JP2902405B2 - Process for producing biologically active oligogalacturonide - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION (a) Industrial application field The present invention is an oligogalacturo which is a substance which enhances the defense mechanism against microbial infection of plants and enables cultivation of crops safely and efficiently. Nido (Oligogalacturoni
de) is provided with high purity and high yield.

(B) Conventional technology and its problems Originally, plants are provided with various infection defense functions.
Although it is known to be resistant to many microbial infections, they are by no means perfect, and in today's crop cultivation aiming for high yield and high quality, a large amount of Pesticide spraying is needed. For example, a chemical is applied to seeds at the time of sowing to promote healthy germination, and the chemicals are further sprayed during growing and fruiting to reduce damage from microbial infection.

However, conventional methods have many problems in terms of safety, such as effects of pesticides remaining on crops on the human body and contamination due to the diffusion of pesticides into the environment.

On the other hand, various types of polysaccharides that construct the cell wall of higher plants are known, and recently, in the dispersion of these polysaccharides, factors regulating plant physiological functions such as infection defense response and differentiation induction are included. Have been discovered and reported.

For example, a specific oligogalacturonic acid has a function of promoting the induced production of antibacterial substances (phytoalexin) and protease inhibitors of soybean, imparting resistance to infection, and further has an effect of promoting flowering of tobacco callus. It is known that It is also known that xyloglucan oligosaccharides prepared from sycamore maple tissue culture cells have an effect of suppressing the growth-promoting effect of auxin on pea sprouts.

Such physiological activities of oligosaccharides are considered to greatly contribute to the innovation of crop cultivation in the future, and there is a need for more efficient and higher yielding methods for preparing oligosaccharides having these physiological activities. I have.

(C) Means for Solving the Problems The present inventor has proposed an antibacterial substance-inducing effect (elicitor (elic)
A method for producing oligogalacturonide having itor activity) in high yield was studied. As a result, by allowing pectic acid lyase to act on pectin containing high methoxyl poly α-1,4-D-galacturonide as a main component,
The inventors have found that oligogalacturonide can be produced in high yield, and that the methyl ester of this oligosaccharide is hydrolyzed, thereby facilitating separation and purification by ion-exchange chromatography. .

Furthermore, the present inventors have found that among the isolated oligogalacturonides, those having a polymerization degree of 6, 9, 10, or 11 have particularly strong elicitor activity. Above all, with regard to oligogalacturonide having a polymerization degree of 6, no report has been made on the elicitor activity at all, and its strong activity was confirmed only by highly pure isolation by the method of the present invention. Things.

The high methoxyl α-1,4-D-galacturonide-based pectic polysaccharide used in the present invention includes:
It may be derived from any plant. For example, apple pectin, citrus pectin, love egg pectin and the like are used, and love egg pectin having high purity and containing substantially no rhamnose or branch in the main chain is particularly preferable. Love egg pectin, after heating the seeds of love egg Itabi (Ficus awkeotsang Makino) to inactivate the enzyme which acts on pectin, extraction, separation, obtained by purifying a high methoxyl linear alpha-1 , 4-D-galacturonide.

The pectate lyase to act on pectin is required to be substantially free of pectin lyase or pectin esterase activity.In the present invention, the culture filtrate of Erwinia carotovora , a kind of plant pathogenic bacteria, is used. Purified by ion exchange chromatography using CM-Sephadex C-50 was used.

According to the present invention, pectate lyase acts on the non-methylesterified galacturonic acid portion in the high methoxyl pectin main chain, the glycosidic bond is cleaved by β-elimination action, and oligogalacturo with an appropriate chain length is produced. Nides are produced. The resulting oligogalacturonide is easily obtained as a white precipitate by adding ethanol. The oligogalacturonide obtained in this way is a mixture containing various degrees of polymerization and can be used as it is as a physiologically active substance. It is preferable to use galacturonide by isolation.

According to the present invention, oligogalacturonide having a degree of polymerization of 1 to 12 can be purified to a high purity by adding sodium hydroxide to an aqueous solution of the oligogalacturonide mixture and demethylating the solution, followed by separation by ion exchange chromatography.
It can be obtained in high yield.

(D) Action Oligogalacturonide obtained by the present invention can be applied to plants by methods such as application to seeds, addition to soil, spraying to leaves, and addition to liquid fertilizer during hydroponics. Can be used as a substance promoting a biological defense function against microbial infection of cultivated plants.

(E) Examples Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention should not be limited thereto.

Example 1 Method for Producing Oligogalacturonide (1) Preparation of Love Egg Pectin Love egg pectin used in the present invention is disclosed in
It was prepared according to the method described in 63-209553.

(2) Preparation of pectate lyase Erwinia carotovor isolated from soft rot lesions of vegetables
and under conditions 30 ° C. in buffer of enzyme preparation 5,000 units (pH 9.0 obtained from the culture filtrate of a wild strain of a, the amount of enzyme which can liberate the galacturonic acid of 1μM per minute, 1 ) With 10 mM phosphate column buffer (pH
7.3) After dissolving in 200 ml and removing insolubles by centrifugation, the mixture was added to a CM-Sephadex C-50 column (2.6 × 70 cm) equilibrated with the same buffer, and washed with the same buffer. For elution, sodium chloride was added to the same buffer, and elution was performed with a sodium chloride concentration of 0 to 0.8M.

(3) Enzymatic degradation of pectin 1 g of Aitama pectin is added to 0.1 M ammonia buffer (pH 9.0) 4
After dissolving in 00 ml, 115 units of Erwinia carotovora pectate lyase was added and incubated at 30 ° C. for 1 hour. After completion of the reaction, insolubles were removed by centrifugation, an equal amount of ethanol was added, and the precipitate was dried to obtain 610 mg of a decomposed product.

(4) Separation and purification of oligogalacturonide NaOH solution was added to the pectin solution to make 0.05N NaOH.
After deesterification treatment at 90 ° C. for 90 minutes, twice the amount of ethanol was added, the resulting precipitate was dissolved in deionized water, and pectic acid sodium salt was prepared by freeze-drying in vacuo. This pectic acid sodium salt (0.94 g) was dissolved in 500 ml of 100 mM imidazole buffer (pH 7.0), and QA was equilibrated with the same buffer.
It was added to a column of E-Sephadex A-25 (2.5 × 20 cm), washed with the same buffer, and eluted with a 0.2-0.8 M gradient of the same buffer 2. The galacturonic acid determination method is the m-hydroxydiphenyl method (Blumenkrantz, N. and Asboe-H
ansen, G. (1973) Anal. Biochem., 54 , 484). As a result, as shown in FIG. 1, 12 fractions could be separated, and oligogalacturonides having a degree of polymerization of 12 or less could be isolated. The degree of polymerization of oligogalacturonide is FAB-M
Confirmed using S. For example, in the case of the sixth fraction in FIG. 1, as shown in FIG. 2, 1055,879,7
03, 527, 351 and an ion peak due to fragmentation were observed, which confirmed that the product was oligogalacturonide having a polymerization degree of 6.

[Example 2] Measurement of elicitor activity (phytoalexin-inducing activity) Soybean cotyledons 8-9 days after germination were washed with tap water, the water content of the surface was removed on filter paper, and then subsurface using a scalpel. It was cut at a depth of 1 mm. The 10 cotyledons were placed as a group on a petri dish covered with filter paper, and the oligogalacturonide solution was dropped at 50 μg / 90 μ / cotyledon. 3m water with 0.02% streptomycin to prevent bacterial contamination
l was soaked in filter paper, moistened and incubated at 26 ° C. for 24 hours.

After the incubation, 10 cotyledons were suspended in 10 ml of deionized water to extract phytoalexin, and this extract was further diluted 10-fold. Glyceolins, which are the main components of soybean phytoalexins, have a strong absorption maximum near 286 nm. Therefore, the absorption at 286 nm was measured to determine the elicitor activity. The above experiment was repeated twice, and the average value was shown as the measured value.

For each of the isolated oligogalacturonides having a degree of polymerization of 4 to 12, the elicitor activity measured by this method is shown in Table 1.

From the above results, the degree of polymerization of oligogalacturonide
6,9,10 or 11 were found to have particularly strong elicitor activity.

[Brief description of the drawings]

FIG. 1 shows the separation pattern of oligogalacturonides having a polymerization degree of 1 to 12 eluted using a column of QAE-SEPHADEX A-25. FIG. 2 shows the results of FAB-MS of oligogalacturonide having a degree of polymerization of 6.

────────────────────────────────────────────────── ─── Continuation of the front page (56) References Journal of the Japanese Society of Agricultural Chemistry, Vol. 62, No. 3, published March 15, 1988, p. 365 (58) Fields investigated (Int. Cl. ⁶ , DB name) C12P 19/14 CA (STN)

Claims (1)

(57) [Claims] 1. Isolation of a biologically active oligogalacturonide having a degree of polymerization of 6 from an oligogalacturonide mixture obtained by allowing pectate lyase to act on pectin obtained from the seeds of loved eggs. A method for isolating oligogalacturonide having a polymerization degree of 6 and having biological activity, comprising demethoxylating the cuturonide mixture with an aqueous alkali solution and separating the mixture by ion exchange chromatography.