JP2015192665A - Microorganisms having cellulose and starch resolution - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method which can perform cellulose and starch saccharification, and subsequent alcoholic fermentation by a method with high efficacy and low environmental load, and to establish a biomass ethanol production method with high efficacy, low cost, and low environmental load.SOLUTION: Provided is a novel Paenibacillus bacteria which has a high cellulose resolution and starch resolution, further produces antibacterial substances showing wide antibacterial spectrum, and has plant growth-promoting actions.

Description

  The present invention relates to a newly identified microorganism belonging to the genus Paenibacillus, an antibacterial extract produced by the microorganism, and a method for producing ethanol using the microorganism.

Biomass ethanol is ethanol produced using plant-derived biomass such as corn and sugarcane as a raw material. Carbon contained in biomass is CO ₂ in the atmosphere fixed by plants through photosynthesis, and the amount emitted by combustion is equal to the amount absorbed by plants (carbon neutral), so biomass ethanol can be regenerated Expected as an energy resource.

  However, since biomass ethanol production raw materials can compete with food, development of biomass ethanol production technology using biomass that is non-food such as soft cellulose such as rice straw and waste woody biomass is strongly desired. ing. The saccharification of biomass raw material, which is the pre-stage of ethanol production by alcohol fermentation, is currently carried out by hydrolysis with strong acid in many cases, and it is pointed out as a problem that the environmental load is high. Furthermore, biomass ethanol is expensive to produce in Japan and is still in the trial production stage. Therefore, development of a low-cost and low environmental load saccharification technology using raw materials with low edible value is desired.

  By the way, seed disinfection is an important treatment for preventing many diseases occurring in the seedling raising period. Pesticides are mainly used for seed disinfection, but recently, organic cultivation without the use of pesticides and chemical fertilizers has been welcomed by consumers in order to improve consumer safety awareness of food. In addition, since the added value of crops is improved for producers, the production volume is increasing year by year. Seed disinfection methods that can be used in organic farming methods that are not counted as pesticides are limited to hot water disinfection and microbial pesticides (eco-hope, tough block). However, it is difficult to control the temperature of hot water disinfection, and a special device is required to accurately perform the heat treatment. On the other hand, microbial pesticides have a narrow range of applicable crops and are mainly used in rice. Therefore, development of microbial pesticides that can be applied to a wide range of crops is desired.

  The present inventors have identified and disclosed a novel microorganism belonging to the genus Paenibacillus that can efficiently decompose chitin having a strong crystal structure (Patent Document 1).

Japanese Patent No. 5447761

  The problem to be solved by the present invention is to develop a technology capable of performing saccharification of cellulose and starch and the subsequent alcohol fermentation by a method with high efficiency and low environmental load, and is a low-cost and low environmental load type biomass. Establish an ethanol production method.

As a result of intensive studies to solve the above problems, the present inventors have identified a novel bacterium belonging to the genus Paenibacillus having a high cellulose resolution and starch resolution. In addition, the present inventors have found that the bacterium produces an antibacterial substance having a broad antibacterial spectrum in the process of screening the bacterium. Furthermore, it has also been found that the antibacterial substance does not exhibit antibacterial activity against yeast. By co-culturing the yeast of the genus Paenibacillus and yeast, or by continuously cultivating it, while suppressing contamination by the antibacterial substance produced by the bacterium of the genus Paenibacillus, saccharification of cellulose and starch by the bacteria and by yeast Alcohol fermentation could be performed efficiently.
Furthermore, the present inventors have found that the bacterium is a solubilization of a poorly soluble phosphorus compound in the soil (supply of phosphorus to the plant), production of plant growth hormone, and removal of iron in the environment by siderophore. It has been found that it has a growth promoting effect on plants by inhibiting growth (plants can also produce siderophores), sterilizing phytopathogenic microorganisms with hydrogen cyanide and antibiotics, inhibiting biosynthesis of ethylene to suppress plant growth, nitrogen fixation, etc. .
Moreover, it discovered that the said bacteria which produce the antibacterial substance which has a wide antibacterial spectrum can be used also as a seed disinfectant, an agrochemical, a soil improvement agent, etc.

The present invention has been completed based on the above findings. That is, the present invention is as follows.
[1] (a) having cellulose resolution and starch resolution;
(B) exhibits antibacterial activity against fungi other than bacteria and yeast,
(C) A bacterium belonging to the genus Paenibacillus or a mutant thereof, which does not have gelatin degradability and nitrate reducing ability.
[2] The fungus other than bacteria and yeast is one or more bacteria belonging to any one of phytopathogenic fungi, Staphylococcus, Streptococcus, Enterococci, Escherichia and Fusarium, Bacteria or its mutants.
[3] The bacterium according to [1] or [2] or a mutant thereof, wherein the bacterium belonging to the genus Paenibacillus is Paenibacillus sp. FPU-37 strain (NITE P-01802).
[4] An antibacterial extract of the bacterium or variant thereof according to any one of [1] to [3].
[5] The antibacterial extract according to [4], produced using acetonitrile as an extraction solvent.
[6] The antibacterial extract according to [4], produced using a lower alcohol as an extraction solvent.
[7] An antibacterial composition containing the antibacterial extract according to any one of [4] to [6].
[8] A culture obtained by culturing the bacterium according to any one of [1] to [3] or a mutant thereof.
[9] A composition comprising the bacterium according to any one of [1] to [3] or a mutant thereof, and cellulose and / or starch.
[10] The composition according to [9], further comprising yeast.
[11] A kit for producing ethanol comprising the bacterium according to any one of [1] to [3] or a mutant thereof, yeast, and cellulose and / or starch.
[12] A method for producing cellulose and / or starch degradation product, comprising culturing the bacterium according to any one of [1] to [3] or a mutant thereof in a medium containing cellulose and / or starch. .
[13] The method according to [12], wherein the cellulose and / or starch degradation product is glucose.
[14] (a) The bacterium according to any one of [1] to [3] or a mutant thereof is cultured in a medium containing cellulose and / or starch, whereby cellulose and / or starch is converted into glucose. Converting,
(B) converting glucose into ethanol by culturing yeast in a medium containing glucose obtained in (a),
A method for producing ethanol comprising:
[15] A method for producing ethanol, comprising culturing the bacterium according to any one of [1] to [3] or a mutant thereof, and yeast in a medium containing cellulose and / or starch.
[16] A method for producing an antibacterial extract, comprising a step of obtaining an extract of the bacterium or variant thereof according to any one of [1] to [3] by extraction with acetonitrile.
[17] A method for producing an antibacterial extract, comprising a step of obtaining an extract of the bacterium or variant thereof according to any one of [1] to [3] by extraction with a lower alcohol.
[18] The bacterium according to any one of [1] to [3] or a mutant thereof, the culture according to [8], or the antibacterial extract according to any one of [4] to [7] Pesticides.
[19] The bacterium according to any one of [1] to [3] or a mutant thereof, the culture according to [8], or the antibacterial extract according to any one of [4] to [7] Seed disinfectant.
[20] A plant growth promoter containing the bacterium according to any one of [1] to [3] or the culture according to [8].
[21] A soil conditioner containing the bacterium according to any one of [1] to [3], the culture according to [8], or the antibacterial extract according to any one of [4] to [7]. .
[22] A method for controlling diseases caused by fungi other than bacteria or yeast, comprising a step of treating a plant with the agricultural chemical according to [18].
[23] A seed disinfection method including a step of treating seeds with the seed disinfectant according to [19].
[24] A method for promoting plant growth, comprising a step of treating a plant with the plant growth promoter according to [20].
[25] A soil improvement method comprising a step of treating soil with the soil improver according to [21].

  The present invention provides novel Paenibacillus bacteria that produce antibacterial substances with high cellulose and starch degradability and having a broad antibacterial spectrum. By using the bacterium, glucose can be produced from various raw materials including cellulose raw material or starch raw material having low edible value, and the glucose can be used as a raw material for ethanol production. Further, since the bacterium produces an antibacterial substance, it is possible to improve the efficiency of the ethanol production process, and the antibacterial substance itself can be usefully used as an agricultural material or a medicine.

FIG. 1 is a diagram showing a screening method for microorganisms having cellulose resolving power. FIG. 2 is a diagram showing a screening method for microorganisms having starch-degrading ability. FIG. 3 is a diagram showing the results of molecular phylogenetic analysis using a 16s rRNA gene base sequence of a newly identified microorganism belonging to the genus Paenibacillus. The microorganism is Paenibacillus sp. Shown as FPU-37. FIG. 4 is a view showing the expression induction of cellulase derived from FPU-37 strain by cellulose and its localization. FIG. 5 is a view showing the induction of expression of amylase derived from FPU-37 strain by starch and its localization. FIG. 6 is a diagram showing the antibacterial activity of FPU-37 strain against various phytopathogenic fungi. FIG. 7 is a graph showing that the extract of FPU-37 strain with acetonitrile has antibacterial activity and that the production of a substance exhibiting the antibacterial activity is induced by addition of crystalline cellulose and fungi. FIG. 8 is a diagram showing an antibacterial spectrum of an extract of FPU-37 strain with acetonitrile or butanol. FIG. 9 is a diagram showing the results of barley alcohol fermentation test using FPU-37 strain and yeast. FIG. 10: is a figure which shows the growth promotion effect with respect to the seedling of rice (Koshihikari) by FPU-37 strain | stump | stock. FIG. 11 is a diagram showing the growth promoting effect on tomato seedlings by FPU-37 strain. (The effect was compared with the soil bacteria 10-1 strain and IK-5 strain, which had already been confirmed to have a growth promoting effect) FIG. 12 is a diagram showing the results of a solubilization ability test of a poorly soluble phosphate with an organic acid of FPU-37 strain. On Pikovskaya'Agar, clear dissolution spots were formed around the cells of the FPU-37 strain, confirming the ability to solubilize poorly soluble phosphorus compounds. FIG. 13 is a diagram showing the results of a siderophore production test of FPU-37 strain. Since clear disappearance of blue decoloration was confirmed on the siderophore assay medium, the siderophore production ability of the FPU-37 strain was confirmed. FIG. 14 is a diagram showing the high hydrogen cyanide (HCN) production ability of the FPU-37 strain. FIG. 15 is a diagram showing the results of verifying the chitin degradation activity of the FPU-37 strain. Since no halo was formed on the chitin-containing medium, it was determined that the FPU-37 strain was not capable of degrading chitin.

The present invention
(A) having cellulose resolution and starch resolution;
(B) exhibits antibacterial activity against fungi other than bacteria and yeast,
(C) not having gelatin resolution and nitrate reducing ability,
Provided is a bacterium belonging to the genus Paenibacillus or a mutant thereof.

  The compounds described herein also include all isomers thereof.

The bacterium belonging to the genus Paenibacillus of the present invention (hereinafter sometimes abbreviated as the bacterium of the present invention) or a variant thereof can degrade cellulose and starch into monosaccharides or oligosaccharides. That is, the bacterium of the present invention or a mutant thereof has an activity of saccharifying cellulose and starch. The degradation of cellulose by the bacterium of the present invention or a mutant thereof can be an endo degradation mode in which the inside of the cellulose molecule is randomly cleaved, or an exo degradation mode in which cellobiose is produced by cleaving from the end of the cellulose molecule. The degradation reaction may take place inside the cell or outside the cell. As examples of the degradation products, glucose that is a monosaccharide, cellobiose that is a disaccharide, and oligosaccharides of 3 to 6 sugars can be produced. The degradation product is preferably glucose, cellobiose or trisaccharide, and more preferably glucose.
In addition, the degradation of starch is an endo-type degradation mode in which its constituent molecules, amylose molecules and amylopectin molecules, are cleaved at random, and / or exo-type that cleaves from the end of amylose and amylopectin molecules to produce maltose or glucose. It can be a decomposition mode. Amylase showing endo-type degradation activity is called α-amylase, amylase showing exo-type degradation activity and producing maltose is called β-amylase, and amylase showing exo-type degradation activity and producing glucose is called glucoamylase. As will be described later, since the amylase produced by the bacterium of the present invention has both the catalytic domain of α-amylase and the catalytic domain of β-amylase, preferred degradation modes include endo-type degradation mode and exo-type degradation mode. The decomposition mode which produces maltose by decomposition | disassembly of is illustrated. Furthermore, as a preferable starch decomposition mode of the present invention, a maltose is produced from starch by the amylase of the present invention, and then the maltose is decomposed by α-glucosidase to produce glucose. These starch degradation reactions may occur inside cells or outside cells. Examples of the degradation products of starch include glucose, which is a monosaccharide, maltose, which is a disaccharide, and oligosaccharides having 3 to 6 sugars. The degradation product is preferably glucose, maltose or trisaccharide, and more preferably glucose.

  A representative example of the cellulase possessed by the bacterium of the present invention or a variant thereof is a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 1, and a nucleotide sequence represented by SEQ ID NO: 2 as an example of a polynucleotide encoding the same. The polynucleotide containing is mentioned. The cellulase gene of SEQ ID NO: 2 shows a high homology with Paenibacillus polymyxa β-1.4-glucanase, a catalytic domain (GH5) classified as a family 5 of carbohydrate hydrolases, N-terminal It has a secretory signal sequence on the side and a family 3 (CBM3) domain of carbohydrate-binding module (CBM) which is a carbohydrate binding module on the C-terminal side. The cellulase produced by the bacterium of the present invention or a mutant thereof can be a substrate-induced secretory enzyme whose expression is induced by addition of cellulose and secreted into the culture supernatant.

  A representative example of the amylase possessed by the bacterium of the present invention or a variant thereof is a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 3, and a nucleotide sequence represented by SEQ ID NO: 4 as an example of a polynucleotide encoding the same. The polynucleotide containing is mentioned. The amylase gene of SEQ ID NO: 4 shows high homology with Paenibacillus polymixa β / α-amylase and has a secretory signal sequence on the N-terminal side. Further, the polypeptide comprising the amino acid sequence represented by SEQ ID NO: 3 has a β-amylase catalytic domain (GH14) and two CBM25s on the N-terminal side, and an α-amylase catalytic domain (C-terminal side). GH13) and Aami-C module, which is a kind of CBM, are considered to exhibit both α-amylase activity and β-amylase activity. The amylase polypeptide possessed by the bacterium of the present invention or a variant thereof has an α, 1, 2, 3 or 4 amino acid substitution, insertion, or deletion made in the amino acid sequence represented by SEQ ID NO: 3. -Also provided are polypeptides having amylase activity and β-amylase activity, and polynucleotides encoding the same. The amylase produced by the bacterium of the present invention can be a substrate-induced secretory enzyme whose expression is induced by addition of starch and secreted into the culture supernatant.

  A representative example of the α-glucosidase possessed by the bacterium of the present invention or a variant thereof is a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 5, and an example of a polynucleotide encoding it is represented by SEQ ID NO: 6. A polynucleotide comprising a nucleotide sequence is mentioned. The α-glucosidase gene of SEQ ID NO: 6 is an enzyme classified into the glycolytic enzyme family 31 (GH31). The α-glucosidase produced by the bacterium of the present invention may be an enzyme localized in a cell.

  The cellulase, amylase, and α-glucosidase polynucleotides and polypeptides possessed by the bacterium of the present invention or variants thereof are useful as markers for the bacterium of the present invention or variants thereof. The cellulase, amylase and α-glucosidase polynucleotides possessed by the bacterium of the present invention or a variant thereof can be used for preparing a nucleic acid probe for detecting the bacterium of the present invention or a variant thereof. Cellulase, amylase and α-glucosidase polypeptides possessed by the bacterium of the present invention or a variant thereof can be used for preparing an antibody for detecting the bacterium of the present invention or a variant thereof. The probe and antibody can be suitably used to identify the bacterium of the present invention or a mutant thereof.

  Furthermore, the present invention also provides a vector comprising a polynucleotide encoding a cellulase, amylase or α-glucosidase polypeptide possessed by the bacterium of the present invention or a mutant thereof, and a transformant transformed with the vector. obtain.

  The bacterium of the present invention or a mutant thereof exhibits antibacterial activity against other bacteria and fungi other than yeast. The other bacteria and fungi other than yeast are inhibited from being killed or grown by the antibacterial activity exhibited by the bacterium of the present invention or a mutant thereof. Specific examples include phytopathogenic microorganisms and human pathogenic microorganisms (eg, Staphylococcus, Streptococcus, Enterococcus, Escherichia, Trichophyton, Candidiasis, Cryptococcosis, and Fusarium. More specifically, tomato leaf mold (Fulvia fulvum (Cooke) Ciferri), rice blast fungus (Magnaporthe grisea), rice blast fungus (Magnaporthe oryzae), omrae ), Strawberry anthracnose fungus (Colletotrichum acutatum), tomato dwarf fungus (Fusarium oxysporum Schechtendal f. Sp. Lycopersici), taro dry rot fungus (Fusarium oxysporum Schechtendal f. Sp. Colocasiae), cucumber vinesporium fungus (i) boiled rice fungus (Fusarium oxysporum f. , Soybean purpura fungus (Cercospora kikuchii), bacterial wilt fungus (Ralstonia solanacearum (formerly known as Pseudomonas solanacearum)), and soft rot (Erwinia carotovora subsp.) Bacteria (Staphylococcus aureus), Staphylococcus epidermidis, and Staphylococcus Staphylococcus bacteria such saprophyticus, Streptococcus pyogenes, Streptococcus pneumoniae, Streptococcus viridans, Streptococcus agalactiae, and Streptococcus dysgalactiae like streptococcal bacteria, Enterococcus faecalis, Enterococcus faecium, Enterococcus avium, Enterococcus caseiflavus, Enterococcus gall Narum, and Enterococcus flavescens like enterococci, E. (Escherichia coli) Esukerikia genus bacteria such as, Trichophyton rubrum, Trichophyton mentagrophytes, Trichophyton tonsurans, Microsporum canis, Microsporum gypseum, ringworm fungi such as Trichophyton verrucosum, candidiasis, such Candida albicans Causative bacteria, Cryptococcus neoformans and other cryptococcosis-causing bacteria, Neisseria gonorrhoeae and other Neisseria meningitidis such as Neisseria meningitidis, Shige la dysenteriae, Shigella flexneri, Shigella boydii, dysentery bacteria such as Shigella sonnei, Salmonella Typhi, Salmonella Paratyphi, Salmonella Typhimurium, such as Salmonella Enteritidis Salmonella, cholera bacteria such as Vibrio cholerae, Pseudomonas aeruginosa, such as Pseudomonas aeruginosa, such as Bordetella pertussis Bordetella pertussis, Haemophilus influenzae and other influenza bacteria, Mycobacterium tuberculosis and other tuberculosis bacteria, Clostridium tetani and other tetanus bacteria, Coryneba terium diphtheriae such diphtheriae and Fusarium oxysporum,, Fusarium solani, Fusarium graminearum, Fusarium asiaticum, Fusarium culmorum, and Fusarium fujikuroi (full (sexual) generation: Gibberella fujikuroi) Fusarium genus fungi, and the like. The bacterium of the present invention or a variant thereof has antibacterial activity, preferably against at least one selected from these bacteria.

  In the present specification, the “antibacterial activity” means an activity of killing bacteria and an activity of suppressing the growth of the bacteria.

  Unlike the related species Paenibacillus polymyxa, Paenibacillus jamilae, Paenibacillus peeriae, and Paenibacillus kribbensis, the bacterium of the present invention or a mutant thereof does not have gelatin resolution and nitrate reducing ability. Gelatin resolution means the ability to dissolve solidified gelatin at room temperature when bacteria are inoculated into a medium containing gelatin. The nitrate reducing ability means the ability to cultivate bacteria in a medium containing nitrate and produce nitrite by a reduction reaction. A person skilled in the art can easily test the gelatin resolution and nitrate reducing ability by a known method. Alternatively, a commercially available kit for testing gelatin degradation and nitrate reducing ability (for example, API20E kit (bioMerieux, Lyon, France)) and the like can be suitably used. One feature of the bacterium of the present invention is that it does not have gelatin degradability and nitrate reducing ability.

Other examples of the characteristics of the bacterium of the present invention or mutants thereof include the following.
-Cell shape is rod-shaped, has peripheral flagella, and has active motility.
-The colony has a cream color close to white (slightly different depending on the culture medium).

  In a further aspect, the bacterium of the present invention is capable of solubilizing poorly soluble phosphorus compounds in soil (supplying phosphorus to plants), producing plant growth hormone, and causing plant pathogens by depriving the environment of iron by siderophores. It has the ability to suppress the growth of microorganisms (plants can also produce siderophores), the ability to kill phytopathogenic microorganisms with hydrogen cyanide and antibiotics, the ability to inhibit the biosynthesis of ethylene to suppress plant growth, and the ability to fix nitrogen.

(Solubility of poorly soluble phosphorus compounds)
The bacterium of the present invention can solubilize poorly soluble phosphorus compounds.
Phosphorus is one of the essential elements of plants and plays an important role as a constituent element such as nucleic acid, phospholipid, NADP, ATP and the like. Some of the bacteria contained in the soil can solubilize poorly soluble phosphorus compounds such as calcium phosphate, aluminum phosphate, and iron phosphate contained in the soil with an organic acid. Although not bound by theory, it is considered that the bacterium of the present invention can also solubilize poorly soluble phosphorus compounds and supply solubilized phosphorus to plants, and thus has a plant growth promoting action.

(Plant growth hormone production ability)
The bacterium of the present invention can produce indole-3-acetic acid (IAA), which is a plant growth hormone. Indole-3-acetic acid (IAA) is one of the plant growth hormones (auxin). Without being bound by theory, it is considered that the bacterium of the present invention has an effect of promoting plant growth by producing IAA.

(Siderophore production capacity)
A siderophore is an iron chelator secreted by bacteria and fungi. Siderophores function by binding to trace amounts of iron in the environment. Without being bound by theory, it is believed that the bacterium of the present invention can suppress the growth of phytopathogenic microorganisms by producing siderophores and depriving the environment of iron.

(Production capacity of hydrogen cyanide)
The bacterium of the present invention produces hydrogen cyanide (HCN). Without being bound by theory, it is considered that the production of hydrogen cyanide (HCN) sterilizes phytopathogenic microorganisms and thus has an effect of promoting plant growth.

(Ethylene biosynthesis inhibiting ability)
Ethylene is synthesized through the route of methionine → S-adenosylmethionine (SAM) → 1-aminocyclopropane-1-carboxylic acid (ACC) → ethylene. The bacterium of the present invention has an ACC deaminase gene, and the growth rate is increased by the addition of ACC.
In general, ethylene inhibits growth and suppresses flower bud formation and budding. Without being bound by theory, it is considered that the bacterium of the present invention has an action of degrading ACC, and therefore can promote plant growth.

(Nitrogen fixing ability)
Nitrogen fixation refers to a process in which stable nitrogen molecules present in large amounts in air are converted into inorganic nitrogen compounds such as ammonia, nitrate, and nitrogen dioxide. The bacterium of the present invention has a nitrogenase (flavodoxin) gene and can grow on a nitrogen-free medium.
Therefore, without being bound by theory, it is considered that the bacterium of the present invention has a nitrogen fixing ability and can supply a nitrogen compound into the soil, so that it can promote the growth of plants.

  Another characteristic of the bacterium of the present invention is that it does not have a chitinolytic activity. The bacterium of the present invention does not have a chitinase (chitinase) gene and does not form a halo when cultured on a chitin-containing medium.

  In a preferred embodiment, the bacterium of the present invention is Paenibacillus sp. FPU-37 strain represented by accession number NITE P-01802. The bacteria have been deposited with the Patent Microorganism Depositary Center of the National Institute of Technology and Evaluation (trusted date: February 20, 2014).

  The bacterium of the present invention or a variant thereof is preferably an isolated bacterium. “Isolated” means that the bacteria have been separated from the natural material and thus have a higher purity than if they existed in nature. When the bacterium of the present invention or a mutant thereof is isolated, the purity thereof is 50% or more, preferably 80% or more, more preferably 90% or more, with respect to the total number of bacteria contained. Particularly preferred is 95% or more, and most preferred is 100%.

  The bacterium of the present invention may be a mutant of the bacterium of the present invention. The mutant is not particularly limited as long as it is a strain derived from the bacterium of the present invention. For example, morphological characteristics, staining, growth, auxotrophy, genetic characteristics, physiological characteristics, and biochemistry It may be a bacterial strain that exhibits one or more similar properties to the bacterium of the present invention in terms of its characteristic characteristics and retains the desired properties of the bacterium of the present invention. The desired properties include cellulase degradation, starch degradation, antibacterial activity, plant growth promoting action, solubilization ability of poorly soluble phosphorus compounds, indole-3-acetic acid (IAA) production ability, siderophore production ability, hydrogen cyanide (HCN) production Ability, ethylene biosynthesis inhibition ability, nitrogen fixation ability. Alternatively, the mutant introduces a gene derived from the bacterium of the present invention (eg, the cellulase and amylase genes of the bacterium of the present invention) necessary for exerting the desired property into another host cell. Can also be produced.

The mutant of the bacterium of the present invention can be obtained by subjecting the bacterium of the present invention to a known mutagen treatment and selecting a strain having a desired effect equivalent to or higher than that of the parent strain. The cellulase resolution can be tested using, for example, a method described in Examples described later, but is not limited thereto, and any method known per se may be used.
The mutant of the present invention can be produced, for example, by modifying the bacterium of the present invention. Examples of such modification treatment include culture in the presence of a mutagenic substance, introduction of a gene that can enhance the usefulness of the bacterium of the present invention, and a gene that can enhance the usefulness of the bacterium of the present invention (eg, Indole-3-pyruvic acid (IPA) biosynthetic gene group, Siderophore biosynthetic gene, Fe ³⁺ -siderphore ABC transporter permease gene, 1-aminocyclopropane-1-minboxylate (ACC) gene, (A) ), Antibiotic production system gene group, gene modification such as enhancement of ribosome function for the purpose of enhancing the biosynthesis ability of proteins such as enzymes, etc., and possessed by the bacterium of the present invention Disruption of that gene, as well as a combination of these operations. The present invention also provides such a production method.
Examples of mutagens include alkylating agents (N-methyl-N′-nitro-N-nitrosoguanidine (NTG), ethylmethanesulfonic acid (EMS), etc.), nucleotide base analogs (bromouracil, etc.), Examples include, but are not limited to, nitroso compounds, DNA intercalators, DNA cross-linking agents, radiation, and ultraviolet rays. Preferably, the mutant has an increased desired effect (eg cellulase degradation, starch degradation, antibacterial activity or growth promoting action) compared to the parent strain, or is equivalent in terms of their effects, eg Strains that can grow in a wide pH range, have a high growth rate, and can grow even at low temperatures.

Whether or not the obtained mutant produces an antibacterial substance can be verified by a method known per se.
For example, it is possible to test by placing a paper disc spotted with a mutant culture medium and an unspotted paper disc on an AM agar medium, inoculating a phytopathogenic fungus into each, and culturing at 28 ° C. When the number of the phytopathogenic fungi on the paper disc spotted with the culture medium of the mutant is small compared to the paper disc on which nothing is spotted, the mutant has antibacterial activity against the phytopathogenic fungus.

Whether or not the obtained mutant has a plant growth promoting action can be verified by a method known per se.
For example, whether or not a mutant can solubilize a poorly soluble phosphorus compound is determined by, for example, culturing on a Pikovskaya agar medium in which calcium phosphate is suspended, forming colonies, and forming a halo-zone. It can be tested by examining the presence or absence. In the case of a mutant capable of solubilizing a poorly soluble phosphorus compound, a halo (zone) is formed around the mutant colony by dissolution of calcium phosphate.

Whether a mutant can produce indole-3-acetic acid (IAA) can be assessed, for example, by the salkowski's test.
Whether or not a mutant produces a siderophore can be tested, for example, by a color reaction using a Chrome Azurol S-iron complex.
Whether a mutant produces hydrogen cyanide (HCN) can be tested, for example, with a picric acid-sodium carbonate test paper.
Whether or not the mutant inhibits biosynthesis of ethylene can be evaluated, for example, by measuring ACC deaminase activity.
Whether or not the mutant is nitrogen-fixed can be evaluated, for example, by measuring nitrogenase activity.

The bacterium of the present invention or a mutant thereof can be cultured by a method known per se used for culturing Paenibacillus bacteria in general. A person skilled in the art can appropriately select a medium to be used for culture. Examples of the medium include M9 medium (minimum medium), KL medium, BHI medium, 2YT medium, YPD medium, AM3 medium, and LB medium. Can be mentioned. Since the bacterium of the present invention has a nitrogen fixing ability, it can also be cultured in a medium containing no nitrogen. The culture time, culture scale, etc. can be appropriately set according to the use of the culture. The culture temperature is preferably 15 to 40 ° C, more preferably 28 to 37 ° C, and most preferably 37 ° C. The medium is preferably adjusted to pH 3-10.
The bacterium of the present invention or a mutant thereof can be cultured, for example, by placing a certain amount of medium in a culture vessel such as a test tube or culture flask, inoculating the bacterium of the present invention or a mutant thereof, It can be carried out by shaking culture at 30 to 42 ° C. under aerobic conditions using a calshaker, a rotary shaker or the like. Larger scale culture can be carried out by aeration and agitation culture using a large-scale culture tank such as a jar fermenter of several liter scale or an industrial tank of several hundred liter to several hundred t scale. The culture time is not particularly limited.

  The present invention provides an antibacterial extract of the bacterium of the present invention or a variant thereof. The antibacterial extract may be extracted by any method as long as it is derived from the bacterium of the present invention or a mutant thereof. In the method, for example, a culture solution containing the bacterium of the present invention or a mutant thereof is subjected to centrifugation, the bacterium is pelleted, and a solution obtained by resuspending the bacterium in an organic extraction solvent such as acetonitrile or lower alcohol is antibacterial. Extract, or suspended in an organic extraction solvent such as acetonitrile or lower alcohol and then subjected to further centrifugation, and the supernatant can be used as an antibacterial extract. In the present specification, “lower alcohol” means an alcohol having 1 to 5 carbon atoms, and preferred lower alcohols include methanol, ethanol, propanol, butanol and pentanol, and more preferably butanol.

  The antibacterial extract contains an antibacterial substance, so that phytopathogenic microorganisms and human pathogenic microorganisms (for example, Staphylococcus bacteria, Streptococcus bacteria, Enterococci, Escherichia bacteria, ringworm fungi, candidiasis More specifically, tomato leaf mold (Fulvia fulvum (Cooke) Ciferri), rice blast fungus (Magnaporthe grisea), rice blast fungus (Magnegorp), etc. Barley red mold fungus (Fusarium gramminearum), strawberry anthracnose fungus (Colletotrichum acutatum), tomato wilt disease fungus (Fusarium oxysporum Schechtendal f. Sp. Lycopersic) i) Fusarium oxysporum Schechtendal f. sp. Colocasiae, Fusarium oxysporum f. sp. Watermelon anthracnose fungus (Colletotrichum orbiculare), soybean purpura fungus (Cercospora kikuchii), bacterial wilt fungus (Ralstonia solanacearum) (formerly known as Pseudomonas solanacerum), and soft rot aero tora vora cerato v. Bacteria fungi, Staphylococcus aureus (Staphylococcus aureus), Staphylococcus epidermidis, and Staphylococcus Staphylococcus bacteria such saprophyticus, Streptococcus pyogenes, Streptococcus pneumoniae, Streptococcus viridans, Streptococcus agalactiae, and Streptococcus dysgalactiae like streptococcal bacteria, Enterococcus faecalis, Enterococcus faecium, Enterococcus avium, Enterococcus casseriflavus, Entero coccus gallinarum, and Enterococcus Enterococcus such flavescens, E. (Escherichia coli) Esukerikia genus bacteria such as, Trichophyton rubrum, Trichophyton mentagrophytes, Trichophyton tonsurans, Microsporum canis, Microsporum gypseum, ringworm fungi such as Trichophyton verrucosum, Candida, such as Candida albicans Disease-causing bacteria, cryptococcosis-causing bacteria such as Cryptococcus neoformans, Neisseria gonorrhoeae and other Neisseria meningitidis, etc. Neisseria meningitidis, Shigella dysenteriae, Shigella flexneri, Shigella boydii, Shigella, such as Shigella sonnei, Salmonella Typhi, Salmonella Paratyphi, Salmonella Typhimurium, Salmonella, such as Salmonella Enteritidis, cholera bacteria such as Vibrio cholerae, Pseudomonas aeruginosa, such as Pseudomonas aeruginosa Bordetella pertussis, etc. Bordetella pertussis, Haemophilus influenzae and other influenza bacteria, Mycobacterium tuberculosis and other tuberculosis bacteria, Clostridium tetani etc. Fungi, diphtheria such as Corynebacterium diphtheriae, and selected from Fusarium oxysporum, Fusarium solani, Fusarium graminearum, Fusarium asiaticum, Fusarium curum, and Fusarium curum. Has antibacterial activity against one type of bacteria.

  In addition, the antibacterial extract can exhibit antibacterial activity against different bacteria depending on the type of solvent used for extraction. For example, an antibacterial extract extracted using acetonitrile may have antibacterial activity against phytopathogenic fungi and fungus belonging to the genus Fusarium, and an antibacterial extract extracted using lower ethanol May have antibacterial activity against bacteria, Streptococcus, Enterococci, and Escherichia.

  Since the antibacterial extract exhibits antibacterial activity against a wide range of bacteria and fungi other than yeast, it can be suitably used for agricultural chemicals, pharmaceuticals, seed disinfectants, soil improvers and the like.

  When the antibacterial extract is used as an agrochemical, the target crops are, for example, rice, barley, tomato, watermelon, cucumber, strawberry, taro, soybean, wheat, corn, potato, kidney bean, pea, peanut, Sugar beet, sugar cane, eggplant, pepper, melon, pumpkin, radish, cabbage, cabbage, komatsuna, onion, leek, leek, garlic, asparagus, burdock, lettuce, garlic, carrot, spinach, okra, apple, pear, ume, grape , Oysters, figs, kiwi and the like.

  When the antibacterial extract is used as a medicine, it is orally or parenterally administered to humans and other mammals (eg, rats, rabbits, sheep, pigs, cows, cats, dogs, monkeys, etc.) It can be safely administered. The medicament may have a therapeutic effect against bacterial and fungal infections. Specific examples of such infections include infection with staphylococcal bacteria, streptococcal bacteria, enterococci, escherichia bacteria, ringworms, candidiasis bacteria, cryptococcosis bacteria, or fusarium bacteria. Examples include infectious diseases that develop.

  In one aspect, the present invention provides an antibacterial composition containing the antibacterial extract. As long as the antibacterial composition contains the antibacterial extract of the present invention as an active ingredient, other polysaccharides, oligosaccharides, glycoproteins, peptides, proteins, nucleic acids, which do not impair the effect of the antibacterial extract, Components such as lipids, inorganic compounds, organic compounds, and minerals may be contained. In addition, the antibacterial composition may further contain a physiologically acceptable carrier such as a diluent, an excipient, a stabilizer, and a preservative as long as the desired effect is not impaired. The content of the antibacterial extract of the present invention contained in the antibacterial composition can be adjusted as appropriate according to the extraction method, use, etc., but is 0.1 to 100 (w / w)% as an example. obtain.

  Since the antibacterial composition of the present invention exhibits a broad antibacterial spectrum, it can be suitably used as agricultural chemicals such as bactericides, pharmaceuticals such as antibiotics, seed disinfectants, soil improvers and the like. Alternatively, the bacterium of the present invention or a mutant thereof can be used as a biopesticide, a seed disinfectant, a soil conditioner, etc. without performing the above extraction operation.

  When the antibacterial composition of the present invention is used as a pesticide, and when the bacterium of the present invention or a mutant thereof is used as a biopesticide, target crops include, for example, rice, barley, tomato, watermelon, cucumber, Strawberry, taro, soybean, wheat, corn, potato, kidney bean, pea, peanut, sugar beet, sugar cane, eggplant, pepper, melon, pumpkin, radish, cabbage, cabbage, komatsuna, onion, leek, leek, garlic, asparagus, burdock , Lettuce, garlic, carrot, spinach, okra, apple, pear, ume, grape, oyster, fig, kiwi and the like.

  When the antimicrobial composition of the present invention is used as a pharmaceutical, it is orally or parenterally administered to humans and other mammals (eg, rats, rabbits, sheep, pigs, cows, cats, dogs, monkeys, etc.) Can be administered safely. The medicament may have a therapeutic effect against bacterial and fungal infections. Specific examples of such infections include infection with staphylococcal bacteria, streptococcal bacteria, enterococci, escherichia bacteria, ringworms, candidiasis bacteria, cryptococcosis bacteria, or fusarium bacteria. Examples include infectious diseases that develop.

  In another aspect, the present invention provides a culture obtained by culturing the bacterium of the present invention or a mutant thereof. In the present specification, “culture” means cultured cells and culture supernatant.

  The culture is useful for producing the antibacterial extract and antibacterial composition of the present invention described above. Since it is considered that the culture also contains α-glucosidase, it can be suitably used for the purpose of degrading maltose to produce glucose. The culture can be used in the method for producing the cellulose and / or starch degradation product of the present invention, the method for producing ethanol of the present invention, and the method for producing the antibacterial extract of the present invention, which will be described later. Since the bacterium of the present invention or a mutant thereof releases α-glucosidase in the culture supernatant by autolysis by long-term culture, the culture supernatant is used to decompose maltose and produce glucose. You can also.

  Moreover, since it is thought that the culture of this invention contains the cellulase and amylase produced by the bacterium of this invention, or its variant, it can be used suitably for saccharification of a cellulose or starch.

  A person skilled in the art can appropriately select the medium used for the culture. Examples of the medium include M9 medium (minimum medium), BHI medium, 2YT medium, YPD medium, AM3 medium, LB medium, and the like. . The culture time, culture scale, etc. can be appropriately set according to the use of the culture. The culture temperature is preferably 15 to 40 ° C, more preferably 28 to 37 ° C, and most preferably 37 ° C. The medium is preferably adjusted to pH 3-10.

The culture obtained by culturing the bacterium of the present invention or a mutant thereof is, for example, an appropriate bacterial cell concentration in water or an appropriate diluent (eg, isotonic buffer or medium). Can be diluted. Alternatively, the culture can be filtered or centrifuged to recover the cells and resuspended in a suitable dispersion medium (eg, isotonic buffer or fresh medium). Furthermore, the recovered cells can be freeze-dried by a conventional method. Alternatively, 10-20% glycerol can be added to the culture and stored frozen at −80 ° C., thawed at the time of use, and resuspended in a medium or the like. In addition, the bacteria form spores (endospores), which are resistant to environmental changes such as heat and dryness and can be stored stably for a long period of time. Bacteria may be grown.
On the other hand, when an effective substance that provides a desired effect is secreted outside the microbial cells (culture supernatant), the culture supernatant can be used as it is or after being concentrated or diluted.

(Pesticide)
As one embodiment of the present invention, the present invention provides an antibacterial extract of the present invention, an antibacterial composition, a bacterium of the present invention or a mutant thereof, or an agrochemical containing the culture thereof (herein, the present invention). (Also called pesticides). The agrochemical of the present invention has a control effect against diseases caused by phytopathogenic microorganisms in which the antibacterial extract, antibacterial composition or bacterium or variant thereof of the present invention has antibacterial activity.
Examples of crops that are targets of the agricultural chemical of the present invention are as described above, but are not limited to the above as long as they have a desired effect.

As long as it has a desired effect, the method for treating agricultural products with the agricultural chemical of the present invention is not particularly limited. For example, the agricultural chemical of the present invention diluted with an aqueous solvent may be sprayed on a target crop with a sprayer, and the agricultural chemical of the present invention is directly mixed in a soil where the target agricultural product is grown or suspended in water or the like. Irrigation treatment may be performed using the agrochemical of the present invention.
When a crop is treated with the agrochemical of the present invention, the crop may be directly treated with the agrochemical of the present invention, and a substance that can contact the crop with the agrochemical of the present invention (eg, agricultural water, root permeation sheet, etc.) May be processed indirectly. Furthermore, in one embodiment, a substance that can come into contact with crops can be used after the treatment with the pesticide of the present invention, followed by removal of the pesticide of the present invention.
When treating agricultural products by spraying the agricultural chemical of the present invention, the amount of the agricultural chemical is preferably 100 to 500 ml if spraying is liquid per about 5 liters of soil used, and the bacterial cell concentration is 1 ml spray liquid. Usually 10 ³ to 10 ⁶ cfu.
The timing for treating the crop with the agricultural chemical of the present invention is not particularly limited as long as it has a desired effect, and can be used at any timing.
There is no restriction | limiting in particular with respect to the application frequency of the agrochemical of this invention. Before and after the plant is treated with the agricultural chemical of the present invention, the plant can be cultivated by a usual method.

  When the mutant of the bacterium of the present invention is used for the agricultural chemical of the present invention, the mutant is preferably a bacterium that produces an antibacterial substance.

(Seed disinfectant)
As one embodiment of the present invention, the present invention provides a seed disinfectant containing the antibacterial extract, antibacterial composition of the present invention, the bacterium of the present invention or a variant thereof, or a culture thereof (herein, the present invention The seed disinfectant of the invention is also provided. The seed disinfectant of the present invention has a control effect on the phytopathogenic microorganisms of which the antibacterial extract, antibacterial composition or bacterium of the present invention has antibacterial activity.

  The seed disinfectant of the present invention can be used for disinfecting seeds, seed meal, seed pods, bulbs and the like.

The seed disinfectant of the present invention can be used by a conventionally known seed disinfecting method as long as the desired effect is not impaired.
Methods for disinfecting seeds include, but are not limited to, wet dressing, dipping, spraying, and smearing. In one embodiment, the seed disinfectant of the present invention is used in a dipping method in which seeds are immersed in a liquid liquid of the seed disinfectant of the present invention. As another embodiment, the seed disinfectant of the present invention in powder form is used for a powder coating method in which seeds are applied. The dipping method can be used both in the seed soaking pretreatment and in the seed germination treatment. The powder coating method can be used for dried seeds. In another embodiment, the seed disinfectant of the present invention can be used by attaching the seed disinfectant of the present invention to a seed whose surface has been moistened.

When seed disinfection is performed, it is preferable to apply 2 to 50% by mass of the preparation with respect to the seed mass, and the bacterial cell concentration is usually 10 ³ to 10 ⁹ cfu per 1 g of seed mass.
When seed sterilization is performed by the dipping method, for example, seed sterilization can be performed by infiltrating seeds of the present invention for 0.5 to 48 hours, preferably 1 to 24 hours.

  When the mutant of the bacterium of the present invention is used for the seed disinfectant of the present invention, the mutant is preferably a bacterium that produces an antibacterial substance.

(Plant growth promoter)
As one embodiment of the present invention, a plant growth promoter (also referred to as a plant growth promoter of the present invention in the present specification) comprising the bacterium of the present invention or a mutant thereof or a culture thereof is provided.
The bacterium of the present invention or a mutant thereof or a culture thereof is obtained by solubilizing poorly soluble phosphorus compounds in soil (supplying phosphorus to plants), producing plant growth hormones, and depriving the environment of iron by siderophores. Has the effect of promoting the growth of plants by suppressing the growth of plant pathogenic microorganisms (plants can also produce siderophores), sterilizing plant pathogenic microorganisms with hydrogen cyanide and antibiotics, inhibiting the biosynthesis of ethylene to suppress plant growth, and fixing nitrogen .

  Examples of plants that can be targeted by the plant growth promoter of the present invention include solanaceous plants (tomatoes, eggplants, potatoes, tobacco, peppers, peppers, etc.), legumes (green beans, soybeans, groundnuts, cowpeas, azuki beans, etc.), rapes. Family plants such as Chinese cabbage, turnip, cabbage, radish, rape, etc., Gramineae plants (rice, wheat, corn, barley, rye, sugarcane, etc.), Cucurbitaceae plants (cucumber, melon, watermelon, pumpkin, eugan, etc.), lily Citrus plants (tulips, lilies, onions, asparagus, leeks, leek, garlic, etc.), citrus plants (especially citrus fruits: citrus: yuzu, lemon, buntan, iyokan, hassaku, grapefruit, etc.) Preferred examples include solanaceous plants and gramineous plants. , But it is not limited to these.

As long as it has a desired effect, the method for treating plants with the plant growth promoter of the present invention is not particularly limited. For example, the plant growth promoter of the present invention diluted with an aqueous solvent may be sprayed on the target plant with a sprayer, and the plant growth promoter of the present invention is directly mixed in the soil where the target plant grows. Irrigation treatment may be performed using the plant growth promoter of the present invention suspended in water or the like.
When the plant growth promoting treatment is carried out by directly mixing the plant growth promoter into the soil, it is preferable to apply 100 to 500 ml if the spraying is liquid per about 5 liters of the soil used. Usually 10 ³ to 10 ⁹ cfu, preferably 10 ⁴ to 10 ⁸ cfu per ml of liquid.

  There is no restriction | limiting in particular with respect to the application frequency of the plant growth promoter of this invention. Before and after using the plant growth promoter of the present invention, the plant can be cultivated by a usual method.

  When the mutant of the bacterium of the present invention is used for the plant growth promoter of the present invention, the mutant is a solubilizing ability of a poorly soluble phosphorus compound, an ability to produce a plant growth hormone, an ability to produce a siderophore, an ability to produce hydrogen cyanide, Preferably, the bacterium has at least one ability selected from the group consisting of the ability to inhibit ethylene biosynthesis, the ability to fix nitrogen, and the ability to produce an antibacterial substance, more preferably two or more, and even more preferably three. As described above, a bacterium having 7 abilities is most preferable.

(Soil conditioner)
As one embodiment of the present invention, a soil conditioner (in the present specification, the soil conditioner of the present invention) comprising the antibacterial extract, the antibacterial composition of the present invention, the bacterium of the present invention or a mutant thereof or a culture thereof Also referred to as an agent).
Since the bacterium of the present invention or a mutant thereof or a culture thereof has a growth promoting effect on plants and an antibacterial action, it can be used as a soil conditioner. Since the antibacterial extract and antibacterial composition of the present invention also have an antibacterial action, they can be used as a soil conditioner.
In the present specification, the soil includes a solid medium such as soil used for soil cultivation, a liquid medium used for hydroponics or the like, or a semi-fluid medium. In the present specification, the soil improver refers to an agent having an action of improving physical or chemical properties of soil. The soil improver of the present invention can be used for any of soil for soil cultivation, soil for hydroponics, and soil for hydroponics (hydroponics, spray cultivation, solid culture).
Examples of soil improvement include, but are not limited to, improvement to soil with low pathogenicity, improvement to soil having growth promoting action, and improvement to soil having antiseptic action.

As long as it has a desired effect, the method for treating soil with the soil improver of the present invention is not particularly limited. For example, the soil conditioner of the present invention may be directly mixed with soil, or the soil conditioner of the present invention may be irrigated after being suspended in water or the like. In the case of hydroponics or hydroponics, the soil conditioner of the present invention may be added directly to the culture solution or hydroponic solution. In one Embodiment of this invention, the soil which processed soil with the soil improvement agent of this invention previously, and removed the soil improvement agent of this invention after that can also be used for cultivation of a plant. The removal of the soil conditioner can be performed, for example, by filtration with a filtration membrane, but is not limited thereto as long as the desired effect is obtained.
In one embodiment of the present invention, an antiseptic effect against bacteria and fungi other than yeast can be imparted to the liquid medium by treating the liquid medium for hydroponics with the soil improver of the present invention.

In the case of soil improvement using the soil improver of the present invention, it is preferable to apply 100 to 500 ml if the spraying is liquid per about 5 liters of soil used, and the cell concentration is usually 10 ³ per 1 ml of sprayed liquid. -10 ⁹ cfu, preferably 10 ⁴ -10 ⁸ cfu.

The timing at which the soil conditioner of the present invention is sprayed is not particularly limited as long as it has a desired effect, and can be used at any timing such as before planting or during plant breeding.
There is no restriction | limiting in particular with respect to the application frequency of the soil improvement agent of this invention.

  If the seed disinfectant of the present invention, the soil improver of the present invention, the agricultural chemical of the present invention or the plant growth promoter of the present invention does not impair the desired effect, it is a powder, granule, wettable powder, granule water. It can be provided in various dosage forms, such as a summing agent (dry flowable), a flowable agent (suspension), an emulsion, an emulsion, and a microcapsule.

The seed disinfectant of the present invention, the soil improver of the present invention, the agricultural chemical of the present invention or the plant growth promoter of the present invention may contain a carrier in addition to the active ingredient as long as the desired effect is not impaired. As a carrier that can be used, any solid or liquid can be used as long as it is commonly used for agricultural chemicals or seed disinfectants, and is not limited to a specific one.
As the solid carrier, mineral carriers (clay, talc, calcium carbonate, diatomaceous earth, zeolite, bentonite, acid clay, activated clay, attapulgus clay, vermiculite, perlite, pumice, silica sand, silica, white carbon, titanium dioxide Etc.), plant-based carriers (wood flour, corn stalk (cob), walnut shell (nut husk), fruit core, rice bran, sawdust, bran, soybean flour, powdered cellulose, starch, dextrin, sugars (glucose, maltose, lactose Inert powders or granules such as sucrose), water-soluble polymer gels such as agar, chlorinated polyethylene, chlorinated polypropylene, polyvinyl acetate, polyvinyl chloride, ethylene-vinyl acetate copolymer, Various polymer powders such as urea-aldevid resin) That.
Examples of the liquid carrier include water, alcohols, polyhydric alcohols, polyhydric alcohol derivatives, ketones, esters, nitrogen-containing carriers, fats and oils, specifically, ethanol, isopropanol, Cyclohexanol, ethylene glycol, diethylene glycol, propylene glycol, hexylene glycol, polyethylene glycol, polypropylene glycol, propylene glycol ether, cyclohexanone, γ-butyrolactone, fatty acid methyl ester (coconut oil fatty acid methyl ester), dibasic acid methyl ester (succinic acid) Acid dimethyl ester, glutamic acid dimethyl ester, adipic acid dimethyl ester), N-alkylpyrrolidone, coconut oil, soybean oil, rapeseed oil and the like.

If the seed disinfectant of the present invention, the soil improver of the present invention, the agricultural chemical of the present invention or the plant growth promoter of the present invention does not impair the desired effect, a surfactant, emulsifier, grinding aid, binding Auxiliary agent, slip aid, dissolution aid, suspension dispersion aid, powdering aid, liquidity modifier, thickener, disintegrating dispersant, antioxidant, spreading agent, spreading agent, wetting agent, A stabilizer, a desiccant, an ultraviolet absorber, an anti-drift agent, an adjuvant, a physical property improver, an active ingredient stabilizer or an antifreeze agent may be included.
Further, the seed disinfectant of the present invention, the soil improver of the present invention, the agricultural chemical of the present invention or the plant growth promoter of the present invention includes other fungicidal components, insecticidal components, plant growth regulating components in addition to the active ingredients. It may be.

  Furthermore, the present invention provides a composition comprising the bacterium of the present invention or a variant thereof, and cellulose and / or starch.

  As the cellulose and / or starch contained in the composition of the present invention, any one containing cellulose and / or starch can be used. From the viewpoint of preventing an increase in the price of edible crops, it is preferable to use a cellulose and / or starch raw material that does not compete with the food supply. Suitable examples of raw materials that do not compete with food supply include non-edible parts of crops (eg, bagasse after sugarcane juice, soft cellulose such as rice straw, corn stalks) and wood waste. In addition, agricultural crops with low edible value (for example, non-standard agricultural products, resource crops, barley, food waste, etc.) can be suitably used as raw materials. From the viewpoint of availability, commercially available starch raw materials such as barley powder and corn starch, and purified products such as crystalline cellulose can also be suitably used. The content of cellulose and / or starch in the composition of the present invention can be appropriately set according to the type of cellulose raw material, the type of starch raw material, the purpose of use of the composition, and the like.

  The content of the microorganism contained in the composition of the present invention can be appropriately set according to the purpose of use.

  As long as the composition of the present invention contains the bacterium of the present invention or a mutant thereof, and cellulose and / or starch as active ingredients, other polysaccharides, oligosaccharides, glycoproteins that do not impair the desired effects, Components such as peptides, proteins, nucleic acids, lipids, inorganic compounds, organic compounds, and minerals may be contained. In addition, the composition may further contain a physiologically acceptable carrier such as a diluent, an excipient, a stabilizer, and a preservative as long as the desired effect is not impaired.

  The composition of this invention can be used suitably for the manufacturing method of the cellulose and / or starch degradation product of this invention mentioned later, and the ethanol manufacturing method of this invention.

  The composition of the present invention may further contain yeast. In the present specification, “yeast” means a yeast capable of performing alcoholic fermentation, and preferable examples include Saccharomyces cerevisiae, Schizosaccharomyces pombe, Saccharomyces pastricus, Saccharomyces cerevisiae, Saccharomyces cerevisiae, The composition of this invention containing the said yeast can be used suitably for the manufacturing method of the ethanol of this invention mentioned later.

  By incubating the composition of the present invention under appropriate conditions under which bacteria can grow, cellulose and / or starch can be decomposed, resulting in glucose, cellobiose, maltose, trisaccharide to hexasaccharide oligosaccharide, etc. Further, ethanol can be produced by subjecting it to alcohol fermentation with yeast. Since the bacterium of the present invention or a mutant thereof exhibits antibacterial activity against a wide range of microorganisms, it is possible to decompose cellulose and / or starch while suppressing contamination, and antibacterial against yeast. Since it does not show activity, the sugar solution after the decomposition step can be used as it is for alcohol fermentation. Therefore, the present invention also provides a kit for producing ethanol comprising the bacterium of the present invention or a mutant thereof, yeast, and cellulose and / or starch, and ethanol can be produced efficiently by using the kit. .

  In one aspect, the present invention provides a method for producing a degradation product of cellulose and / or starch, comprising culturing the bacterium of the present invention or a variant thereof in a medium containing cellulose and / or starch. By performing this culture, cellulose and / or starch is saccharified by the bacterium of the present invention or a mutant thereof, and degradation products such as glucose, cellobiose, maltose, and oligosaccharides of 3 to 6 sugars are generated in the medium.

  In the production method, the definition of the bacterium of the present invention or a variant thereof, and preferable cellulose and / or starch raw materials are the same as described above.

  The culture method in the production method is not particularly limited as long as saccharification of cellulose and / or starch by the bacterium of the present invention or a mutant thereof is possible. Examples of the medium that can be used include M9 medium (minimum medium), BHI medium, 2YT medium, YPD medium, AM3 medium, and LB medium. The culture temperature is preferably 10 to 40 ° C, more preferably 28 to 37 ° C, most preferably 37 ° C, and the medium is preferably adjusted to pH 3 to 10. The culture time, culture scale, and the like can be appropriately set according to the purpose of use of the degradation product obtained by the culture.

  “Cellulose and / or starch degradation product” means a degradation product of cellulose, a degradation product of starch, or a mixture of these degradation products. The size of the sugar chain of the degradation product obtained by the production method is not particularly limited, and examples thereof include glucose that is a monosaccharide, cellobiose and maltose that are disaccharides, oligosaccharides of 3 to 6 sugars, and the like. The degradation product is preferably glucose.

Furthermore, the present invention provides
(A) converting cellulose and / or starch into glucose by culturing the bacterium of the present invention or a variant thereof in a medium containing cellulose and / or starch;
(B) converting glucose into ethanol by culturing yeast in a medium containing glucose obtained in (a),
A method for producing ethanol comprising

  In the step (a), the definition of the bacterium of the present invention or a mutant thereof, preferred cellulose raw material and starch raw material, preferred medium and culture method are the same as described above. The culture time is preferably the time required to decompose at least the cellulose raw material and starch raw material into glucose and obtain the required amount of glucose. The culture time can be appropriately set according to the type and amount of the cellulose raw material and starch raw material used, the culture scale, and the like.

  The yeast used in the step (b) is not particularly limited as long as it is a yeast capable of performing alcoholic fermentation. is there.

  The culture of the yeast is not particularly limited as long as it is performed under conditions suitable for alcohol fermentation. For example, the culture temperature when yeast needs to be grown is 10 to 40 ° C, preferably 28 ° C. The temperature at the time of fermentation may be 10-30 degreeC, Preferably it is 15-25 degreeC, More preferably, it may be 15 degreeC. In addition, the pH of the medium can be adjusted to preferably pH 3 to 10, more preferably pH 4.9. The types of media that can be used are as described above.

  The steps (a) and (b) can be performed simultaneously. That is, this invention also provides the manufacturing method of ethanol including the process of culture | cultivating the bacterium of this invention, its variant, and yeast in the culture medium containing a cellulose and / or starch.

  In the method for producing ethanol of the present invention, since the starch raw material can be efficiently saccharified without the starch raw material being pregelatinized, the pretreatment of the raw material can be simplified. In addition, since the bacterium of the present invention exhibits antibacterial activity against a wide range of microorganisms, it is possible to saccharify the raw material while suppressing contamination even if the sterilization treatment of the raw material is omitted. Can be saved. Therefore, the ethanol production method of the present invention can provide a low-cost and low environmental load ethanol production method by omitting the raw material pretreatment and sterilization treatment steps. On the other hand, since the bacterium of the present invention or a mutant thereof does not exhibit antibacterial activity against yeast, the culture solution after saccharification treatment is used as it is for alcoholic fermentation by yeast, or the bacterium of the present invention or a mutant thereof. It is also possible to co-culture the body and yeast and simultaneously carry out saccharification treatment of the raw material and alcohol fermentation.

  Ethanol produced by the above method can be purified by a method known to those skilled in the art and used for a desired application such as fuel.

  In another aspect, the present invention provides a method for producing an antibacterial extract comprising the step of obtaining an extract of a bacterium of the present invention or a variant thereof by extraction with acetonitrile or extraction with a lower alcohol. .

  In this method, for example, a culture solution containing the bacterium of the present invention or a mutant thereof is subjected to centrifugation, the bacterium is pelleted, and a solution obtained by resuspending the bacterium in acetonitrile or lower alcohol is used as an antibacterial extract. Alternatively, after suspending in acetonitrile or lower alcohol, it can be further centrifuged, and the supernatant can be used as an antibacterial extract. The extraction using acetonitrile and the extraction using lower alcohol may be performed alone or continuously. When performing continuously, either acetonitrile extraction or lower alcohol extraction may be performed first.

  The antibacterial extract obtained by the method exhibits antibacterial activity against a wide range of bacteria and fungi other than yeast. The fungi other than the bacteria and yeast are as described above. Therefore, the antibacterial extract can be suitably used for agricultural chemicals, pharmaceuticals and the like.

  When the antibacterial extract is used as an agrochemical, the target crops are, for example, rice, barley, tomato, watermelon, cucumber, strawberry, taro, soybean, wheat, corn, potato, kidney bean, pea, peanut, Sugar beet, sugar cane, eggplant, pepper, melon, pumpkin, radish, cabbage, cabbage, komatsuna, onion, leek, leek, garlic, asparagus, burdock, lettuce, garlic, carrot, spinach, okra, apple, pear, ume, grape , Oysters, figs, kiwi and the like.

  When the antibacterial extract is used as a medicine, it is orally or parenterally administered to humans and other mammals (eg, rats, rabbits, sheep, pigs, cows, cats, dogs, monkeys, etc.) It can be safely administered. The medicament may have a therapeutic effect against bacterial and fungal infections. Specific examples of such infections include infection with staphylococcal bacteria, streptococcal bacteria, enterococci, escherichia bacteria, ringworms, candidiasis bacteria, cryptococcosis bacteria, or fusarium bacteria. Examples include infectious diseases that develop.

(Control method)
As one embodiment of the present invention, the present invention provides an antibacterial extract of the present invention, an antibacterial composition, a bacterium of the present invention or a mutant thereof, or an agrochemical containing the culture thereof (herein, the present invention). A method for controlling diseases caused by fungi other than bacteria and yeast, which comprises a step of treating a plant with a pesticide. The concentration of the agricultural chemical of the present invention to be used, the target crop, the application method, the frequency of use, and the like are the same as those described for the agricultural chemical of the present invention.

(Seed disinfection method)
In one embodiment of the present invention, the present invention comprises a seed disinfection step comprising treating a seed with the antibacterial extract, antibacterial composition of the present invention, the bacterium of the present invention or a mutant thereof, or a culture thereof. Provide a method. Seed disinfection methods are the same as those described for seed disinfectants.

(Plant growth promotion method)
As one embodiment of the present invention, there is provided a method for promoting plant growth, comprising the step of treating a plant with the bacterium of the present invention or a mutant thereof or a culture thereof.
About a method, it applies to what was described about the plant growth promoter.

(Soil improvement method)
As one embodiment of the present invention, there is provided a soil improvement method comprising a step of treating soil with the antibacterial extract, the antibacterial composition of the present invention, the bacterium of the present invention or a mutant thereof, or a culture thereof.
About a soil improvement method, it applies to what was described about the soil improvement agent.

  Hereinafter, the present invention will be described more specifically with reference to examples, but it goes without saying that the present invention is not limited thereto.

(Example 1) Screening of novel microorganisms having cellulose and starch degrading ability Selection of microorganisms capable of degrading cellulose (Figure 1)
First, a minimal medium containing rice straw powder as the only carbon source was prepared. The rice straw powder was obtained by cutting the rice straw to a length of about 3 to 5 cm with scissors, drying it with a dryer at 70 ° C. for about one week, and then pulverizing it into a powder form with a mixer. The 7% rice straw medium was prepared by dissolving 100 mL of M9 medium (Na ₂ HPO ₄ ; 6 g, KH ₂ PO ₄ ; 3 g, NaCl; 0.5 g, NH ₄ Cl; 1 g in distilled water to 1 L). ) And 7 g of rice straw powder were put into a bottle, autoclaved (121 ° C., 20 minutes), and after cooling, 1 mL of sterilized 100 mM CaCl ₂ and 1 mL of 1M MgSO ₄ were added.
To the rice straw medium, soil sampled from 40 locations inside and outside Fukui Prefecture was added, and enrichment culture was repeated to identify microorganisms having cellulose-degrading ability. The yeast contained in the soil fermented using glucose produced by the decomposition of cellulose to generate carbon dioxide, and therefore the target microorganisms were concentrated using carbon dioxide foaming as an index (FIG. 1A).
Furthermore, microorganisms were seeded on an agar medium supplemented with Congo red and carboxymethylcellulose (CMC), and microorganisms were selected using the size of dissolution spots as an index (FIG. 1B).
Thus, microorganisms having high cellulose resolution were selected.

2. Selection of microorganisms with starch degradability (Fig. 2)
About 300 g of sampling soil was added to a solution obtained by mixing 1 L of distilled water and 50 g of Rokujo barley powder (Fukui barley club, stone grinding, βg), and the mixture was incubated at room temperature (about 23 ° C.) for 4 days. Mix 10 g of Rokujo barley powder and 200 mL of distilled water, make a new medium adjusted to pH 2.5-3.0 using lactic acid, add 5 mL of the culture solution, and leave it at 50 ° C. for 3 days. Incubated. By lowering the pH of the medium, it is possible to suppress the growth of various bacteria and prevent the medium from being spoiled. Thereafter, 100 μL of the culture broth was prepared by mixing 6.25% (w / v) Rojo barley liquid medium (0.5 g of Rojo barley and 8 mL of distilled water mixed and autoclaved at 121 ° C. for 20 minutes) (about It was added to a test tube containing pH 5.6) (FIG. 2, primary screening).
Microorganisms that produce starch-degrading enzymes (amylases) are considered to be highly assimilating (metabolic ability) to the disaccharide (maltose), which is a degradation product of starch, and are used on a minimal agar medium with maltose as the sole carbon source. Using the growth rate as an index, microorganisms that form large colonies were selected (FIG. 2, secondary screening). Maltose-containing agar medium was prepared by dissolving 6 g of Na ₂ HPO ₄ , 3 g of KH ₂ PO ₄ , 0.5 g of NaCl, 1 g of NH ₄ Cl and 15 g of Agar in 500 mL of distilled water and autoclaving at 121 ° C. for 20 minutes. This was prepared by mixing 500 mL of an aqueous solution in which 20 g maltose hydrate was dissolved, 1 mL of 100 mM CaCl ₂ , and 1 mL of 1M MgSO _4, which were sterilized separately after cooling, and flowing into a petri dish.
Furthermore, microorganisms with high starch saccharification ability were selected on the minimal agar medium supplemented with 6-joo barley flour containing abundant starch, using the size of dissolved spots as an index (FIG. 2, tertiary screening). When inoculating the Rojo Barley Agar medium, colonies with different shapes were selected and inoculated as much as possible. Six-joule barley agar was prepared by dissolving 6 g Na ₂ HPO ₄ , 3 g KH ₂ PO ₄ , 0.5 g NaCl, 1 g NH ₄ Cl, 20 g six-joo barley powder, 15 g Agar in 1 L distilled water, 121 It was prepared by mixing 1 mL of 100 mM CaCl ₂ and 1 mL of 1M MgSO ₄ , autoclaved at 20 ° C. for 20 minutes, sterilized separately after cooling, and flowing into a petri dish.
Single colonies that formed dissolution spots in the third screening were each dissolved in 2YT liquid medium (Polypepton 16 g, Yeast Extract 10 g, and NaCl 5 g in distilled water, adjusted to pH 7.5 with NaOH, and made 1 L with distilled water. Inoculated into 5 mL) (prepared by autoclaving at 121 ° C. for 20 minutes) and pre-cultured overnight. 20 μL of the preculture was mixed with 10 mL of M9 liquid medium containing 1% (w / v) corn starch, and cultured with shaking at 28 ° C. and 150 rpm for 4 days. Thereafter, the culture solution is centrifuged at 15000 rpm for 5 minutes, and 20 μL of the culture supernatant is subjected to a glucose rapid colorimetric method (Anal. Sci., 29 (11), 1021-5) to efficiently decompose starch into glucose. Microorganisms that can be identified were identified (Figure 2, 4th screening).

(Example 2) Identification test of novel microorganisms having cellulose resolution and starch resolution Interestingly, microorganisms having high cellulose resolution and microorganisms having high starch resolution selected as described above were sampled from the same area. It was. Thus, in order to determine the genus of both microorganisms, a molecular phylogenetic analysis was performed using the base sequence of the 16S rRNA gene (about 1.5 kb). As a result, the base sequence of the 16S rRNA gene of these microorganisms was 100. % Matched. Furthermore, as a result of comparing the crystalline cellulose resolution, starch resolution, physiological properties, biochemical properties, and morphological characteristics of both microorganisms, they were found to be the same microorganism. The characteristics of the microorganism are listed below.
-Cell shape is rod-shaped, has peripheral flagella, and has active motility.
-The colony has a cream color close to white (some differences depending on the culture medium).
Produces amylase.
-Produces glucanase.
• Produce antibiotics against bacteria and fungi.
・ Do not hydrolyze gelatin.
・ Nitrate is not reduced.
Moreover, the result of the molecular phylogenetic analysis using the base sequence of 16S rRNA gene is shown in FIG. As a result of molecular phylogenetic analysis, it was clarified that the microorganism was a microorganism belonging to the genus Paenibacillus and formed a gene cluster with AJ320493, AJ271157, AB680894, and AF391123 which are Painebacillus genus strains (type strain) (FIG. 3). Since the microorganism is different from the related species in that it does not have gelatin degradability and nitrate reducing ability, the microorganism was identified as a new species of Paenibacillus genus and named FPU-37 strain.

(Example 3) Identification of saccharide-degrading enzyme of FPU-37 strain As a result of genome draft analysis by a next-generation sequencer (Illumina Hiseq), the genome size of FPU-37 strain is about 5.9 Mbp, It was found to have a relatively large genome size. Based on the gene sequences of known cellulase, amylase and α-glucosidase already reported in the genus Paenibacillus, homology search of these gene sequences existing on the genome of FPU-37 strain was performed. A variety of genes were hit.
The cellulase gene (SEQ ID NO: 1) of FPU-37 strain shows high homology with Paenibacillus polymyxa β-1.4-glucanase, and is classified as a family 5 of Glycoside Hydrolases (GH). It was revealed that it has a domain (GH5), a secretory signal sequence on the N-terminal side, and a carbohydrate-binding module (CBM) family 3 (CBM3) domain on the C-terminal side, which is a carbohydrate binding module.
The amylase gene (SEQ ID NO: 3) of the FPU-37 strain showed high homology with Paenibacillus polymixa β / α-amylase and had a secretory signal sequence on the N-terminal side. Furthermore, the amylase gene has a β-amylase catalytic domain (GH14) and two CBM25s on the N-terminal side, and an α-amylase catalytic domain (GH13) and a kind of CBM Aamy- It became clear to have a C module. Therefore, the amylase gene of FPU-37 strain is considered to have both α-amylase activity and β-amylase activity.
In addition, it was revealed that the α-glucosidase gene (SEQ ID NO: 5) of the FPU-37 strain is an enzyme classified into the carbohydrate degrading enzyme family 31 (GH31).

(Example 4) Localization and expression induction of cellulase, amylase and α-glucosidase of FPU-37 strain Localization and expression induction of cellulase of FPU-37 strain (1) BHI medium (prepared by dissolving 3.7 g of BHI in distilled water to 100 mL and then autoclaving (121 ° C., 20 minutes)) And (2) 1% (w / v) starch, (3) 1% (w / v) crystalline cellulose, or (4) 1% (w / v) Fusarium powder added to 100 mL of BHI medium. did.
From the pre-culture of FPU-37 cultured in 20 ml of BHI liquid medium at 37 ° C. for 1 day, add 5 ml of the culture solution to each of the above media (1) to (4) and swirl culture at 37 ° C. for 3 days. did. Thereafter, 1.5 mL of each culture was transferred to a tube, centrifuged (15000 rpm, 5 minutes), and the supernatant was transferred to a new tube. The supernatant was used for the following enzyme reaction.
On ice, a substrate solution (p-Nitrophenyl-β-D-Glucoside (PNP-Glu) or p-Nitrophenyl-β-D-Cellobioside (PNP- (Glu) ₂ )) 40 μL, buffer ( 30 μL of citrate buffer (pH 5.6)) and 30 μL of enzyme solution (supernatant obtained above) were added to prepare a reaction solution. As a negative control, a reaction stop solution (0.2 M sodium hydroxide solution) 200 μL was immediately added without reacting the reaction solution. The reaction solution was allowed to react at 37 ° C. for 3 hours, immediately after the reaction, transferred to ice, and 200 μL of a reaction stop solution was added to stop the reaction.
Thereafter, the reaction solution was transferred to a 96-well plate at 200 μL / well, and the absorbance at 405 nm was measured with an absorbance plate reader.
The results are shown in FIG. Since cellulase activity was observed only in the culture supernatant cultured with the addition of crystalline cellulose, it became clear that the cellulase of the FPU-37 strain was induced by cellulose. Further, when the localization of the cellulase activity was examined, the activity was found only in the culture supernatant (FIG. 4B). From these results, it was revealed that the cellulase of the FPU-37 strain was induced to be expressed by cellulose and secreted outside the cell.

2. FPU-37 Strain Amylase Localization and Expression Induction In 20 mL each of 2YT, 2YT + 3% (w / v) soluble starch liquid medium, the FPU-37 strain was cultured with shaking at 30 ° C. and 120 rpm for 3 days. Thereafter, 2 mL of each culture was dispensed, centrifuged at 15000 rpm for 5 minutes, and the supernatant was transferred to a new tube. Suspension buffer (MES buffer (48 mL, 1 M, pH 6.2), EDTA2Na (20 mM), bovine serum albumin (10 mg / mL, 0.2% (w / v)) 2) each of which was made up to 500 mL with distilled water), vortexed to suspend the precipitate, centrifuged at 15000 rpm for 5 minutes, discarded the supernatant, and then further 15000 rpm The suspension was centrifuged for 1 minute, the supernatant was discarded, 2 mL each of the suspension buffer was added, and the precipitate was suspended by vortex to obtain a cell suspension.
α-amylase of the above-mentioned culture supernatant and cell suspension obtained from 2YT medium or 2YT + 3% (w / v) soluble starch medium using an α-amylase measurement kit (Kikkoman Biochemifa Co., Ltd.) Activity was measured (FIG. 5A). The α-amylase activity was observed in the culture supernatant (FIG. 5A, culture supernatant) of the FPU-37 strain, and the activity was low in the bacterial cell suspension (FIG. 5A, bacterial cell fraction). Furthermore, in the culture supernatant to which soluble starch was added (FIG. 5A, culture supernatant, starch +), the activity of α-amylase was remarkably higher than that to which it was not added (FIG. 5A, culture supernatant, starch−). From this, it was confirmed that the addition of starch induced the expression of α-amylase.
Β-amylase activity of the above culture supernatant and cell suspension obtained from 2YT medium or 2YT + 3% (w / v) soluble starch medium using BETA-AMYLASE BETAMYL-3 ASSAY KIT (MEGAZYME) Was measured (FIG. 5B). β-amylase activity is strongly observed in the culture supernatant (FIG. 5B, culture supernatant, starch +) of the FPU-37 strain in which soluble starch is added to the medium, and FPU− in which soluble starch is not added to the medium. Although it was weak in 37 culture supernatants (FIG. 5B, culture supernatant, starch), the activity was low in the cell suspension (FIG. 5B, cell fraction). Furthermore, the activity of β-amylase is significantly higher in the culture supernatant to which soluble starch was added (FIG. 5B, culture supernatant, starch +) compared to the culture supernatant not added (FIG. 5B, culture supernatant, starch-). From this, it was confirmed that the addition of starch induced the expression of β-amylase.

3. Localization of α-glucosidase of FPU-37 strain In 10 mL of 2YT + 3% (w / v) soluble starch liquid medium, the FPU-37 strain was cultured with shaking at 30 ° C for 3 days. Thereafter, the culture solution was transferred to a 15 mL tube, centrifuged at 8000 rpm for 10 minutes, and the supernatant was transferred to a new tube. 10 mL of PBS buffer solution (NaCl 8 g / L, KCl 0.2 g / L, Na ₂ HPO ₄ × 12H ₂ O 2.9 g / L, KH ₂ PO ₄ 0.24 g / L) is added to the precipitated cells. In addition, the cells were washed by vortexing. This bacterial cell suspension is centrifuged at 8000 rpm for 10 minutes, the supernatant is discarded, and further centrifuged at 8000 rpm for 1 minute. After the supernatant is completely discarded, 10 mL of PBS buffer is added and vortexed. Was suspended to obtain a cell suspension. Transfer 1 mL of the microbial solution to another tube, add lysozyme lysozyme to 1 mg / mL to the remaining microbial solution, react at 30 ° C. for 30 minutes, perform ultrasonic disruption, and centrifuge at 15000 rpm for 10 minutes. The supernatant was used as a cell lysate.
The α-glucosidase activity of the culture medium (control), the culture supernatant (extracellular fraction = secretory enzyme), the cell suspension (cell surface enzyme), and the cell lysate (cell enzyme) was measured as sample 0. After adding 1% 3% maltose aqueous solution to 5 mL and reacting at 37 ° C. for 1 hour, glucose rapid colorimetry (Anal. Sci., 29 (11), 1021-5) is used as an indicator of glucose production. Measured. As a result, α-glucosidase activity was detected only in the cell lysate.
From the above results, it can be seen that α-amylase and β-amylase of the FPU-37 strain are extracellular enzymes that are induced by addition of starch, and α-glucosidase is localized in the cell. confirmed.

(Example 5) Analysis of antibacterial activity of FPU-37 strain Antibacterial activity against phytopathogenic fungi In the process of screening the FPU-37 strain, the inventors have found that the FPU-37 strain exhibits a strong antibacterial activity that eliminates surrounding microorganisms. Therefore, the present inventors confirmed the antibacterial activity of the FPU-37 strain against agriculturally important plant pathogenic fungi by counterculture (FIG. 6).
On the AM3 agar medium, a paper disk spotted with 40 μL of the culture solution of FPU-37 strain and a paper disk spotted with nothing were placed, and the phytopathogenic bacteria shown in FIG. 6 were inoculated and cultured at 28 ° C.
FPU-37 strain was shown to have strong antibacterial activity against any phytopathogenic fungus.
2. Localization and induction of antibacterial substances 500 mL of culture medium in which FPU-37 strain was cultured for 3 days in BHI medium containing 1% (w / v) crystalline cellulose was centrifuged (8000 rpm, 10 minutes), 1 mL of the supernatant was taken and sterilized with a filter to obtain a culture supernatant sample. The remaining supernatant was discarded, and 20 mL of PBS was added to the cells and resuspended. The bacterial cell suspension was centrifuged (6000 rpm, 15 minutes), and 5 times the amount of bacterial cell acetonitrile (eg, 5 μL of acetonitrile per 1 mg of bacterial cell) was added and resuspended. Thereafter, the suspension is stirred (145 rpm, 30 minutes), centrifuged (6000 rpm, 15 minutes), the supernatant is collected, and the supernatant is further centrifuged (8000 rpm, 5 minutes). Collected. The supernatant was used as an acetonitrile extraction fraction.
When antibacterial activity against cucumber pathogen (Fusarium oxysporum f. Sp. Cucumerinum) was confirmed, antibacterial activity was not confirmed in the culture supernatant, medium alone, and control (acetonitrile only), and only the acetonitrile extract fraction was antibacterial activity. (FIG. 7A). This result shows that the antibacterial substance produced by the FPU-37 strain is not secreted into the culture supernatant and is attached to the cells.
Further, it was revealed that the antibacterial activity was induced when the FPU-37 strain was cultured in a medium containing crystalline cellulose or fungal cell wall (1% (w / v) Fusarium powder) (FIG. 7B). ).
3. Analysis of antibacterial activity of acetonitrile extracted fraction and butanol extracted fraction Acetonitrile extracted fraction was obtained by the same method as above. A butanol extract fraction was obtained in the same manner except that butanol was used instead of acetonitrile. The antibacterial activity of these extracted fractions against microorganisms was analyzed.
Antibacterial activity against Staphylococcus aureus, Fusarium spp., And yeasts (using the Japan Brewing Association, Association No. 901 yeast) was confirmed, but the acetonitrile extract fraction was extracted with butanol only against Fusarium spp. The fraction showed antibacterial activity only against S. aureus (FIG. 8A). Therefore, it was suggested that FPU-37 strain produces at least two types of antibacterial substances. Further, it was revealed that the antibacterial substance does not exhibit antibacterial activity against yeast. This property makes it possible to co-cultivate the FPU-37 strain and yeast, and to easily and efficiently produce ethanol from cellulose raw materials and / or starch raw materials.
Furthermore, when the antibacterial spectrum of the butanol extract fraction was confirmed, it was revealed that both the gram positive bacteria and the gram negative bacteria showed wide antibacterial activity (FIG. 8B).

(Example 6) Ethanol production using FPU-37 and yeast Since FPU-37 strain can efficiently saccharify starch, it produces a strong antibacterial substance exhibiting antibacterial activity against a wide range of microorganisms other than yeast. By utilizing this feature, the raw material required for starch saccharification with amylase is not subjected to the alpha conversion and sterilization, and the starch-rich six-joo barley flour is used as the sole carbon source as it is, and the FPU-37 strain and Parallel double fermentation with yeast (Association Yeast 901) was performed (FIG. 9).
Sixty barley flour (70 g) and M9 medium (100 mL) were mixed, adjusted to pH 4.9 with lactic acid, and used as a fermentation medium without sterilization by autoclave. The FPU-37 strain is cultured in 400 mL of 2YT medium for 2 days (28 ° C.), centrifuged at 5000 rpm for 15 minutes, the supernatant is discarded, 10 mL of M9 medium adjusted to pH 4.9 with lactic acid is added, The body was suspended and 10 mL of the suspension was added to the fermentation medium. Then, the saccharification process was performed for 2 days in a 25 degreeC thermostat. Association yeast 901, cultured for 2 days in 400 mL of YPD medium (28 ° C), was centrifuged at 5000 rpm for 12 minutes, the supernatant was discarded, and 5 mL of M9 medium adjusted to pH 4.9 with lactic acid was added. The cells were suspended, and 5 mL of the suspension was added to the fermentation medium after the saccharification treatment. Static culture fermentation was performed for 40 days in a constant temperature bath at 15 ° C. The fermentation broth was collected every few days, diluted 100 times with MilliQ water, and the ethanol concentration was measured using gas chromatography.
As shown in FIG. 9, the ethanol concentration in the fermentation medium increased greatly for 19 days from the start of fermentation, and the increase became gradual after the 19th day. An increase in ethanol concentration is also observed in the fermentation medium without the addition of FPU-37 strain, but this is considered to be because the amylase derived from barley functioned without being deactivated because Rojo barley flour was used as it was without heating. . In addition, when the 6-joo barley flour gelatinized by the autoclave process was used and the FPU-37 strain was not added to the fermentation medium, it was confirmed that ethanol was not detected even if yeast was added to the fermentation medium. Moreover, although the α-glucosidase produced by the FPU-37 strain is localized in the cell, since the FPU-37 strain is a bacterium that is easily lysed, α-glucosidase is released into the culture solution by lysis, and the FPU-37. It is considered that maltose produced by this amylase was further decomposed into glucose, and yeast produced ethanol from the glucose.
On the 40th day after the start of fermentation, the concentration of ethanol produced when the FPU-37 strain was used was 4.8%, and when the FPU-37 strain was not used, it was 3.8% (Fig. 9). Therefore, it was shown that by using the FPU-37 strain, ethanol can be efficiently produced without subjecting the raw material to a gelatinization treatment and a sterilization treatment.

(Example 7) Seed disinfection (miscellaneous bacteria suppression) effect of FPU-37 strain Seed disinfection is an important treatment for preventing many diseases occurring in the seedling raising period. Pesticides are mainly used for seed disinfection, but recently, organic cultivation without the use of pesticides and chemical fertilizers has been welcomed by consumers in order to improve consumer safety awareness of food. In addition, since the added value of crops is improved for producers, the production volume is increasing year by year. Seed disinfection methods that can be used in organic farming methods that are not counted as pesticides are limited to hot water disinfection and microbial pesticides (eco-hope, tough block). However, it is difficult to control the temperature of hot water disinfection, and a special device is required to accurately perform the heat treatment. On the other hand, microbial pesticides have a narrow range of applicable crops and are mainly used in rice. Therefore, the FPU-37 strain with a broad antibacterial spectrum is considered to be a seed disinfectant that can be used for other than rice (tomato, lettuce, komatsuna, cabbage, spinach, bulbs, seeds, etc.). A seed disinfection test was conducted using
The test was performed on a sterile agar medium so that the effect of the FPU-37 strain on the microorganisms actually attached to the seeds could be clearly determined. The medium is Murashige and Skog Plant Salt Mixture (4.6 g / L, Wako Pure Chemicals 392-00591). Yeast extract (5 g / L) and glucose (5 g / L) so that both plants and microorganisms can grow. Agar (15 g / L) was further added (MSYG medium). The agar medium was dispensed into a petri dish after autoclaving. Antibiotic (streptomycin) used to suppress the growth of miscellaneous bacteria was added so as to reach a concentration of 100 μg / ml after the temperature dropped to about 50 ° C. after autoclaving. Tomato seeds were germinated by directly placing 60 seeds on MSYG medium or antibiotic-containing MSYG medium. In the seed disinfection (bacteria control) effect of the FPU-37 strain, seeds were impregnated with 1 mL (2 × 10 ⁸ CFU / mL) of an overnight culture solution of the FPU-37 strain, and then placed on the MSYG medium for germination.

  Many of the tomato seeds germinated by placing them on the MSYG medium without seed disinfection did not germinate because germs proliferated in large quantities around the seeds (germination rate 18%). On the other hand, in the antibiotic-containing MSYG medium, the germination rate was high (92%) by suppressing the propagation of various bacteria, and the result that the importance of seed disinfection was realized. Also in the seed impregnated with the FPU-37 strain, the propagation of miscellaneous bacteria was well suppressed, and the germination rate was as high as 83%. From the above results, it is considered that the FPU-37 strain has the effect of suppressing germ propagation and increasing the germination rate. It is also expected as a soil conditioner (soil sterilization).

(Example 8) Growth promotion effect of FPU-37 strain The effect of the FPU-37 strain on plant growth was tested.
In order to test the effect of FPU-37 strain on rice growth, rice (Koshihikari) was divided into two groups. In the house, an FPU-37 strain overnight culture solution (2 × 10 ⁸ CFU / mL) was given to half, the medium only to the other half, and 50 mL per seedling box by irrigation twice a week. FIG. 10 shows the result 20 days after the transfer to the field.
Growth of rice to which the FPU-37 strain was added was promoted compared to rice to which only the medium was given. Therefore, it was shown that the overnight culture solution of FPU-37 strain has a growth promoting effect on rice.
Next, the effect of the FPU-37 strain on tomato growth was tested.
The tomato seedlings are divided into 4 groups, each of which has an overnight culture of FPU-37 strain (2 × 10 ⁸ CFU / mL), an overnight culture of 10-1 strain (2 × 10 ⁸ CFU / mL), and 1K-5 strain. An overnight culture (2 × 10 ⁸ CFU / mL) or medium alone was given to the root by 10 ml each by irrigation. The result 20 days after the addition is shown in FIG.
The growth of the tomato added with the FPU-37 strain was promoted as compared to the tomato fed with the medium alone. Therefore, it was shown that the overnight culture solution of FPU-37 strain has a growth promoting effect on tomato. The effect was comparable to the 1K-5 and 10-1 strains whose growth promoting action was already confirmed.
The FPU-37 strain has strong antibacterial activity against phytopathogenic fungi and the like, but interestingly, it has been found that it has a growth promoting effect on crops such as rice and tomato (FIGS. 10 and 11). ). Then, in order to elucidate the molecular mechanism regarding the plant growth promotion effect of FPU-37 strain, each factor related to growth promotion was analyzed.
The factors listed in Table 2 estimated to be involved in the plant growth promoting effect were analyzed.

Example 8-1. Test for Solubilization of Slightly Soluble Phosphate with Organic Acid 31.3 g of Pikovskaya'Agar (HIMEDIA M520-500G) was dissolved in 1 L of water and dispensed into a petri dish after autoclaving at 121 ° C. for 20 minutes. The FPU-37 strain was cultured at 28 ° C. for 1 week on this agar medium, and the solubilizing ability of the poorly soluble phosphorus compound was tested using transparent dissolution spots formed on the medium clouded with insoluble phosphorus as an index.

  Clear dissolution spots were formed around the cells of the FPU-37 strain, and the solubilizing ability of the poorly soluble phosphorus compound was confirmed (FIG. 12).

Example 8-2. Against 1mM FeCl ₃ · 6H ₂ O solution 10mL dissolved in production test (1) Fe-CAS Indicator ( 10mM HCl siderophores, and 50mL of Chrome Azurol S (TCI TG2VM-TS ) solution (1.21 mg / mL) 60 mL of 40 ml HDTMA (TCI 33H7B-RI) aqueous solution (1.82 mg / ml), (2) 2YT-PIPES agar medium (yeast extract 10 g / L, polypeptone 16 g / L, NaCl 5 g / L, PIPES (DOJINDO GB157) 30.24 g / L, agar 15 g / L) 1 L, and (3) 10% (w / v) casamino acid solution 50 mL were mixed after autoclave sterilization, dispensed into a petri dish, and siderophore assay A medium was prepared. The Chrome Azurol S-iron complex is colored blue, but the color disappears when iron is taken away by the siderophore. The FPU-37 strain was cultured on an agar medium at 28 ° C. for 12 days, and the production of siderophore was evaluated using the disappearance of blue color derived from the Chrome Azurol S-iron complex as an index.
Since clear blue disappearance was confirmed on the siderophore assay medium, the siderophore production ability of the FPU-37 strain was confirmed (FIG. 13).

Example 8-3. Production analysis of hydrogen cyanide (HCN) Agar medium was prepared by adding agar 15 g / L, yeast extract 5 g / L, and glycine 5 g / L to M9 minimal medium (Table 4), and colonies formed on this agar medium were used. The acidity of hydrogen cyanide was tested. In order to test hydrogen cyanide, the test paper should be 2% (w / v) in 0.5% (w / v) Picric Acid (2,4,6-Trinitrophenol) (Wako 207-08671) aqueous solution. The filter was sterilized by adding sodium carbonate, impregnated in Whatman Filter Paper (no. 1) and air-dried (yellow in this state).
This test paper (yellow) was placed on a colony formed on an agar medium, and HCN production was evaluated using the change from yellow to brown as an index. In this method, the color of the test paper changes from yellow to orange, red, and brown, and low, medium, and high levels of HCN production can be evaluated, respectively.
The results are shown in FIG. Since the test paper turned brown, it was confirmed that it has a high HCN production ability.

Example 8-4. Chitin degradation activity The degradation activity of chitin was evaluated using dissolution spot formation on a chitin-containing medium (M9 agar medium containing 0.5% (w / v) colloidal chitin and 0.5% yeast extract) as an index. In addition, the presence of a chitinolytic enzyme (chitinase) gene was also confirmed by genome analysis (FIG. 15).
Since no halo was formed on the chitin-containing medium, it was determined that the FPU-37 strain was not capable of degrading chitin. This result is consistent with the absence of a chitinase gene on the genome by genomic analysis.

Example 8-5. Production test of indole-3-acetic acid (IAA)

Tryptophan was added to the KL medium to a final concentration of 0.1%, the FPU-37 strain was inoculated, and cultured with shaking at 28 ° C. for 3 days. Add 2 drops of orthophosphoric acid to 2 mL of the culture supernatant from which the cells have been removed by centrifugation, and add 4 mL of Salkowski reagent (1 mL of 0.5 M FeCl ₃ to 50 mL of 35% perchloric acid). Preparation) was added and IAA was colored red for 30 minutes at room temperature, and the absorbance at 530 nm was measured. Simultaneously with the measurement of the culture solution, a calibration curve was prepared with the IAA standard, and the amount of IAA produced in the culture solution was quantified.
As a result of the measurement, it was confirmed that the FPU-37 strain produced indole-3-acetic acid (IAA) at a high concentration of “8.41 μg / mL” as a plant growth hormone in the culture supernatant.

Example 8-6. Measurement of nitrogenase (flavodoxin) Nitrogenase (flavodoxin) is an enzyme possessed by bacteria that fix nitrogen, and catalyzes the reaction of converting nitrogen in the atmosphere into ammonia. Nitrogenase activity was evaluated using the growth ability in a minimal medium (a medium obtained by removing ammonium sulfate from a KL medium) as an index. Genome analysis also confirmed the presence of the nitrogenase (flavodoxin) gene.
As a result of the culture test, since the absorbance at OD660 reached 0.65 after 3 days of culture in the culture in a nitrogen source-free medium, it was judged that the FPU-37 strain had a nitrogen fixing ability. Furthermore, the nitrogenase (flavodoxin) gene (SEQ ID NO: 7) present on the genome was also confirmed.

[Base sequence of nitrogenase (flavodoxin) derived from FPU-37 strain (SEQ ID NO: 7)]
ATGAACTTGGGTAAAATCATGATTGCTTATGCTAGCATGACCGGTAATACAGAGGAAATCGCGGAATTGATCGCCGAGGGTGTTCGTCAGGCAGGGCATGAGGCAGAGTTGAAGGCTTCGTATGATTGCAACGCGCAAGAATTGCTCGCATATGACGGATTCCTGTTGGGTGTATATACATGGGGAGATGGTGAGCTTCCTGATGAATTTTTAGATTTTTACGAGGAACTAGACGAGCTTGATCTGTCTGGTAAAAAGACTGCTGTATTTGGCAGTGGAGATACATCTTATACACAATTCTGTGGCGCAGTGGACTTGGTAGAGGGAAAAGTC AAGAACGCGGCGCATCAGTCATTCAGGAGAGTTTGAAGATTGAATTCAATCCACTCGATGATGAGAAGGAGAATTGCCGAGCTTACGGAAGACAATTTGCGCAGGCTGGCATTGGAGTGTCCTGA
[Amino acid sequence of nitrogenase (flavodoxin) derived from FPU-37 strain (SEQ ID NO: 8)]
MNLGKIMIYASMTGGNTEEIIAELIAEGVRQAGHEAELKASYDCNAQELLLAYDGFLLLGVYTWDGGDELPFLFDFYEELDELDLSGKTAVFGSGDTSYTQFCGAVDLVEGKVDGRFGASCRQ

Example 8-7. Measurement of 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase activity The ability to inhibit the synthesis of ethylene by measuring the ability to assimilate ACC as a precursor in ethylene biosynthesis (ACC deaminase is a metabolic enzyme) Evaluated. Specifically, a minimal medium (a medium obtained by removing ammonium sulfate from a KL medium) containing ACC as the only nitrogen source was prepared, and the growth ability was evaluated by quantifying it as turbidity. The above test revealed that the FPU-37 strain has a nitrogen-fixing ability. Therefore, the ACC metabolic ability was actually evaluated by increasing the growth ability by adding ACC. In addition, the presence of the 1-aminocyclopropane-1-carboxylate deaminase (ACC metabolizing enzyme) gene was also confirmed by genome analysis.
As a result of the test, the growth rate of the FPU-37 strain was increased by the addition of ACC. Therefore, it was judged that the FPU-37 strain had ACC metabolic ability and produced ACC metabolic enzyme. Furthermore, the 1-aminocyclopropan-1-carboxylate deaminase (ACC metabolizing enzyme) gene (SEQ ID NO: 9) present on the genome was also confirmed.

[FPU-37 strain-derived 1-aminocyclopropane-1-carboxylate deaminase nucleotide sequence (SEQ ID NO: 9)]
ATGAACAGAATTTATCTGGATCATGCCGCATCGACTCCTATGCATCCTGAGGTTGCGCAGACTATGATGGATATCATGACCGGCCAATTCGGCAACGCTTCGAGCGTTCATGCCTTTGGCCGCGATGCCAAGCGCACCGTCAGCGCAGCGCGGGATGCCGTTGCGGCCTCTTTGGGCTGCAAGCCTGAAGAAATTATATTTACGAGCGGAGGGACAGAGAGTGATAATCTGGCCCTGTTTGGGACGGCTACGGCAGATGGTCGTACTTCCGGCCATATTATTACAACCGCGATTGAACATCACGCCGTGCTTCATGCTTGCGATGAGCTGGAA AAGCAGGATACCAGGTGACCTATGTCCCAGTCAATGGTTTTGGGCGTGTGAATCCTGCGGACATTGAGGCAGCTATTCGCCCGGATACTTTTTTGATCAGCATGATGTATGCCAACAATGAGGTGGGCGTCATTCAGCCGGTCCAAGAAGTAGGACAGATTGCGAGAGAGCATGGAATTATATTTCATGTAGATGCAGTCCAAGCCTTCGGGCATATATCCATCGACTGCGGGAACCTCCCGATTGACTTACTTAGTGTGTCTGGCCATAAGATTAATGGACCGCAAGGAGTGGGTGCATTGTATATCCGTCAGGGTACACGCATTCAGCCG TAATGCATGGGGGACTTCAGGAGAAGAAACGCCGTGCAGGAACAGAAAATATCGCCGGCATTGCTGGGCTGGCTAAAGCCGCAACTCTTGCCAATGCTTCCATTGAAGAGCGAGGAGCACACGATATGGCTCTTCGCCAAACGCTGCTAAAAGCGTTGGAAAACTCTCTGGGCAGTCATGAGTTTATAATCAATGGCGATCCTGAACATTTTGTGCCTAATGTGCTAAATATTAGCTTTCCAGCCATTGGTACAGAGACCATGCTGATGAATCTCGACATGGAAGGAATCGCAGCGGCCAGCGGATCAGCCTGCACTTCGGGGTCTCTAGAGC TATCGCATGTGTTGCAGGCGGATGGATCTTCCTGGAAGATGTTTGAATTTCAGCGATTCGATTTAGCTTCGGTTTGGGTAATATACTACGAGAAAAATGGAATAACGGGCCATGAATAGATTGAGATC
[Amino acid sequence of FPU-37 strain-derived 1-aminocyclopropane-1-carboxylate deaminase (SEQ ID NO: 10)]
MNRIYLDHAASTPMHPEVAQTMMDIMTGQFGNASSVHAFGRDAKRTVSAARDAVAASLGCKPEEIIFTSGGTESDNLALFGTATADGRTSGHIITTAIEHHAVLHACDELEKAGYQVTYVPVNGFGRVNPADIEAAIRPDTFLISMMYANNEVGVIQPVQEVGQIAREHGIIFHVDAVQAFGHISIDCGNLPIDLLSVSGHKINGPQGVGALYIRQGTRIQPLMHGGLQEKKRRAGTENIAGIAGLAKAATLANASIEERGAHDMALRQTLLKALENSLGSHEFIINGDPEHFVPNVLNISFPAIGTETMLMNLDMEGIAAASGSACTSGSLE SHVLQAMDLPEDVLNSAIRFSFGLGNTTEEMEYTAKKIETIWKRLRTRY

Example 8-8. Antibiotics As described in Example 5, strain FPU-37 produces antibiotics.

Summary of Growth Promotion Effect of FPU-37 Strain From the above results, the molecular mechanism of the growth promotion effect on plants possessed by FPU-37 strain is “solubilization of poorly soluble phosphorus compounds in soil (supply of phosphorus to plants)” ”And“ production of plant growth hormones ”,“ suppression of the growth of phytopathogenic microorganisms by depriving the environment of iron with siderophores (plants can also produce siderophores) ”,“ sterilization of phytopathogenic microorganisms with hydrogen cyanide and antibiotics ”, It was suggested that this is a combined effect of multiple factors such as “inhibition of biosynthesis of ethylene that suppresses plant growth” and “nitrogen fixation”. The results are shown in Table 7.
The excellent antibacterial activity of the FPU-37 strain is expected to be applied not only to seed disinfectants but also to soil conditioners and biopesticides.

  ADVANTAGE OF THE INVENTION According to this invention, the novel Paenibacillus bacterium which has a high cellulose resolution and starch resolution | decomposability and produces a strong antimicrobial substance is provided. By using the bacterium, glucose can be produced from various cellulose raw materials and starch raw materials, and the glucose is useful for producing ethanol. Moreover, the efficiency of the ethanol production process can be improved by the effect of the antibacterial substance produced by the bacteria. The antibacterial substance itself can be usefully used as an agricultural material or a medicine.

Claims (25)

(A) having cellulose resolution and starch resolution;
(B) exhibits antibacterial activity against fungi other than bacteria and yeast,
(C) not having gelatin resolution and nitrate reducing ability,
A bacterium belonging to the genus Paenibacillus or a mutant thereof.   The bacterium according to claim 1 or a bacterium according to claim 1, wherein the fungus other than bacteria and yeast is one or more of phytopathogenic fungi, Staphylococcus, Streptococcus, Enterococcus, Escherichia and Fusarium. Mutant.   The bacterium according to claim 1 or 2, wherein the bacterium belonging to the genus Paenibacillus is Paenibacillus sp. FPU-37 strain (NITE P-01802).   An antibacterial extract of the bacterium according to any one of claims 1 to 3 or a mutant thereof.   The antibacterial extract according to claim 4, which is produced using acetonitrile as an extraction solvent.   The antibacterial extract according to claim 4, which is produced using a lower alcohol as an extraction solvent.   The antibacterial composition containing the antibacterial extract as described in any one of Claims 4-6.   The culture obtained by culture | cultivating the bacterium as described in any one of Claims 1-3, or its variant.   A composition comprising the bacterium according to any one of claims 1 to 3 or a variant thereof, and cellulose and / or starch.   The composition according to claim 9, further comprising yeast.   A kit for producing ethanol comprising the bacterium according to any one of claims 1 to 3, or a mutant thereof, yeast, and cellulose and / or starch.   A method for producing a cellulose and / or starch degradation product, comprising culturing the bacterium according to any one of claims 1 to 3 or a variant thereof in a medium containing cellulose and / or starch.   The method according to claim 12, wherein the cellulose and / or starch degradation product is glucose. (A) A step of converting cellulose and / or starch into glucose by culturing the bacterium according to any one of claims 1 to 3 or a variant thereof in a medium containing cellulose and / or starch. ,
(B) converting glucose into ethanol by culturing yeast in a medium containing glucose obtained in (a),
A method for producing ethanol comprising:   A method for producing ethanol, comprising culturing the bacterium according to any one of claims 1 to 3 or a mutant thereof, and yeast in a medium containing cellulose and / or starch.   The manufacturing method of an antimicrobial extract including the process of obtaining the extract of the bacterium as described in any one of Claims 1-3, or its variant by extraction using acetonitrile.   The manufacturing method of an antimicrobial extract including the process of obtaining the extract of the bacterium as described in any one of Claims 1-3, or its variant by extraction using a lower alcohol.   An agrochemical containing the bacterium according to any one of claims 1 to 3 or a mutant thereof, the culture according to claim 8, or the antibacterial extract according to any one of claims 4 to 7.   Seed disinfection containing the bacterium according to any one of claims 1 to 3 or a variant thereof, the culture according to claim 8 or the antibacterial extract according to any one of claims 4 to 7. Agent.   A plant growth promoter containing the bacterium according to any one of claims 1 to 3 or the culture according to claim 8.   A soil conditioner containing the bacterium according to any one of claims 1 to 3, the culture according to claim 8, or the antibacterial extract according to any one of claims 4 to 7.   A method for controlling a disease caused by a fungus other than bacteria or yeast, comprising a step of treating a plant with the agricultural chemical according to claim 18.   A seed disinfection method comprising a step of treating seeds with the seed disinfectant according to claim 19.   A method for promoting plant growth, comprising a step of treating a plant with the plant growth promoter according to claim 20.   The soil improvement method including the process of processing soil with the soil improvement agent of Claim 21.