JP6407688B2 - Plant cell wall thickening promoter and plant cell wall degradation promoter - Google Patents

Description

  The present invention relates to a plant cell wall thickening promoter, a plant cell wall degradation promoter, a plant cell wall thickening promotion method, a plant biomass raw material production method, and a plant biomass degradation / saccharification method using these.

  In recent years, in order to solve the progress of the depletion of fossil resources and the global warming problem, a shift to a society utilizing bioenergy and biomaterials has been attempted.

  Plants are the most abundant biomass on land. In plants, the cell wall plays a major role in maintaining cell morphology, but the cell wall is rich in polysaccharides that serve as biomass materials such as cellulose and hemicellulose, and is an important structure as a biomass material. These polysaccharides are finally converted into sugars for use, but a technique for changing the cell wall is desired for more efficient use of biomass. Specifically, there is a demand for a compound capable of quantitatively and qualitatively controlling the cell wall, such as increasing the amount of the cell wall itself, changing the structure, or making it easy to saccharify.

  The cell wall is retained by almost all prokaryotes and most eukaryotes and is present in plants without exception. The function of the plant cell wall has various roles such as recognition of cells, exchange of substances inside and outside of cells, and resistance to pests, in addition to determining the size and shape of cells and increasing the structural strength of plants. Has been revealed. The growing cell consists of a thin cell wall called the primary cell wall, and many cells form several layers of thick secondary cell walls as they mature. It is mainly the secondary cell wall that gives the structural strength of the plant body.

  Patent Document 1 describes a secondary thickening promoter containing cytokinin as an active ingredient that promotes secondary thickening of xylem in plants, but there are few reports on chemical substances that cause thickening of plant cell walls. .

  Rasaroside A (Lasaroside) is a polyether ionophore obtained by culturing Streptomyces genus actinomycetes and is known to have a controlling effect against root-knot disease (Patent Document 2). unknown. Rasaroside A has already been totally synthesized (Non-patent Document 1).

  In addition, in Lazaroside A, there are Lasaloside B, Lazaroside C, Lazaroside D, and Lazaroside E, which are analogues in which one of the methyl groups present in Lazaroside A is replaced with an ethyl group, and these analogues are also Streptomyces genus radiation. It is obtained by culturing fungi and is known to have antibacterial activity equivalent to or higher than that of Lazaroside A (Non-patent Document 2).

JP 2007-197355 A JP 2004-277370 A

Ireland R.E. et al., J. Am. Chem. Soc., 105, 1988-2006 (1983) Westley J.W. et al., Journal of Antibiotics, 27 (10), 744-753 (1974).

  The subject of this invention is changing a plant cell wall with a chemical substance.

The gist of the present invention is as follows.
(1) A plant cell wall thickening promoter containing a culture of Streptomyces genus Actinomycetes or a mutant thereof, or a processed product thereof, containing lasaloside A or a salt thereof, or a salt thereof.

（式中、Ｒ_１、Ｒ_２、Ｒ_３及びＲ_４は、同一又は異なって、水素原子又はＣ_１−３アルキル基である。）
で示される化合物又はその塩を含有する植物細胞壁肥厚促進剤。 (2) The following formula (I):

(In formula, R < ₁ >, R < ₂ >, R < ₃ > and R < ₄ > are the same or different and are a hydrogen atom or a _C1-3 alkyl group.)
The plant cell wall thickening promoter containing the compound or its salt shown by these.

(3) A plant cell wall degradation promoter containing a culture of Streptomyces genus actinomycetes or a mutant thereof, or a processed product thereof, containing lasaloside A or a salt thereof, or a salt thereof.

(4) A plant cell wall degradation promoter containing the compound represented by the formula (I) or a salt thereof according to (2).

(5) A method of promoting plant cell wall thickening, comprising treating a plant or a tissue or cell thereof with the plant cell wall thickening promoter according to (1) or (2).

(6) A method for producing a plant biomass raw material, comprising treating a plant or a tissue or cell thereof with the plant cell wall thickening promoter according to (1) or (2).

(7) A method for decomposing and saccharifying plant biomass, comprising treating a plant or tissue or cell thereof with the plant cell wall degradation promoter according to (3) or (4), and then decomposing and saccharifying with a cellulose-degrading enzyme. .

  According to the present invention, a plant cell wall can be changed by a chemical substance. Moreover, according to the plant cell wall degradation promoter of the present invention, ethanol productivity can be improved by increasing the degradation efficiency of the plant cell wall.

FIG. 1 shows the results of observation of a sample with a laser scanning confocal microscope (LSM700; manufactured by Carl Zeiss). In FIG. 1, BF shows the result by the bright field observation method, and Calcofluor (UV) shows the result by the fluorescence observation method (ultraviolet irradiation). FIG. 2 shows the result of observation of the sample with a transmission electron microscope. FIG. 3 shows the results of measuring the cell wall thickness using image analysis software. FIG. 4 shows the results of gene ontology (GO) annotation analysis using TAIR online software. FIG. 5 shows the results of a plant cell wall degradation / saccharification test.

で示される化合物である。ラサロシドＡ及び前記式（Ｉ）で示される化合物の塩としては、製薬上許容可能な塩であればよく、例えば、ナトリウム、リチウム、カリウム等のアルカリ金属塩；マグネシウム、カルシウム等のアルカリ土類金属塩；鉄、銅、亜鉛、マンガン等の重金属塩；アンモニア、メチルアミン、ジメチルアミン、トリメチルアミン、エチレンジアミン、テトラメチルエチレンジアミン、アニリン、Ｎ，Ｎ−ジメチルアニリン、ピリミジン、ルチジン、コリジン、ヒドラジン、Ｎ−メチルグルカミン等のアンモニウム又はアミン塩；グアニジンとの塩等が挙げられる。 Lazaroside A used in the present invention has the following formula (Ia):

It is a compound shown by these. The salt of lasaloside A and the compound represented by the formula (I) may be a pharmaceutically acceptable salt, for example, an alkali metal salt such as sodium, lithium or potassium; an alkaline earth metal such as magnesium or calcium; Salt; Heavy metal salt such as iron, copper, zinc, manganese; ammonia, methylamine, dimethylamine, trimethylamine, ethylenediamine, tetramethylethylenediamine, aniline, N, N-dimethylaniline, pyrimidine, lutidine, collidine, hydrazine, N-methyl Examples include ammonium or amine salts such as glucamine; salts with guanidine.

  Rasaroside A is a substance obtained by culturing Streptomyces genus actinomycetes, and cultures of Streptomyces genus actinomycetes or mutants thereof or processed products thereof also contain lasaloside A or a salt thereof and can be used as an active ingredient of the present invention. it can.

  Streptomyces genus actinomycetes or mutants thereof used in the present invention are not limited as long as they have the ability to produce lasaloside A. For example, Streptomyces lasaliensis, particularly Streptomyces lasaliensis ATCC 31180, Streptomyces lasaliensis NRRL 3382R, or mutants thereof are Can be mentioned.

  The method for mutagenizing the microorganism is not particularly limited as long as it induces mutation. For example, chemical methods using mutation agents such as N-methyl-N′-nitro-N-nitrosoguanidine, ethyl methanesulfonate, physical methods such as ultraviolet irradiation and X-ray irradiation, biological methods such as genetic recombination and transposon A method or the like can be used. This mutagenesis treatment may be performed once, or two or more mutagenesis treatments may be performed such that the mutant obtained by this mutagenesis treatment is further mutagenized.

  The method of culturing the microorganism and collecting Lazaroside A from the culture, if necessary, can be carried out generally according to a method for culturing bacteria belonging to the genus Streptomyces.

  First, the strain is aerobically cultured in a medium containing nutrients that can be utilized by actinomycetes. As a nutrient source, known ones conventionally used for culturing actinomycetes can be used. For example, as a carbon source, glucose, galactose, sucrose, glycerin, starch syrup, dextrin, starch, molasses, animal / vegetable oil Etc. Moreover, as a nitrogen source, soybean powder, wheat, wheat germ, corn steep liquor, peptone, yeast extract, meat extract, ammonium sulfate, sodium nitrate, urea and the like can be used alone or in combination. Moreover, you may add inorganic salts which can produce | generate sodium, cobalt, chlorine, a sulfuric acid, phosphoric acid, and another ion as needed. Furthermore, an organic or inorganic substance that helps the growth of the fungus and promotes the production of Lazaroside A can be added as appropriate.

  The culture method is not particularly limited, but a liquid culture method is preferable. A suitable temperature for culturing is 15 to 37 ° C. In many cases, the culturing is performed at around 26 to 30 ° C. The production of lasalocid A varies depending on the medium and culture conditions, but the accumulation reaches the maximum in 1 to 10 days for both shaking culture and tank culture. When the accumulated amount of lasaloside A in the culture reaches the maximum, the culture is stopped, and the target substance is collected from the culture as necessary.

  In the present invention, depending on the purpose, a culture solution, a culture such as a dried product thereof, or a treatment after removing microbial cells from the culture solution and then drying, a solution obtained by removing microbial cells from the culture solution, or the like. A thing can be used suitably.

  In order to purify and isolate Lazaroside A from the culture after completion of the culture, generally means commonly used for collecting microbial metabolites can be appropriately used. For example, various ion exchange resins, nonionic adsorption resins, gel filtration chromatography, thin layer chromatography, high performance liquid chromatography, or means such as crystallization, solvent extraction, concentration under reduced pressure, and lyophilization may be used alone or in combination as appropriate. Or iterative use. These purified products are also included in the processed product of the present invention.

  Lazaroside A is a substance that is soluble in organic solvents and insoluble in water. Accordingly, examples of the extraction solvent include alcohols such as methanol, ethanol, propanol, and ethylene glycol; ethers such as diethyl ether, dioxane, and tetrahydrofuran; ketones such as acetone and methyl ethyl ketone; aromatics such as benzene, toluene, and xylene. Hydrocarbons; Halogenated hydrocarbons such as dichloromethane, chloroform and carbon tetrachloride; Amides such as N, N-dimethylformamide and N, N-dimethylacetamide; Esters such as ethyl acetate, butyl acetate and fatty acid glycerin ester Nitriles such as acetonitrile and propionitrile; dimethyl sulfoxide and the like are used. These may be used as appropriate by mixing one or more of them in an appropriate ratio. Moreover, the said solvent, for example, the mixed solvent which mixed other solvents, such as acetic acid, in a small amount, can also be used.

  Rasaroside A includes Lasaroside B, Lasaroside C, Lasaroside D, and Lasaroside E, which are analogs in which one of the methyl groups present in Lasaroside A is replaced with an ethyl group. These analogs are also Streptomyces actinomycetes. It is obtained by culturing and is known to have antibacterial activity equivalent to or better than lasaroside A (Non-patent Document 4). Furthermore, a method for synthesizing Lazaroside A is also known.

（式中、Ｒ_１、Ｒ_２、Ｒ_３及びＲ_４は、同一又は異なって、水素原子又はＣ_１−３アルキル基である。）
で示される化合物も本発明の有効成分として用いることができる。 Therefore, the following formula (I):

(In formula, R < ₁ >, R < ₂ >, R < ₃ > and R < ₄ > are the same or different and are a hydrogen atom or a _C1-3 alkyl group.)
The compounds represented by can also be used as the active ingredient of the present invention.

Examples of the C _1-3 alkyl group represented by R ₁ , R ₂ , R ₃ or R ₄ in the formula (I) include a methyl group, an ethyl group, a propyl group, an isopropyl group, and a cyclopropyl group.

In the formula (I), a compound in which R ₁ , R ₂ , R ₃ and R ₄ are methyl groups is a lasaloside A, a compound in which R ₂ , R ₃ and R ₄ are methyl groups, and a compound in which R ₁ is an ethyl group is lasaloside B, R ₁ , R ₃ and R ₄ are methyl groups, R ₂ is an ethyl group, a compound is Lazaroside C, R ₁ , R ₂ and R ₄ are methyl groups, and a compound where R ₃ is an ethyl group is Lazaroside D, A compound in which R ₁ , R ₂ and R ₃ are methyl groups and R ₄ is an ethyl group is lasaloside E.

  The compound represented by the formula (I) can be prepared by subjecting an aldehyde compound (II) and a zinc enolate of a ketone compound (III) to an aldol condensation in accordance with a known method, for example, as described below. Can be synthesized.

（式中、Ｂｎはベンジル基であり、Ｒ_１、Ｒ_２、Ｒ_３及びＲ_４は前記と同義である。）

(In the formula, Bn is a benzyl group, and R ₁ , R ₂ , R ₃ and R ₄ are as defined above.)

  By treating a plant or a tissue or cell thereof with the plant cell wall thickening promoter of the present invention, thickening of the plant cell wall is promoted.

  After treating a plant or tissue or cell thereof with the plant cell wall degradation promoter of the present invention, decomposition and saccharification with cellulose degrading enzyme can increase the degradation efficiency of the plant cell wall and improve ethanol productivity. .

  Plants to which the present invention is applicable include dicotyledonous plants, monocotyledonous plants, gymnosperms, and trees. For example, cruciferous, gramineous, leguminous, eggplant, beech, lily, akaza, myrtaceae, willow, palm, etc. can be targeted for production. More specifically, Arabidopsis, Western rape, broccoli, cauliflower, cabbage, cabbage, rice, wheat, barley, corn, soybean, tomato, eggplant, potato, leek, onion, garlic, spinach, sugar cane, eucalyptus, poplar, jatropha , Oil palm, wasabi, leek and the like.

  The plant cell wall thickening promoter and plant cell wall degradation promoter of the present invention may be applied without adding any components to the active ingredient, but may be dissolved or dispersed in a suitable liquid carrier or solvent depending on the purpose of use. It is preferably used as a dosage form such as an emulsion, oil, aqueous suspension, emulsion, liquid, wettable powder, powder, granule, flowable, etc. with admixture or admixture with a suitable solid carrier. .

  Suitable liquid carriers include, for example, alcohols such as methanol, ethanol, propanol and ethylene glycol; ethers such as dioxane and tetrahydrofuran; ketones such as acetone and methyl ethyl ketone; fats such as kerosene, kerosene, machine oil and edible oil Aromatic hydrocarbons such as benzene, toluene and xylene; halogenated hydrocarbons such as dichloromethane, chloroform and carbon tetrachloride; amides such as N, N-dimethylformamide and N, N-dimethylacetamide Esters such as ethyl acetate, butyl acetate and fatty acid glycerin esters; nitriles such as acetonitrile and propionitrile; dimethyl sulfoxide and the like are used. These may be used as appropriate by mixing one or more of them in an appropriate ratio.

  Examples of the solid carrier include plant powders such as rice bran, bran, soybean powder, wheat flour, and wood powder; clays such as kaolin, bentonite, and acid clay; talc such as talc powder and wax stone powder; diatomaceous earth, mica powder, vermiculite Silica mineral powders such as calcium silicate, calcium carbonate, alumina, activated carbon and the like are used. These may be used as appropriate by mixing one or more of them in an appropriate ratio.

  The plant cell wall thickening promoter of the present invention can be used, for example, by direct administration so as to act on an organ in which secondary thickening is to be promoted. Specifically, in the case of roots and rhizomes, the plant cell wall thickening promoter of the present invention can be used by adding it to a medium or soil, and in the case of ground stems or stems, the plant cell wall thickening promoter of the present invention is directly sprayed. It can be used by coating or wrapping the stem or stem with a cloth soaked with the plant cell wall thickening promoter of the present invention.

  When the plant cell wall thickening promoter of the present invention is used in the form of a solution, the content of lasaloside A or the compound represented by the formula (I) can be appropriately adjusted according to the applied plant, but is usually 0.01 ˜150,000 mg / L, preferably 0.05 to 150,000 mg / L.

  By treating a plant or a tissue or cell thereof with the plant cell wall thickening promoter of the present invention, thickening of the plant cell wall is promoted. A plant biomass raw material can be obtained by recovering a cellulosic component from a plant in which thickening of the plant cell wall has been promoted or a tissue or cell thereof.

  The cellulosic component is a main component of the plant cell wall and refers to a cellulosic polysaccharide composed of cellulose and / or hemicellulose. In the present specification, the term “cellulosic component” refers to cellulose as a main component (for example, 50% or more). 60% or more, 70% or more). If it is a herbaceous plant, a cellulosic component can be collect | recovered by drying a plant body. If it is a tree, after crushing to a chip | tip and performing the removal process (digestion) of a lignin, a cellulosic component can be collect | recovered.

  Cellulosic components can be used as raw materials for the production of saccharides, bioethanol and the like.

  The plant cell wall degradation promoter of the present invention, for example, is added to a culture medium or soil to culture and cultivate plants or tissues or cells thereof, and then decomposes and saccharifies with a cellulose-degrading enzyme, so that the degradation efficiency of plant cell walls is increased. The ethanol productivity can be improved.

  The cellulose-degrading enzyme is not particularly limited, and a known one can be used. For example, cellulase, pectinase, hemicellulase, β-glucanase, xylanase, mannase, amylase, mecerase, acremonium cellulase (from Acremonium cellulolyticus bacterium) Obtained cellulase). These may be used alone or in combination of two or more.

  The addition amount of the cellulolytic enzyme is preferably 0.1 to 10 parts by mass, more preferably 0.3 to 5 parts by mass with respect to 100 parts by mass of biomass, for example.

  In addition, after adding a cellulolytic enzyme to a mixture, it is preferable to stir and to advance saccharification. The stirring time is, for example, 1 to 24 hours.

  Although the temperature of the mixture in this saccharification process can be suitably set according to the kind of enzyme, etc., it is 30-70 degreeC, for example, Preferably it is 40-60 degreeC.

  In addition, the pH of the mixture in this saccharification step may be appropriately adjusted depending on the type of enzyme and the like, and is preferably set to pH 4.5 to 7.5, for example. This pH adjustment can be performed by addition to a known acid or base mixture.

  Since lignin coexisting with plant biomass inhibits enzymatic degradation, it is necessary to remove lignin before the reaction, so far it has been common to pre-process raw biomass with a strong acid / high pressure reaction or lignin degrading enzyme However, there were problems in terms of environment and cost. As described above, by culturing and cultivating plants or tissues or cells thereof in the presence of the plant cell wall degradation promoter of the present invention, the cellulose degradation rate is increased without pretreatment as described above, and ethanol Productivity can be improved.

  EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples, but the scope of the present invention is not limited thereto.

[Example 1]
(1) Plant cell culture This is the most widely used plant culture cell line in the world, and because it has the fastest growth rate and can be easily cultured in large quantities. Tobacco BY-2 cell line as the 'HeLa' cell (Nigataiana tobacum L. cv. Bright Yellow 2) (Nagata, T., Nemoto, Y. and Hasezawa, S. (1992) Int. Rev. Cytol. 132: 1-30) was adopted.

  According to the description in the above literature, the cultured cells were grown in tobacco BY-2 cells in a modified Linsmaier-Skoog medium under continuous dark shaking at 27 ° C. and 130 rpm. Cultured cells were diluted 1:95 every 7 days and maintained in subculture with fresh medium.

(2) Rasaroside treatment On the day after the dilution of tobacco BY-2 cells, rasaloside A sodium (manufactured by BioAustralis Fine Chemicals) was added to the culture solution of tobacco BY-2 cells at a final concentration of 0 μM, 0.1 μM, 0.25 μM or 1. Added at 0 μM (Lasaroside A sodium was dissolved in DMSO). After culturing for 48 hours, tobacco BY-2 cells were stained with 0.001% calcofluor white (BD, New Jersey, USA) for 20 minutes. (Calcofluor white is a fluorescent stain that binds strongly to structures containing cellulose or chitin.) After staining, the sample was observed with a laser scanning confocal microscope (LSM700; manufactured by Carl Zeiss).
The results are shown in FIG. Lazaroside A sodium thickened the plant cell wall in a concentration-dependent manner.

[Example 2]
The day after the dilution of tobacco BY-2 cells, rasaloside A sodium (manufactured by BioAustralis Fine Chemicals) was added to the culture solution of tobacco BY-2 cells at a final concentration of 0 μM, 0.1 μM or 1.0 μM and cultured for 48 hours ( Lazaroside A sodium was dissolved in DMSO).

  For transmission electron microscopy (TEM) observation, the sample was fixed with 2% glutaraldehyde buffered with 20 mM sodium cacodylate (pH 7.0, 20 hours, 4 ° C.) and washed with the same buffer ( 2 hours, 4 ° C.). Samples were then post-fixed with 2% osmium tetroxide in 20 mM sodium cacodylate buffer for 2 hours at 4 ° C. Fixed samples were dehydrated with a series of alcohol concentrations and embedded in Spurr resin. Ultracut E ultramicrotome diamond knives (Leica, Wien, Austria) were used to make ultrathin sections as small as 70 nm and placed on a Formvar-coated grid. Subsequently, double staining was performed by treatment with a 1% uranyl acetate solution for 20 minutes and a lead citrate solution for 15 minutes. The sample was washed with distilled water and then observed with a transmission electron microscope JEM-1200 EX (JEOL Ltd.).

The results are shown in FIG.
Moreover, the result of having measured the thickness of the cell wall using image analysis software (ImageJ software, National Institutes of Health, MD, USA) is shown in FIG.

[Example 3]
As a plant material, the whole genome has been decoded and has many advantages as a model organism, so it is easy to use as a research material. Therefore, Arabidopsis thaliana (L.) Heynhold, Col-0 ecotype Wild type) was used.

  The surface of the seed was sterilized with a sterilized solution composed of 0.025% Triton X-100 and 5% NaClO, washed with sterilized water, and then the seed was incubated at 4 ° C. for 4 days under dark conditions to break the dormancy. The dormant seeds were transferred to a germination medium (MS inorganic salts, Gamborg vitamins, 1% sucrose, 0.8% agar, pH 5.8) and incubated at 22 ° C. for 3 days under dark conditions. Germinated cotyledons were placed in a liquid solution (MS inorganic salts, 1% sucrose, pH 5.8) containing no rasaloside A sodium or 1 μM lasalocid A sodium and incubated at 22 ° C. for 24 hours in the dark. . Next, cotyledons were collected, and total RNA was extracted using Plant RNeasy Mini Kit (QIAGEN). 1 μg of total RNA was subjected to Cy3 labeling reaction using Quick Amp Labeling Kit and RNA Spike-In Kit (Agilent Technologies) according to the attached protocol. The cRNA labeled with Cy3 was hybridized with Arabidopsis oligo DNA microarray Ver.4.0 (manufactured by Agilent Technologies) according to the attached protocol.

  Online software from TAIR (Thea; The Arabidopsis Information Resource; a database of Arabidopsis genome, gene, and molecular biological data; http://www.arabidopsis.org/tools/bulk/go/index.jsp) Then, gene ontology (GO) annotation analysis was performed.

The results are shown in FIG. In FIG. 4, the horizontal axis represents the classification of protein subcellular localization, and the vertical axis represents the ratio of the number of genes. Of the pair of left and right columns in FIG. 4, the left (light color) column represents the entire gene group encoding proteins, and the right (dark color) column is more than doubled by the addition of lasalocid A sodium. The gene group which became. This bar graph is obtained by dividing the gene group in which the expression level is doubled instead of the expression level by the total number of intracellular organs that play the role. For example, if the gene A is classified as a cytosol (cytosol), one is added to the cytosol (cytosol), while if the B gene functions in both the cell wall and the endoplasmic reticulum (ER). , Both of which are classified twice.
The use of lasalocid A sodium increased the proportion of genes encoding cell wall proteins.

[Example 4]
The day after the dilution of tobacco BY-2 cells, 1.0 μM of rasaloside A sodium (manufactured by BioAustralis Fine Chemicals) was added to the culture solution of tobacco BY-2 cells and cultured for 48 hours (sodium rasaloside A was dissolved in DMSO. ). A control group was prepared by adding DMSO not containing lasaloside A sodium and culturing in the same manner.

  After culturing, the collected cells were obtained by sequentially performing phosphate buffer (pH 7.0) extraction, chloroform / ethanol mixture (1: 1) extraction, phenol / acetic acid / water mixture (2: 1: 1) extraction. In the enzyme solution obtained by washing the precipitate with ethanol and water, followed by amylase treatment (0.1 M phosphate buffer (pH 7.0) 20 ml, 4 mg / ml amylase 2 ml, 0.2% Na-azide 1 ml) For 25 hours at 25 ° C.) to obtain a cell wall fraction. The obtained cell wall fraction was pulverized with a ball mill, washed with water and methanol three times, and then subjected to amylase treatment and saccharifying enzyme treatment under the following conditions.

(Amylase treatment)
Amylase: α-amylase (Aspergillus oryzae) SIGMA A6211
Reaction time: 25 ° C., 48 hours Enzyme added amount: total amount of 460 μl of reaction solution 40 mg of 4 mg / ml α-amylase added pH: treated in buffer of pH 7.0

(Saccharification enzyme treatment)
(a) Cellulase from Trichoderma reesei ATCC 26921 (C2730 SIGMA)
(b) Cellobiase from Aspergillus niger (C6105 SIGMA)
Enzyme addition amount: 5 μl of (a) and (b) were mixed to prepare 1 mg / ml, and 100 μl was added to 1 ml enzyme reaction solution (final concentration 0.1 mg / ml)
Reaction time: 45 ° C., 0, 3, 6, 24 hours, pH during measurement treatment: treatment in a buffer of pH 5.0 FIG. 5 shows the results of measuring saccharification efficiency. FIG. 5 shows that the saccharification efficiency is increased by treatment with rasaloside A sodium.

Claims (7)

  A plant cell wall thickening promoter containing a culture of Streptomyces genus Streptomyces or a mutant thereof, or a processed product thereof, containing lasaloside A or a salt thereof, or them. Formula (I):

(In formula, R < ₁ >, R < ₂ >, R < ₃ > and R < ₄ > are the same or different and are a hydrogen atom or a _C1-3 alkyl group.)
The plant cell wall thickening promoter containing the compound or its salt shown by these.   A plant cell wall degradation promoter containing lasaloside A or a salt thereof, or a culture of Streptomyces actinomycetes or a mutant thereof, or a processed product thereof, containing them.   A plant cell wall degradation promoter comprising the compound represented by the formula (I) or a salt thereof according to claim 2.   A method of promoting plant cell wall thickening, comprising treating a plant or a tissue or cell thereof with the plant cell wall thickening promoter according to claim 1 or 2.   A method for producing a plant biomass raw material, comprising treating a plant or a tissue or cell thereof with the plant cell wall thickening promoter according to claim 1 or 2.   A method for decomposing and saccharifying plant biomass, comprising treating a plant or a tissue or cell thereof with the plant cell wall degradation promoter according to claim 3 or 4 and then decomposing and saccharifying with a cellulose degrading enzyme.

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