroponically at a temperature of 28 to 30 ° C. for 6 to 7 days under a dark condition. (3) From the yellowed rice seedlings obtained in this way, select a seedling with a straight leaf blade that does not yet have the third leaf, and remove the leaf blade / leaf sheath part about 1 cm above and below the lamina joint of the second leaf. cut out. (4) Floating the rice slices in distilled water, 28 to 30 ° C
When left in a dark condition at a temperature of up to 24 hours, a slight bending appears between the leaf blade and the leaf sheath after 24 hours. About 30 from this
A section bent to 40 degrees is selected for the test. (5) Put the test substance in a petri dish with a diameter of 9 cm and put it in 20 m
Dissolve in 1 l of 2.5 mM potassium maleate buffer, float 10 of the above sections, and leave for 48 hours at a temperature of 28 to 30 ° C and in total darkness. (6) Take out the rice section and measure the resulting leaf blade inclination angle with a protractor.

[0023]

According to the production method of the present invention, a brassinosteroid-like substance having a useful physiological activity can be efficiently produced in high yield and in large quantities.

[Brief description of the drawings]

BRIEF DESCRIPTION OF DRAWINGS FIG. 1 is a graph showing the amount of brassinosteroid produced by the cultured cells of the strain used in the present invention, the culture solution, and the crown gall cell V208 strain cell as a control.

Continuation of the front page (56) References JP-A-3-191793 (JP, A) JP-A-1-269500 (JP, A) (58) Fields investigated (Int. Cl. ⁷ , DB name) C12P 33 / 00 BIOSIS (DIALOG) CA (STN)

Claims (4)

(57) [Claims] 1. A brassinosteroid-like substance obtained by culturing a photosynthetic bacterium of the genus Rhodopseudomonas and isolating or extracting the brassinosteroid-like substance from the culture. Production method. 2. The method of claim 1 wherein the brassinosteroid-like substance is rhodosh
The method for producing a brassinosteroid-like substance according to claim 1, wherein the brassinosteroid-like substance is obtained by isolating or extracting from a culture cell and / or a culture medium of a photosynthetic bacterium of the genus Domonas . 3. The method for producing a brassinosteroid-like substance according to claim 1, wherein the photosynthetic bacterium is Rhodopseudomonas spheroides IFO-12203 strain. 4. A claim that medium brassinosteroid-like substance is isolated or extraction is characterized by a culture
4. The method for producing a brassinosteroid-like substance according to 2 or 3 .