KR102638957B1 - Starch content enhancer, agricultural composition and plant fertilizer containing the same - Google Patents

Abstract

The present invention relates to a starch content enhancer and pesticide and fertilizer compositions containing the same. The starch content enhancer according to the present invention can be applied to plants without genetic modification and improve the starch content in plants by excellently inhibiting the activity of SEX4, which is involved in starch decomposition.

Description

Starch content enhancer, agricultural composition and plant fertilizer containing the same}

The present invention relates to a starch content enhancer and pesticide and fertilizer compositions containing the same, and more specifically, to a starch content enhancer containing an inhibitor of an enzyme involved in starch decomposition in plants without genetic modification.

Starch, the main stored carbohydrate in plants, is one of the most widely produced food resources in the world, and plants that are important sources of starch include corn, wheat, potatoes, cassava, and sweet potatoes. Starch can be used on its own in the food field, or starch alone can be extracted to give various functions to foods as a substitute for gelatin or gum. Because starch is easy to modify physically and chemically, and modified starch has new functions, starch is extracted and purified from plants through various processes and used as an industrial raw material in industries such as construction, pharmaceuticals, and textiles in addition to the food field. . For example, starch is being applied not only to eco-friendly plastics, but also to eco-friendly fibers and eco-friendly detergents, following the trend to prevent environmental pollution caused by plastics. Recently, starch has been used as biofuel as an alternative to fossil fuels that cause environmental pollution. It is attracting attention. Accordingly, the demand for increasing the starch content in plants is increasing, and the need for technology development is emerging.

Starch degradation in plants requires reversible glucan phosphorylation by dikinases and phosphatases on the surface of starch granules in plant chloroplasts. Among phosphatases, SEX4 (Starch Excess 4) dephosphorylates the C3 and C6 positions of starch glycosyl residues to allow starch decomposition, so dephosphorylation of starch by SEX4 is essential for starch decomposition. Accordingly, plants with loss of SEX4 function are able to contain more starch due to suppressed starch decomposition and starch accumulation. To date, technology has been known to modify plant genes to reduce the activity of SEX4, but the side effects of genetic modification have not yet been fully examined, leading to concerns about safety and increased consumer resistance. Therefore, there is a need for technology to increase starch content in plants by reducing SEX4 activity without genetic modification.

Patent Document 1. U.S. Patent No. 9410133 Patent Document 2. U.S. Patent Publication No. 2014-0251317

In order to solve the conventional problems, the present inventors completed the present invention by selecting compounds having inhibitory activity of SEX4.

Therefore, the object of the present invention is to provide a starch content enhancer containing a compound that has the ability to inhibit SEX4 activity.

Additionally, the present invention aims to provide a pesticide composition containing a starch content enhancer.

Additionally, the present invention aims to provide a fertilizer composition containing a starch content enhancer.

Another object of the present invention is to provide a method for cultivating plants with improved starch content, which includes treating the plants with a starch content enhancer.

Another object of the present invention is to provide plants grown by a method for cultivating plants with enhanced starch content, which includes treating the plants with a starch content enhancer.

In order to achieve the above-described object, the present invention provides a starch content enhancer comprising a compound represented by the following formula (1) or formula (2).

[Formula 1]

[Formula 2]

The plant may be any one or more selected from the group consisting of oats, millet, buckwheat, barley, sorghum, rice, corn, and rye.

The plant may be any one or more selected from the group consisting of potatoes, sweet potatoes, konjac, and cassava.

The compound represented by Formula 1 or Formula 2 may have inhibitory activity on SEX4 in plants.

The compound represented by Formula 1 or Formula 2 can inhibit the activity of SEX4 against amylopectin with an IC ₅₀ of 5 μM to 15 μM.

Additionally, the present invention provides a pesticide composition containing the starch content enhancer.

Additionally, the present invention provides a fertilizer composition containing the starch content enhancer.

Additionally, the present invention provides a method for cultivating plants with improved starch content, comprising treating the plants with the starch content enhancer.

The treatment may be either soil fertilization or foliar fertilization.

Additionally, the present invention provides plants grown by the above cultivation method.

The starch content enhancer of the present invention can increase the starch content in plants by reducing the activity of SEX4 and inhibiting starch decomposition.

In addition, it has the advantage of being safe as it has an excellent SEX4 activity inhibition effect without genetic modification.

Figure 1 is a graph showing the inhibition performance of compound library compounds obtained by pNPP assay.
Figure 2 is a concentration-response graph showing the activity inhibition performance of SEX4 of Examples 1 to 2 and Comparative Examples 1 to 5 on amylopectin substrate obtained by malachite green analysis.
Figure 3 is a graph showing the activity of SEX4 against amylopectin substrate in the absence and IC ₅₀ concentration of Examples 1 to 2 and Comparative Examples 1 to 5.

Hereinafter, the present invention will be described in detail.

Since the present invention can be subject to various modifications and can have various embodiments, specific embodiments will be illustrated in the drawings and described in detail in the detailed description. However, this is not intended to limit the present invention to specific embodiments, and should be understood to include all transformations, equivalents, and substitutes included in the spirit and technical scope of the present invention. In describing the present invention, if it is determined that a detailed description of related known technologies may obscure the gist of the present invention, the detailed description will be omitted.

The terminology used herein is only used to describe specific embodiments and is not intended to limit the invention. Singular expressions include plural expressions unless the context clearly dictates otherwise. In this application, terms such as “comprise” or “have” are intended to designate the presence of features, numbers, steps, operations, components, or a combination thereof described in the specification, but are not intended to indicate the presence of one or more other features or It should be understood that this does not exclude in advance the possibility of the presence or addition of numbers, steps, operations, components, or combinations thereof.

The present invention provides a starch content enhancer comprising a compound represented by Formula 1 or Formula 2 below.

[Formula 1]

[Formula 2]

The compound represented by Formula 1 or Formula 2 may be included in the starch content enhancer in an amount of 0.01 to 95% by weight, preferably 0.05 to 90% by weight, and more preferably 0.1 to 85% by weight, but is not limited thereto. No.

Starch is the main storage carbohydrate of plants and is composed of amylopectin and amylose. Depending on the content ratio of amylopectin and amylose, there are differences in gelatinization properties and gelling properties. Among the components of starch, amylopectin, similar to glycogen, is composed of α-1,6-glycosidic branches and α-1,4-glycosidic bonds, and amylose is a glucose moiety connected by α-1,4-glycosidic bonds. It is a linear molecule composed of

Starch degradation requires reversible glucan phosphorylation by dikinases and phosphatases on the surface of starch granules in plant chloroplasts (Baunsgaard, L et al, A novel isoform of glucan, water dikinase phosphorylates pre-phosphorylated α-glucans and is involved in starch degradation in Arabidopsis. The Plant Journal 2005 , 41 (4), 595-605.). The glucan phosphorylation mechanism is initiated by α-glucan water dikinase (GWD) and phosphoglucan water dikinase (PWD). Glucan water dekinase phosphorylates the C6 position of a starch glycosyl residue, providing pre-phosphorylation priming for phosphoglucan water dekinase to phosphorylate the C3 position. Phosphorylation of starch by glucan water dekinase and phosphoglucan water dekinase disrupts the packing of the amylopectin double helix, leading to maltose degradation by β-amylase, BAM1, BAM3, isoamylase, and ISA3. ) and allow the release of oligosaccharides. SEX4 dephosphorylates the C3 and C6 positions to remove phosphates that interfere with the movement of BAM (Oliver Kotting et al., STARCH-EXCESS4 Is a Laforin-Like Phosphoglucan Phosphatase Required for Starch Degradation in Arabidopsis thaliana,he Plant Cell Jan 2009 , 21 (1) 334-346.). These enzymes work in an organized manner to break down starch.

Starch Excess 4 (SEX4) is a dual-specificity phosphatases (DSP) among protein tyrosine phosphatases (PTPs), and like Sex Four 2 (Lsf2) is an enzyme involved in starch degradation like SEX4. ). SEX4 contains an amino-terminal chloroplast targeting peptide (cTP), a dual specificity dephosphorylase (DSP) domain and a carbohydrate binding module (CBM). SEX4 is essential for starch decomposition, and without SEX4 activity, starch decomposition is inhibited. Therefore, plants with loss of SEX4 function have delayed starch decomposition, accumulate starch, and contain three times more starch than normal plants. Therefore, compounds that inhibit the activity of SEX4 can inhibit starch decomposition in plants and increase starch content when applied to plants.

The plant may be any one or more selected from the group consisting of oats, millet, buckwheat, barley, sorghum, rice, corn, and rye, and may preferably be selected from rice or corn, but is not limited thereto.

Additionally, the plant may be any one or more selected from the group consisting of potatoes, sweet potatoes, konjac, and cassava, preferably potatoes, but is not limited thereto.

The SEX4 is conserved in green algae, the ancestors of land plants, and land plants (Gentry, The phosphatase laforin crosses evolutionary boundaries and links carbohydrate metabolism to neuronal disease, The Journal of Cell Biology, Vol. 178, No. 3, July 30, 2007 477-488), compounds that have inhibitory properties on SEX4 activity can be applied to grains and grains to inhibit SEX4 activity, thereby inhibiting starch decomposition and increasing starch content.

The compound represented by Formula 1 or Formula 2 according to the present invention can inhibit the activity of SEX4 in plants.

In addition, the compound represented by Formula 1 or Formula 2 can inhibit the activity of SEX4 against amylopectin with an IC ₅₀ of 5 to 15 μM, preferably 7 to 12 μM.

Amylopectin is a major component of starch, accounting for 70 to 80% of starch, and the compound represented by Formula 1 or Formula 2 can increase the starch content in plants by inhibiting the activity of SEX4 on amylopectin.

The present invention provides a pesticide composition containing the starch content enhancer.

Pesticides are chemicals used to disinfect the soil of farmland for growing crops, to disinfect seeds, to protect crops from diseases and pests that occur during the crop cultivation period, and to prevent loss due to pests and diseases when storing harvested agricultural products. In addition, it includes all agents that promote or inhibit the growth of crops and agents that improve the quality of crops by improving coloring.

In the pesticide composition, the starch content enhancer may be included in an amount of 0.01 to 99% by weight, preferably 0.05 to 95% by weight, and more preferably 0.1 to 85% by weight. When the content of the starch content enhancer satisfies the above more preferable range, the starch content enhancement effect can be significantly improved without inhibiting starch decomposition for growth.

In addition to the starch content enhancer, the pesticide composition of the present invention may contain auxiliaries for ease of use, and may specifically include spreaders, extenders, solvents, and emulsifiers. In addition, plant growth regulators, insecticides, fungicides and pesticide active ingredients may be additionally included.

The present invention provides a fertilizer composition containing the starch content enhancer.

Fertilizers refer to substances that bring about chemical changes in the soil to provide nutrition to plants or to help grow plants, and substances supplied to improve the quality of plants. In the fertilizer composition, the starch content enhancer is contained in the range of 0.01 to 99% by weight, preferably 0.05 to 95% by weight, and more preferably 0.1 to 85% by weight. When the content of the starch content enhancer satisfies the above more preferable range, the starch content enhancement effect can be significant without inhibiting starch decomposition for growth.

The fertilizer composition of the present invention may further include a carrier, diluent, and extender for convenient use in addition to the starch content enhancer. Specific examples of the carrier include inorganic minerals such as calcium carbonate, bentonite, kaolin, and talc.

Additionally, the fertilizer composition of the present invention may contain substances that can promote plant growth or supply nutrients to plants in addition to starch content enhancers. Specifically, it may include sources of nitrogen, phosphoric acid, potassium, calcium, sulfur, magnesium, minerals, organic nutrients, and phosphorus.

The present invention provides a method for cultivating plants with improved starch content, comprising treating the plants with the starch content enhancer.

The treatment may be either soil fertilization or foliar fertilization.

Soil fertilization means applying a chemical solution to the soil, and foliar fertilization means spraying a chemical solution on the leaves.

The present invention provides plants grown by the above cultivation method.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention in more detail, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention. .

Experiment example

experiment material

All compounds used in experimental examples and material examples were purchased from Sigma-Aldrich.

Experimental Example 1. Selection of compounds showing SEX4 inhibitory activity

Compounds with SEX4 inhibition performance were selected from a compound library (Korean Compound Bank Library) consisting of approximately 1,840 drug-like and natural-like compounds.

pNPP (para-nitrophenyl phosphate) is an indicator that can measure the activity of dephosphorylation enzyme. When pNPP (para-nitrophenyl phosphate) is dephosphorylated by a dephosphorylation enzyme, p-nitrophenol (pNP) is produced. The amount of p-nitrophenol (pNP) produced and dephosphorylated as measured at absorbance at 410 nm. Enzyme activity can be measured.

A reaction mixture was created by mixing 2 μg of SEX4, 1X assay buffer at pH 5.5 (20 mM sodium acetate, 10 mM bis-Tris, 10 mM Tris-HCl), 5 mM dithiothreitol (DTT), 50 mM pNPP , and the above compounds. . 20 μl of the reaction mixture was reacted at 37 °C for 10 minutes and the reaction was terminated by adding 200 μl of 0.25 N NaOH. Afterwards, the SEX4 activity inhibition performance was measured based on the absorbance measured at 410 nm with a Beckman Coulter DU 700 Series UV/Vis Spectrophotometer, and the results are shown in Figure 1. In addition, seven compounds showing excellent SEX4 activity inhibition performance were selected and shown in Table 1 below.

Figure 1 is a graph showing the inhibition performance of compound library compounds obtained by pNPP assay, through which it can be seen that seven of the candidates have excellent inhibition performance against SEX4.

division structure Formula 1 (O-000001 F09)
Example 1 Formula 2 (O-000001 G11)
Example 2 Formula 3 (O-000007 F05)
Comparative Example 1 Formula 4 (O-000007 G07)
Comparative Example 2 Formula 5 (O-000004 H08)
Comparative Example 3 Formula 6 (G-000036 D04)
Comparative Example 4 Formula 7 (G-000037 H03)
Comparative Example 5

Experimental Example 2. Malachite Green Assay

The malachite green assay is a method of measuring released inorganic phosphate based on the amount of released inorganic phosphate, malachite green, and complex formed by molybdate, and can measure the activity of dephosphorylation enzyme. In Experimental Example 2, the activity of SEX4 on amylopectin was measured.

2 μg A 20 μl reaction mixture was prepared by mixing SEX4, 100mM sodium acetate, 50mM bis-Tris, 50mM Trsi (pH 6.5), 10mM DTT, and 45μg amylopectin.

The reaction mixture was incubated at 37°C for 15 minutes, followed by 20 μl of 100 mM N-etylmalemide and 40 μl of malachite green reagent (0.15% w/v malachite green and 1.25% w/v Ammonium molybdate Tetrahydrate in 1 M HCl) for 40 minutes. The reaction was terminated by addition. Afterwards, the activity of SEX4 was measured by absorbance measured at 620 nm using a Beckman Coulter DU 700 Series UV/Vis Spectrophotometer.

Experimental Example 3. IC for amylopectin ₅₀ calculation of

IC ₅₀ (half maximal inhibitory concentration) means 50% inhibitory concentration. In Experimental Example 3, when the reaction that occurs when SEX4 is active on the substrate amylopectin is 100%, it means the concentration that can inhibit this reaction by 50%. The larger the IC ₅₀ value, the greater the concentration required to reduce the activity of SEX4 to 50%, indicating a lower inhibitory efficacy. Therefore, it can be said that the lower the IC ₅₀ value, the higher the SEX4 activity inhibition performance of amylopectin.

Without changing the amount of amylopectin, the concentration of the inhibitor is changed from 0 to 0.2 mM, and from the regression curve using PRISM, the concentration that inhibits the activity of SEX4 by 50% (half maximal inhibitory concentration, IC ₅₀ ) was calculated using Equation 1 below, and the results are shown in Figure 2 and Table 2.

[Equation 1]

division IC ₅₀ (μM) Example 1 7.49 Example 2 9.72 Comparative Example 1 21.10 Comparative Example 2 38.61 Comparative Example 3 16.49 Comparative Example 4 57.33 Comparative Example 5 13.05

As shown in Table 2, it was confirmed that Examples 1 and 2 had an IC ₅₀ value of 10 μM or less and that the activity inhibition ability against SEX4 was superior to other compounds with SEX4 inhibition ability.

From these results, it was confirmed that Examples 1 and 2 were very useful ingredients as starch content enhancers by inhibiting the activity of SEX4 in plants.

Experimental Example 4. Calculation of reaction rate constant (kinetic parameter)

The concentration of amylopectin was varied from 5 to 45 μg, and the V _max , K _m , and k _cat values were measured by nonlinear regression analysis of the Michaelis-menten equation at steady state (Table 3). V _max is the maximum enzyme speed, K _m is the dissociation constant of the enzyme-substrate complex, and as the K _m value increases, the affinity between the enzyme and the substrate is lower.

Figure 3 is a graph showing the activity of SEX4 against amylopectin substrate in the absence and IC ₅₀ concentration of Examples 1 to 2 and Comparative Examples 1 to 5.

There are four types of enzyme inhibition: competitive inhibition, non-competitive inhibition, and mixed model inhibition. If the inhibitor is similar to the substrate and competes with the enzyme and binds to the substrate, it is called a competitive inhibition type and the inhibitory effect is affected by the concentration of the substrate. A type of inhibition in which the inhibitor binds to a site different from the enzyme binding site present in the substrate and does not bind to the enzyme binding site is called a non-competitive inhibition type. The non-competitive inhibition type is an inhibition type in which the inhibitor binds only when the substrate and enzyme are combined, and the mixed model inhibition type refers to a type in which the dissociation constant of the inhibitor for the enzyme and the dissociation constant for the enzyme-substrate complex are different.

As shown in Table 3, Example 1 and Comparative Examples 1 to 3 in which both V _max and K _m were reduced were non-competitive inhibition types, and Example 2 and Comparative Examples 4 to 5 were V _max and K _m It can be seen that these are all types of increased competition suppression.

K _i (inhibition constant) value is the dissociation constant of the enzyme-inhibitor complex and means the affinity between the inhibitor and the inhibitor binding site. The smaller the K _i value, the greater the affinity and the greater the inhibitory action, indicating an effective inhibitor.

K _i is the IC ₅₀ concentration The V _max and K _m values obtained by varying the substrate concentration in the presence and absence of the inhibitor were calculated and shown in Table 4 below.

division K _i (μM) Example 1 16.42 Example 2 7.81 Comparative Example 1 20.81 Comparative Example 2 46.40 Comparative Example 3 31.12 Comparative Example 4 28.31 Comparative Example 5 29.84

As shown in Table 4, Examples 1 and 2 had K _i values of 20 μM or less and were confirmed to have more effective inhibitory performance on SEX4 activity against amylopectin than other compounds.

Based on these results, it was confirmed that Examples 1 and 2 could be used as useful ingredients for starch content enhancers by inhibiting the activity of SEX4.

Experimental Example 5. Comparison of LSF2 activity

In order to measure the performance of Examples 1 to 2 and Comparative Examples 1 to 5 to inhibit LSF2 activity, LSF2 activity to amylopectin was inhibited in the same manner as in Experimental Examples 2 to 3, except that LSF2 was used instead of SEX4. Performance was measured and shown in Table 5 below.

division IC ₅₀ (μM) SEX4 LSF2 Example 1 7.49 >50 Example 2 9.72 >50 Comparative Example 1 21.10 >50 Comparative Example 2 38.61 >50 Comparative Example 3 16.49 >50 Comparative Example 4 57.33 >202 Comparative Example 5 13.05 >204

In all examples and comparative examples, the IC ₅₀ value for LSF2 was greater than the IC ₅₀ value for SEX4 and was found to be beyond the measurable range. Therefore, it was confirmed that all examples and comparative examples can act as specific inhibitors for SEX4.

Experimental Example 6. Analysis of starch content according to pesticide treatment containing starch content enhancer

An aqueous solution containing 2% by weight of Examples 1 and 2, respectively, was applied to brown rice grown under the same conditions twice at 5-day intervals. Next, untreated brown rice was set as a control, and after 3 days, the starch content was measured using the Total Starch Assay Kit (Megazyme) (Table 6).

division Example 1 Example 2 control group Processing method Foliar fertilization Untreated 0.4L/ ^m2 Starch content (% by weight) 75 74 69

As shown in Table 6, it was confirmed that the starch content increased significantly in brown rice treated in Examples 1 to 2. Through this, it was confirmed that the starch content enhancer according to the present invention is an effective ingredient in increasing the starch content of plants when included in pesticides.

Experimental Example 7. Starch content analysis according to fertilizer treatment containing a starch content enhancer

An aqueous solution containing 10% by weight of each of Examples 1 and 2 and potato fertilizer (manufactured by KG Chemical) were mixed at a weight ratio of 1:9 and applied to potatoes grown in the same environment, and potatoes treated with only potato fertilizer were used as a control group. The starch content of potatoes harvested one week after soil fertilization was measured using the Total Starch Assay Kit (Megazyme) (Table 7).

division Example 1 Example 2 control group Processing method soil fertilization Untreated 0.4kg/ ^m2 Starch content (% by weight) 17.7 18.2 15.2

As shown in Table 7, Examples 1 and 2 were included in fertilizers and excellently improved the starch content. This shows that the starch content enhancer according to the present invention is a useful ingredient for increasing starch content in fertilizers. Confirmed.

Claims (10)

Contains a compound represented by the following formula (1) or formula (2),
The compound represented by Formula 1 or Formula 2 below is a starch content enhancer in plants, characterized in that it inhibits the activity of SEX4.
[Formula 1]

[Formula 2]
The starch content enhancer according to claim 1, wherein the plant is one or more selected from the group consisting of oats, millet, buckwheat, barley, sorghum, rice, corn, and rye. The starch content enhancer according to claim 1, wherein the plant is at least one selected from the group consisting of potato, sweet potato, konjac, and cassava. The starch content enhancer according to claim 1, wherein the compound represented by Formula 1 or Formula 2 has an inhibitory activity on SEX4 against amylopectin with an IC ₅₀ of 5 to 15 μM. delete A pesticide composition comprising the starch content enhancer according to any one of claims 1 to 4. A fertilizer composition comprising the starch content enhancer according to any one of claims 1 to 4. A method of cultivating plants with improved starch content, comprising treating plants with the starch content enhancing agent according to any one of claims 1 to 4. The method of claim 8, wherein the treatment is one of soil fertilization and foliar fertilization. Plants grown by the cultivation method according to paragraph 8.