JP2665533B2 - Agar-degrading enzyme-producing bacterium and method for softening agar medium of plant tissue culture seedling using enzyme produced by the strain - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[Industrial application field] The present invention softens an agar medium for tissue culture of a plant using a specific microorganism producing an agar-decomposing enzyme and an enzyme produced by this microorganism, and can easily replant a seedling. The method to be performed. [Related Art] Agar is widely used for tissue culture of plants, but when plant seedlings are replanted into soil, it is necessary to cleanly remove the agar from its roots. Conventionally, the agar was manually rubbed and loosened from the roots, but this method is a very time-consuming operation. Therefore, at present, it is desired to establish a method for efficiently softening such agar. [Problem to be Solved by the Invention] In replanting a plant seedling, the operation of removing agar from the root is a very laborious operation. Therefore, in order to replant such plant seedlings more efficiently, there is a need for a method for easily producing a substance that softens agar (agar-degrading enzyme) in a large amount. [Means for Solving the Problems] As the agar-decomposing enzyme, a reagent Agarase commercially available from SIGMA and an enzyme produced by microorganisms of the genus Cytophaga, Pseudomonas and Vibrio have been known. It has been found that the agar medium of plant seedlings can be softened under simple conditions by using such an enzyme. Therefore, the present inventors conducted a search for microorganisms that produce agar-decomposing enzymes. As a result, it has been found that the desired agarase can be obtained by culturing a specific microorganism belonging to the genus Xanthomonas. Furthermore, it has been found that the agar medium of plant seedlings can be softened under extremely simple conditions by using such an enzyme. The present invention has been completed based on such findings. The details of the present invention will be described below. (1) Investigation of microorganisms belonging to the genus Xanthomonas and producing agar-decomposing enzymes, (2) Softening the agar medium for tissue culture of plants using the agar-degrading enzymes produced by the microorganisms belonging to the genus Xanthomonas and culturing the tissue A method for easily and efficiently replanting seedlings. The bacteriological properties of agarase-producing bacteria along the genus Xanthomonas isolated from seawater by the present inventors are shown below. Bacteriological properties of Xanthomonas sp. TK-38 (a) Morphology Microscopic observation (cultured in Nutrient Broth No. 2 medium containing 0.1% agar for 48 hours) Cell shape and size 0.5-0.8 μm × 1 2 μm bacillus Polymorphism of cells No cell polymorphism, motility Polar flagella, motility, (observed after staining according to Leifson's flagellar staining method) Spore presence None Gram staining method negative (b) Growth state in each medium Plate of Nutrient Broth No. 2 (Difco) containing 0.1% agar and 2% faceran (Difco) at 1-2 ° C. Forming, color is pale yellow, glossy, thick colony. Plate culture of Nutrient Broth No. 2 containing 1.5% agar at 30 ° C. for 48 hours causes the cells to dent the agar, and a round dent is formed on the medium according to the colony. The condition is the same as that of marine agar (Difco) plating. In a liquid culture of marine broth (Difco) containing 0.1% agar, at 30 ° C., 48 hours, 150 rpm (rotary shaker), the medium becomes turbid yellow and the agar-free stagnation disappears. (C) Physiological properties a. Nitrate reduction b. MR test negative C. VR test negative d. Indole formation not generated e. Hydrogen sulfide formation not generated f. Starch hydrolysis hydrolyze g. Extrins Hydrolyzes hydrolyzes h. Hydrolyzes gelatin does not hydrolyze i. Urease negative j. Oxidase positive k. Catalase positive l. Produces pigments xanthomonadine (Bergey's Manual of Systematic Bacteriology volme
1) m. Acid production in milk-purple medium negative (milk protein degradation) n. Enzyme aerobic oO-F test Oxidation p. Growth range Temperature 10-40 ° C pH 5-9 Salt concentration 0 3% q. Acid production from saccharides (Andrade Peptone aqueous medium, p
H7.4) Culture for 3 weeks Glucose + mannose + (weak) fructose-galactose + trehalose + sucrose + xylose + lactose + inositol No assimilation Maltose + arabinose + sorbitol No assimilation No assimilation No assimilation Glycerin assimilation This strain is classified into the genus Xanthomonas based on the above mycological properties. The related bacteria include Xanthomonas albilineans, but differ in the results such as the concentration of viable salt and the generation of acid due to the decomposition of sugar. Thus, this strain is a new strain belonging to the genus Xanthomonas (Bergey's Manual of
Syatematic Bacteriology volume 1). The target agar-degrading enzyme can be produced by inoculating the novel agar-degrading enzyme-producing bacterium of the present invention into a nutrient medium and culturing it. Here, a medium containing salt and agar and containing nutrients that can be used by the microorganism was used.
For example, marine broth containing about 0.1% of agar is exemplified. Shaking culture is suitable as the culture method, and the culture temperature is 20 to 40 ° C, preferably 25 to 30 ° C. The culturing days are suitably 1 to 5 days, preferably 1 to 2 days. The culture conditions should be selected so that the production of the target enzyme is maximized in consideration of the properties of the microorganism. The bacterial cells were removed from the culture solution obtained as described above by centrifugation, and a crude enzyme was obtained from the supernatant by a conventional method. Crude enzyme pH6
Lyophilized in the state of ~ 7 and stored at low temperature. When the seedling medium is softened, the crude enzyme is dissolved in distilled water, and an appropriate amount is directly injected into the agar upper layer medium in a test tube.
Left at ℃. As a result, the medium softens into a gel and the seedlings can be easily pulled out. Seedlings were washed lightly and then replanted in soil. After transplanting, the seedlings survived normally, and no physical or physiological disorders due to enzyme action were observed. The crude enzyme was obtained from the supernatant by the following method. That is, the supernatant was salted out with ammonium sulfate, and the precipitated precipitate was collected, desalted by dialysis (at pH 6), and then lyophilized to obtain a crude enzyme. The activity of the obtained crude enzyme was about 8 U / mg. The optimal temperature for enzyme activity is 45
The optimum pH was 6 ° C and the pH was adjusted with a phosphate buffer.
Here, 1 U is defined as an amount of an enzyme that produces a reducing sugar equivalent to 1 μg of galactose per minute under a predetermined condition. Example 1 Example 1 Xanthomonas sp. TK-38 was inoculated into one MA liquid medium (150 ml / Erlenmeyer flask) and cultured at 30 ° C. for 1 day with shaking (150 r).
After that, 9 ml of NC liquid medium (600 ml / Erlenmeyer flask) per 1 ml was inoculated, and cultured for 1 day under the same conditions as the MA liquid medium. After completion of the culture, the cells and insoluble matter are centrifuged (80
(00 rpm, 15 minutes), and a crude enzyme was obtained from the supernatant by the following method. That is, the supernatant was salted out with ammonium sulfate (saturation 80%), and the deposited precipitate was centrifuged (5000 rpm,
15 min) and desalted by dialysis (at pH 6). Then, it was freeze-dried to obtain a crude enzyme. The activity and recovery of the enzyme are as shown in the table below. Enzyme activity was measured by the Somogy Nelson method. Enzyme solution in 0.9 ml of 0.1 M phosphate buffer (pH6) containing 1% agar
0.1 ml was added and reacted at 45 ° C. for 10 minutes. After the reaction, the reaction was stopped by adding 1 ml of a somogy copper solution, and the mixture was heated at 100 ° C. for 15 minutes. After sufficiently lowering the temperature of the reaction solution, 2 ml of Nelson's solution
Was added, the absorbance was measured at a wavelength of 500 nm, and the enzyme activity was calculated from the value. Example 2 The crude enzyme obtained as in Example 1 was dissolved in phosphate buffer (pH 6) to a concentration of 800 U / ml, and the reagent Agarase was used.
(Sigma) was adjusted to a concentration of 800 U / ml by the same method, and an agar dissolution test was performed on the cultured seedlings (asparagus) in the following manner. The seedlings used were grown to about 5 cm.
0 ml of high medium (test tube). 5 ml of the enzyme solution was injected into each cultured seedling and left at 25 ° C. for 2 days. As a result, the crude enzyme obtained in Example 1 and the reagent Agarase (Sigma) were used.
Agar on the seedlings could be easily removed by lightly applying running water. The agar did not dissolve at all in 5 ml of the phosphate buffer used as a control. Example 3 The crude enzyme obtained in the same manner as in Example 1 was dissolved in buffer phosphate (pH 6) to a concentration of 760 U / ml, and 5 ml was directly injected into an agar medium in which plant seedlings were planted. The medium of the seedling used here is a 10-ml high-layer medium in a test tube, and the seedlings are asparagus and gerbera grown to about 5 to 8 cm. After the injection, the mixture was shaken at 25 ° C. and 60 rpm for 2 days. Two days later,
The seedlings were extracted from the softened agar, washed lightly with water, and transferred to the soil. Thereafter, the survival and growth of the seedlings were observed, and normal growth was observed in each case. Example 4 The crude enzyme obtained in the same manner as in Example 1 was dissolved in distilled water to prepare enzyme solutions having a concentration of 0 U / ml, 400 U / ml, and 800 U / ml. Injected into agar medium. After the injection, the mixture was left standing at 25 ° C. for 2 days. Two days later, the seedlings were extracted in the same manner as in Example 2, washed lightly with water, and it was observed whether or not the agar was sufficiently removed. As a result, the agar was sufficiently softened at 800 U / ml and could be easily removed. On the other hand, at 400 U / ml, the agar was slightly softened, but it was difficult to extract the seedlings easily. At 0 U / ml, no softening of the agar was observed. Xanthomonas sp. TK-38 has been accepted by the Japan Fine Industrial Research Institute on December 18, 1987 as (FERM P-9763).

[Attachment of trust certificate]

[Effects of the Invention] As described above, the agarase-degrading enzyme is produced in a large amount by the present invention, and the medium of the seedling is softened very easily. Seedlings such as asparagus have a very large amount of root hair, and it is difficult to rub the agar by hand to damage the root hair and to remove the agar neatly. Insufficient removal of the medium agar may cause contamination of pathogenic bacteria. By using the present invention, it is possible to transfer a large amount of seedlings much more easily and efficiently than before. It has become.

 ──────────────────────────────────────────────────続 き Continuing on the front page (72) Inventor Tsuyoshi Hayami 3-2-9 Tsukishima, Chuo-ku, Tokyo Ocean fishery Inside the Ocean Research Institute Co., Ltd. (72) Inventor Yasuo Iso 3-2-9, Tsukishima, Chuo-ku, Tokyo Ocean fishing Inside the Ocean Research Institute Co., Ltd. (72) Inventor Taku Yoshiwara 2-2-9 Tsukishima, Chuo-ku, Tokyo Ocean Fisheries Inside the Ocean Research Institute Co., Ltd. (56) References Shigeru Minamitake, Bull. Coll. Ag r. & Vet. Med. , Nihon Univ. , No. 43, p. 84-91 (1986)

Claims (3)

(57) [Claims] 1. A method for softening an agar medium of a plant tissue culture seedling so that the agarase is allowed to act on an agar medium for tissue culture of a plant to easily and efficiently replant the tissue culture seedling. 2. Xanthomonas sp. TK-38, a novel microorganism as an agarase-producing enzyme-producing bacterium (Accession No. FERMP-9763) Items to be identified (a) Form: Cell shape and size 0.5 to 0.8 μm × 1 to 2 μm Bacillus subtilis Presence of cell polymorphism No cell polymorphism, motility Presence of flagella, motility, presence of spores None Gram staining negative (b) Growth in each medium State Plate medium of Nutrient Broth No.2 (Difco) containing 0.1% agar and 2% faceran, forms 1-2 mm round colonies at 30 ° C for 48 hours, color is pale yellow, glossy, thick Colony. Plate culture of Nutrient Broth No. 2 containing 1.5% agar at 30 ° C. for 48 hours causes the cells to dent the agar, and a round dent is formed on the medium according to the colony. The condition is the same as that of marine agar (Difco) plating. In a liquid culture of marine broth (Difco) containing 0.1% agar, at 30 ° C., 48 hours, 150 rpm (rotary shaker), the medium becomes turbid yellow and the agar-free stagnation disappears. 3. Xanthomonas sp. TK-38 (FERM P-976)
A method in which the agar-decomposing enzyme produced in 3) is allowed to act on an agar medium for plant tissue culture to soften the agar medium of plant tissue culture seedlings in order to easily and efficiently replant the tissue culture seedlings.