JP3498979B2 - How to increase sugar elicitor activity - Google Patents

How to increase sugar elicitor activity

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Publication number
JP3498979B2
JP3498979B2 JP22026093A JP22026093A JP3498979B2 JP 3498979 B2 JP3498979 B2 JP 3498979B2 JP 22026093 A JP22026093 A JP 22026093A JP 22026093 A JP22026093 A JP 22026093A JP 3498979 B2 JP3498979 B2 JP 3498979B2
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activity
oligosaccharide
elicitor activity
elicitor
reducing end
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JPH0767681A (en
Inventor
昭雄 小林
一儀 河津
章博 田井
浩 神崎
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株式会社紀文食品
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Description

Description: BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for increasing the elicitor activity of a sugar having elicitor activity. The present invention also relates to a method for preparing an oligosaccharide having high elicitor activity by purifying a laminaran hydrolyzate. [0002] When pathogenic bacteria enter a plant, phytoalexin, an antibacterial substance, is synthesized or activated in plant cells. A substance that activates a plant secondary metabolic system to induce phytoalexin is called elicitor, and it is known that many compounds such as cell wall components of pathogenic bacteria and their degradation products have elicitor activity. In particular, oligosaccharides generated by the degradation of polysaccharides in the cell wall of the pathogen by enzymes in the plant cells are degraded in plants.
It is known to activate novo synthesis to induce phytoalexins. [0003] The mechanism of the induction of phytoalexins by elicitors has so far been largely unclear. This is because the secondary metabolic system of the plant is complicated, many enzymes are involved, and the activation of the enzymes occurs in a short time, so that analysis is difficult. [0004] In recent years, in the production of useful substances by plant cultured cells, it has been reported that the amount of production increases when an elicitor or a cell wall component containing the elicitor is added to a culture system. Its usefulness is attracting attention. Furthermore, elicitors are very useful as a means for molecularly analyzing the defense mechanism of infection in plants. However, the research so far has focused only on the search for a substance having elicitor activity, and few attempts have been made to actively increase the elicitor activity of the substance. In addition, many steps and costs are required to isolate a substance having elicitor activity from its cell components by culturing a microorganism in a large amount, so that industrial production of elicitor has been difficult. [0006] Accordingly, an object of the present invention is to provide a simple method for increasing the elicitor activity of a sugar having an elicitor activity. Another object of the present invention is to provide a method for preparing an oligosaccharide having elicitor activity from a degradation product thereof, using a natural polysaccharide which is available at a low cost as a raw material. The present invention provides a method for enhancing the elicitor activity of an oligosaccharide having an elicitor activity by modifying the reducing end of the oligosaccharide. [0008] Modification of the reducing end of the oligosaccharide is carried out by reacting the reducing end with a compound having a nucleophilic substituent. The compound having a nucleophilic substituent refers to a compound that undergoes a nucleophilic reaction on the carbonyl carbon at the reducing end. For example, R-NH 2, R- OH, R-SH, R-
COOM (M is a metal), alkylated metal RM, and the like can be given. R can be selected widely as long as it does not adversely affect the elicitor activity.
Among them, a hydrophobic group capable of increasing the affinity with the receptor is preferable. If the affinity with the receptor is high, the elicitor activity can be further increased. The reaction between the oligosaccharide and the compound having a nucleophilic substituent can be performed under conditions well known to those skilled in the art. A specific example of the reaction between an oligosaccharide and a compound having a nucleophilic substituent is pyridyl amination. Pyridyl amination is one of the methods for labeling a sugar, and refers to introducing an aminopyridyl group at the reducing end of the sugar. Pyridyl amination can be performed using methods well known to those skilled in the art. For example, the reaction can be carried out by reacting an oligosaccharide with a coupling reagent and then reacting it with NaBH 3 CN. By modifying the reducing end as described above, the elicitor activity of the oligosaccharide can be significantly increased (see Examples and FIG. 2). Therefore, by using the method of the present invention, it is possible to prepare a large amount of oligosaccharide having high elicitor activity by a simple method. The present invention also provides a simple method for separating oligosaccharides from natural polysaccharides to enhance elicitor activity. Focusing on the fact that the cell wall of the microorganism is composed of polysaccharides, screening of various natural polysaccharides exhibiting elicitor activity revealed that laminaran derived from the brown alga Eisenia bicyclis showed activity. Was done. First, an oligosaccharide, which is the minimum unit having elicitor activity, is separated and purified from the laminaran. At that time, laminaran is decomposed by an enzyme. As the enzyme, any enzyme that cleaves the β-1,3 bond of the sugar chain may be used, but tunicase is preferably used. By purifying the obtained decomposition product by a usual purification method using a column or the like, a fraction of oligosaccharide having high elicitor activity can be obtained. As the column, a known column such as an activated carbon column or a gel filtration column, or a combination thereof can be used. Next, the reducing end of the oligosaccharide having the elicitor activity is modified. The method for modifying the reducing end is as described above, whereby an oligosaccharide having high elicitor activity can be obtained. The modified oligosaccharide can be further separated and purified by a method such as HPLC. Hereinafter, the present invention will be described in more detail with reference to examples. EXAMPLES (Method of Measuring Elicitor Activity) Elicitor activity was measured using alfalfa as a model plant. Alfalfa cotyledons grown aseptically were cut, placed in a solution containing the sample, and cultured in the dark for 48 hours. afterwards,
The test solution was analyzed using HPLC, and the produced phytoalexin was quantified. HPLC of phytoalexin
One example of the result is shown in FIG. Phytoalexins used as indicators of elicitor activity are 4 ', 7-dihydroxyflavonone and 4', 7-dihydroxyflavone, which are biosynthesis intermediates of medical pins, statibin, and medical pins. The peak areas of these four compounds in HPLC were determined, and those having significantly increased peak areas were defined as active. In quantitative analyses, 2
When the induction was more than doubled, it was evaluated as active (+), and when it was less than that, it was evaluated as no activity (-). (Example 1) Preparation of oligosaccharide having elicitor activity Screening of natural polysaccharides exhibiting elicitor activity revealed activity in laminaran derived from brown alga Eisenia bicyclis. The laminaran was then enzymatically degraded in order to obtain the minimum unit showing activity.
5.5 g of lamina run (manufactured by Tokyo Kasei, LOT.FCY0
1) 500 ml of 0.1 M phosphate buffer (pH 7.0)
And the solution was added to Tunicase (Daiwa Kasei, 50 m
g) was added and reacted at 37 ° C. for 4 hours. This reaction solution was concentrated by an evaporator. Next, the obtained degradation product was fractionated by an activated carbon column and a biogel P-2 column, and the elicitor activity of each fraction was measured to obtain an active fraction. (Example 2) Modification of reducing end of active fraction The reducing end of the obtained active fraction was modified by pyridyl amination. Pyridyl amination was performed by the following method. Coupling reagent (1.08 g of 2-aminopyridine, 0.1 ml of HCl) was added to the active oligosaccharide fraction (120 mg).
7 ml and 6.3 ml of H 2 O) were added, and the mixture was reacted at 80 ° C. for 1 hour. Thereafter, NaBH 3 CN (350 mg) was added, and the mixture was further reacted at 80 ° C. for 3 hours. Then, Ag1
-X8 column (manufactured by Bio-Rad), Toyopearl H
Purification with a W40S column (manufactured by Toso) gave a pyridylaminated oligosaccharide fraction (140 mg). FIG. 2 shows the change in elicitor activity due to pyridyl amination. The elicitor activity was measured by the method described above. 12.5μ by pyridyl amination
g / ml concentration does not change the activity,
At concentrations above g / ml, the activity increased about 6-fold. Also,
Inactive fractions showed no activity by pyridyl amination. That is, it was shown that the elicitor activity of an oligosaccharide having elicitor activity was significantly increased by pyridyl amination. Example 3 Separation of Pyridyl Aminated Oligosaccharide Next, the obtained pyridyl aminated oligosaccharide was subjected to HPLC
And the respective activities were measured. The column is a Toyopearl HW-40S column (manufactured by Tosoh), and three types of pyridylaminated oligosaccharides A and B are used.
And C were obtained (FIG. 3). The HPLC developing solution was a methanol-acetic acid solution, and the methanol concentration was 5% to 1%.
Increased to 2.5%. The flow rate was set to 0.8 ml / min.
Developed at ° C. When the elicitor activity of these oligosaccharides was measured, oligosaccharide A was up to a concentration of 25 μg / ml, oligosaccharide B was up to a concentration of 3.2 μg / ml, and oligosaccharide C was up to a concentration of 0.8 μg / ml. Activity was observed (Table 1). [Table 1] That is, it was shown that an oligosaccharide having high elicitor activity can be prepared by modifying the reducing end of the hydrolysis product of laminaran by the enzyme.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 shows an example of the results of HPLC analysis of phytoalexin used for elicitor activity measurement. FIG. 2 shows changes in elicitor activity due to pyridyl amination. FIG. 3 shows HPLC of pyridylaminated oligosaccharide.
The result is shown.

────────────────────────────────────────────────── ─── Continuation of the front page (56) References Proceedings of the 1992 Annual Meeting of the Japanese Society of Agricultural Chemistry (1992), March 5, 1992, p. 304, 2lp5 (58) Fields investigated (Int. Cl. 7 , DB name) C12P 19/00-19/64 EUROPAT (QUESTEL) JICST file (JOIS) BIOSIS / WPI (DIALOG)

Claims (1)

  1. (57) [Claims] (1) It has an elicitor activity derived from laminaran
    A method for enhancing the elicitor activity of an oligosaccharide , wherein the reducing end of the sugar is pyridylaminated .
JP22026093A 1993-09-03 1993-09-03 How to increase sugar elicitor activity Expired - Fee Related JP3498979B2 (en)

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Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
日本農芸化学会誌1992年度大会講演要旨集(1992),1992年 3月 5日,第304頁、2lp5

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