JP5689560B1 - New microorganism - Google Patents

Abstract

An object of the present invention is to provide a novel microorganism useful for treating organic waste such as human waste and sludge. A new species of the genus Frateribacillus, 1) negative gram staining, forming spores, 2) negative oxidase test, positive catalase test, aerobic 3) 1% When D-galactose or sucrose is added as saccharide to LB medium (pH 8.0) containing 2% of sugar and 0.2% NaCl, acid generation is not performed, but D-glucose, lactose, D- A Frateribacillus flavalbus having characteristics such as acid generation when mannitol, glycerin or N-acetylglucosamine is added is provided. [Selection figure] None

Description

The present invention relates to a novel microorganism that is a new species of the new genus Frateribacillus. More specifically, the present invention relates to Frateribacillus flavalbus having the following characteristics 1) to 10).
1) Gram staining is negative, spore formation 2) Oxidase test is negative, catalase test is positive and aerobic 3) LB medium (pH 8.0) containing 1% sugar and 2.0% NaCl ), When D-galactose or sucrose is added as the sugar, acid generation is not performed, but when D-glucose, lactose, D-mannitol, glycerin or N-acetylglucosamine is added, acid generation is not performed. 4) Optimal growth temperature is 45 ° C. to 55 ° C., and growth is not confirmed at 35 ° C. or lower or 65 ° C. or higher 5) Optimal pH is 7.0 to 8.0, pH 5.7 or lower, or pH 10 Growth is not confirmed at more than 0. 6) Growth is confirmed even when NaCl is added to CYC medium to a final concentration of 7%. 7) The main menaquinone is MK7. The amino acid composition of tidoglycan includes alanine, glutamic acid, and meso-type diaminopimelic acid (meso-DAP) 9) The genomic DNA has a GC content of 51.2% to 52.4% 10) The most important cell fatty acid is iso -C16: 0.

  In the conventional composting process, fungi such as mold, Actinomycetales (actinomycetales) bacteria called actinomycetes, Thermoactinomyces genus Thermoactinomycetaceae (Thermoactinomycetaceae), Thermoactinomycetaceae (Thermoactinomycetaceae) Bacteria from the genus (Bacillus genus) and Geobacillus (Geobacillus genus) such as Bacillusae (Bacillaceae) decompose organic substances and compost them. These microorganisms have the ability to form spores, and in an environment where the temperature is higher than the growth temperature of each individual, they exhibit heat resistance in the spore state and can stably remain in the compost. Even after decomposition and assimilation, it can be repeatedly used as compost as back compost.

  In composting, aerobic fermentation is preferable to anaerobic fermentation because organic matter can be decomposed faster and malodors can be reduced. In the process of composting by aerobic fermentation, the temperature inside the compost exceeds 45 ° C within 10 days after the start of aerobic fermentation by aeration, and the high temperature state is maintained for a long time until the end of composting. The In the high temperature environment in the compost of 45 ° C. to 70 ° C., the micromonosporaceae family (Micromonosporaceae family), the streptomycetaceae family (Streptomycetaceae family), the thermomonosporaceae family (Thermomonosporanceae family), the Streptosporanidae family Cepidae family Cepidae department Actinomycetales (Actinomycetales) Actinomycetes group, thermoactinomycetaceae family (Thermoactinomycetaceae family) bacteria group having thermophilic and filamentous morphology is responsible for the degradation of organic matter (Non-patent Document 1). Moreover, the bacteria group of the Bacillusae family (Bacillaceae family) to which the genus Geobacillus belongs is also present in the compost under a high temperature environment (Non-patent Documents 2 and 3). Within the high temperature compost, fungi belonging to these limited taxonomic groups are mainly responsible for composting, but the species in the family are composed of fungi of similar properties. Therefore, generally, the fungus group in the compost after becoming high temperature is in a state of low diversity.

  Patent Document 1 describes a method for producing compost using a strain of the genus Anoxybacillus, and Patent Document 2 describes a method for producing compost using a strain of the genus Bacillus, both of which are Bacillusae (Bacillaceae family). Of bacteria.

  On the other hand, organic wastes for waste treatment are diverse, such as organic sludge, food residue, animal manure, etc. The pH, salt concentration, humic acid concentration, C / N ratio in compost are different depending on the type of waste materials. It will change greatly. For this reason, in order to efficiently compost various organic wastes, add fungi that are different from existing fungi and grow them in compost, and decompose and assimilate organic matter with various fungi. It is desirable. In addition, it is desirable that the added fungal group has a spore-forming ability, can maintain a dormant state, and can withstand environmental changes. The stable spore state is easy to store and transport, and it is economical because it can be used repeatedly as back compost.

  So far, the present inventors have searched for a novel bacterium that can be used for composting and found a hyperthermophilic bacterium belonging to a novel genus that grows in an environment of 80 ° C. or higher (Patent Document 3).

JP 2011-24569 A JP 2008-278890 A JP 2005-13063 A

Journal of bioscience and bioengineering, Vol. 99, p1-11, 2005 International Journal of Systemic and Evolutionary Microbiology, Vol. 52, p2251-2255, 2002 Systematic and Applied Microbiology, Vol. 34, p419-423, 2011

  An object of the present invention is to provide a novel microorganism useful for the treatment of organic waste such as human waste and sludge.

The present inventors have found a novel microorganism, and isolated and identified it, thereby completing the present invention.
That is, the present invention is as follows.
[1] A new species of the genus Frateribacillus and the following 1) to 10), the Frateribacillus flavalbus.
1) Neisseria gonorrhoeae with negative Gram staining and spore formation 2) Negative oxidase test, positive catalase test and aerobic 3) LB medium (pH 8.0) containing 1% sugar and 2% NaCl ), When D-galactose or sucrose is added as the sugar, acid generation is not performed, but when D-glucose, lactose, D-mannitol, glycerin or N-acetylglucosamine is added, acid generation is not performed. 4) Optimal growth temperature is 45 ° C. to 55 ° C., and growth is not confirmed at 35 ° C. or lower or 65 ° C. or higher 5) Optimal pH is 7.0 to 8.0, pH 5.7 or lower, or pH 10 Growth is not confirmed at more than 0.0 6) Growth is confirmed even when NaCl is added to CYC medium to a final concentration of 7% 7) The main menaquinone is MK7 8) Cell wall 9) The GC content of genomic DNA is 51.2% to 52.4%, including alanine, glutamic acid and meso-type diaminopimelic acid (meso-DAP) as the amino acid composition of ptidoglycan. 10) The most important cell fatty acid is iso -C16: 0 [2] having a 16S rRNA gene comprising a base sequence having a homology of 98.7% or more with respect to the base sequence shown in any of SEQ ID NO: 1 or SEQ ID NO: 8 to SEQ ID NO: Frateribacillus flavalbus described in [1] above.
[3] YM17 strain (NITE BP-0176), YM17-2 strain (NITE BP-01948), YM17-3 strain (NITE BP-01949), YM17-4 strain (NITE BP-01950) or YM17-5 strain ( Frateribacillus flavalbus according to [1] or [2] above, wherein the DNA-DNA hybridization homology value with NITE BP-01925) is 70% or more.
[4] Any one of the above [1] to [3], further including any one or more of anteiso-C15: 0, C16: 0, anteiso-C17: 0, and iso-C17: 0 as bacterial fatty acids Frateribacillus flavalbus described in 1.
[5] Frateribacillus flavalbus subspecies flavalbus including anteiso-C15: 0, anteiso-C17: 0, and iso-C17: 0 in addition to iso-C16: 0 as microbial fatty acids (Frateribacillus flavalbus subsp. Flavalbus) Frateribacillus flavo albus according to any one of [1] to [4] above.
[6] Frateribacillus flavalbus subspecies fimetalium containing anteiso-C15: 0, C16: 0, anteiso-C17: 0 in addition to iso-C16: 0 as a cell fatty acid (Frateibacillus flavalbus subsp. Fimetarium) Frateribacillus flavalbus (Frateribacillus flavalbus) according to any one of [1] to [4] above.
[7] Frateribacillus flavalbus used in the treatment of organic waste according to any one of [1] to [6] above.
[8] The Frateribacillus flavalbus according to [7] above, wherein the organic waste is human waste, organic sludge, food residue, or animal manure.
[9] YM17 strain (NITE BP-0176), NaCl_2013016_04 strain, YM17-2 strain (NITE BP-01948), YM17-3 strain (NITE BP-01949), YM17-4 strain (NITE BP-01950), NaCl_2013016_19 strain Any of the above [1] to [8] Frateribus flavalbus.
[10] A method for treating organic waste using the Frateribacillus flavalbus according to any one of [1] to [9] above.
[11] A protein degradation method using the Frateribacillus flavalbus according to any one of [1] to [9] above.

  The novel microorganism obtained by the present invention is a new species of the new genus Frateribacillus. This novel microorganism is useful for the treatment of organic waste such as human waste and sludge, protein degradation, and the like, and can be used for various applications.

It is the figure which showed the cell photograph of YM17 stock | strain (Example 1). (Example 1) which was the figure which showed the phylogenetic tree created by the neighborhood joint method. It is the figure which showed the phylogenetic tree by the 16S rRNA gene of 12 strains isolate | separated from YM17 strain | stump | stock (Example 2). It is the figure which showed the state of the microbe in compost | cultivation culture (Example 2). It is the figure which showed the result of gelatin zymography (Example 2).

The Frateribacillus flavalbus of the present invention refers to a new species of the genus Frateribacillus having the following characteristics 1) to 10).
1) Gram staining is negative, spore formation 2) Oxidase test is negative, catalase test is positive and aerobic 3) LB medium (pH 8.0) containing 1% sugar and 2.0% NaCl ), When D-galactose or sucrose is added as the sugar, acid generation is not performed, but when D-glucose, lactose, D-mannitol, glycerin or N-acetylglucosamine is added, acid generation is not performed. 4) Optimal growth temperature is 45 ° C. to 55 ° C., and growth is not confirmed at 35 ° C. or lower or 65 ° C. or higher 5) Optimal pH is 7.0 to 8.0, pH 5.7 or lower, or pH 10 Growth is not confirmed at more than 0. 6) Growth is confirmed even when NaCl is added to CYC medium to a final concentration of 7%. 7) The main menaquinone is MK7. The amino acid composition of tidoglycan includes alanine, glutamic acid, and meso-type diaminopimelic acid (meso-DAP) 9) The genomic DNA has a GC content of 51.2% to 52.4% 10) The most important cell fatty acid is iso -C16: 0.

Examples of the “main menaquinone” include 81.8% or more of MK7.
Moreover, what contains 34.1% or more and 42.6% or less of iso-C16: 0 is mentioned as "main microbial cell fatty acid".
Furthermore, as “bacterial fatty acid”, in addition to iso-C16: 0, it may contain any one or more of anteiso-C15: 0, C16: 0, anteiso-C17: 0, and iso-C17: 0 good. The Frateribacillus flavalbus of the present invention preferably contains 86.1% or more of these fatty acids in total.
Further, the Frateribacillus flavalbus of the present invention is 16S rRNA comprising a base sequence having a homology of 98.7% or more with respect to the base sequence shown in SEQ ID NO: 1 or SEQ ID NOs: 8 to 19. It preferably has a gene.
Further, the Frateribacillus flavalbus of the present invention has 70% homology between DNA-DNA hybridization with YM17 strain, YM17-2 strain, YM17-3 strain, YM17-4 strain or YM17-5 strain. Is preferred.

Such a flattery flavus flavalbus of the present invention has the properties of 1) to 10) above, and in addition to iso-C16: 0 as a cell fatty acid, anteiso-C15: 0, anteiso- Examples include Frateribacillus flavoarubus subspecies flavoarubus (Ferrateribacillus flavalbus subsp. Flavalbus) containing C17: 0, iso-C17: 0.
Examples of such flattery flavus flavalbus subspecies flavalbus (Frateribacillus flavalbus subsp. Flavalbus) include the YM17 strain, the YM17-5 strain, and the like.

Further, the present invention has the properties of the above 1) to 10) as Frateribacillus flavalbus, and in addition to iso-C16: 0 as a cell fatty acid, anteiso-C15: 0, C16: 0 Also included is Frateribacillus flavalbus subsp. Fimetalium, including anteiso-C17: 0.
As such Frateribacillus flavalbus subsp. Strains, NaCl_2013016_37 strain, NaCl_2013016_38 strain, and the like.

The YM17 strain mentioned as Frateribacillus flavalbus of the present invention has the above-mentioned characteristics, and the base sequence of 16S rRNA is 100% identical to the base sequence described in SEQ ID NO: 1.
This strain has been deposited internationally based on the Budapest Treaty at the National Institute for Product Evaluation and Technology (NITE) Patent Microorganism Deposit Center upon application of the applicant. The contents specifying the deposit are described below.
(1) Name of depositary institution: National Institute of Technology and Evaluation, Patent Microorganisms Deposit Center (2) Contact: 2-5-8 Kazusa Kamashi, Kisarazu City, Chiba Prefecture 292-0818
Phone number 0438-20-5580
(3) Accession number: NITE BP-0176
(4) Display for identification: YM17
(5) Original deposit date: November 29, 2013

  Similarly, YM17-2 strain, YM17-3 strain, YM17-4 strain and YM17-5 strain mentioned as the Frateribacillus flavalbus of the present invention may also be incorporated by the applicant's application. It is deposited internationally based on the Budapest Treaty at the National Institute for Evaluation Technology (NITE) Patent Microorganism Depositary. The contents specifying these deposits are shown in the examples.

The Frateribacillus flavalbus of the present invention also includes mutant strains of strains corresponding to the Frateribacillus flavalbus. Also, among the Frateribacillus flavalbus, Frateribacillus flavalbus subspecies Frabalibus flavus s mu sp.
This mutant strain in the present invention is one whose physical characteristics and chemical characteristics have been modified by genetic engineering techniques such as chemical mutation introduction, natural mutation, morphological variation, and transfection. It has the characteristic of 10).

The Frateribacillus flavalbus of the present invention can be used for the treatment of organic waste such as organic sludge, food residues, or animal manure. Compost etc. can be produced by adding Frateribacillus flavalbus to these organic wastes.
In the organic waste processing method using the Frateribacillus flavalbus of the present invention, not only the Frateribacillus flavalbus is added to the organic waste, but also other processes useful in the treatment of the organic waste are included. It may be included.

Furthermore, the Frateribacillus flavalbus of the present invention can be used for protein degradation.
In the method for degrading a protein using Frateribacillus flavalbus according to the present invention, not only Frateribacillus flavalbus is added to a protein or protein-containing degradation target, but also other steps useful in the degradation of the protein are included. It may be included.

  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]
1. Isolation and identification of Frateribacillus flavalbus Isolation of the strain The YM17 strain was isolated from the compost of the composting plant in Suo City, Kagoshima Prefecture.
That is, 4 mL of 1/2 Nutrient medium containing 50 μg / mL Novobiocin (peptone: 0.25%, sodium chloride: 0.25%, yeast extract: 0.1%, meat extract: 0.05%, pH 7.4) After adding 400 mg of the compost and culturing at 55 ° C. for 2 days, this liquid culture was applied to 1/2 Nutrient agar medium containing 50 μg / mL Novobiocin and cultured for 4 days to isolate a colony of YM17 strain.

2. Taxonomic properties Test methods The taxonomic properties of the strains are “Shinsei Kagaku Kogaku Koza 17 Volume Microbial Experiments” (published by Tokyo Kagaku Dojin), “Revised Classification and Identification of Microorganisms <below” (published by the Academic Publishing Center), Classification and identification experiment method (issued by Springer Japan) ", BERGEY'S MANUAL OF Systematic Bacteriology. Classification and identification of the isolated strains were performed by comparison with the species described in International Journal of Systemic and Evolutionary Microbiology.
The morphological properties, culture properties, physiological properties, growth ranges, and chemical taxonomic properties obtained by this analysis are shown in Tables 2-1 and 2-5. Moreover, the cell photograph which culture | cultivated YM17 stock | strain at 55 degreeC was shown in FIG. In FIG. 1, it was confirmed that the YM17 strain is a Neisseria gonorrhoeae and forms a spore at the end of the cell. The bar in FIG. 1 represents 10 μm. Table 2 (Tables 2-1 to 2-5) also shows the taxonomic properties of the 12 strains isolated in Example 2.

B. Test medium The pH of each medium is as described in Table 2-1. Each medium except litmus milk was autoclaved at 121 ° C. for 15 minutes. The litmus milk was autoclaved at 110 ° C. for 10 minutes. The urea in the Christensen urea medium, the sugar in the OF test medium, and the saccharides that were examined for acid production from the sugar were filter sterilized and added to each medium to the stated final concentrations. Acid production from sugar was examined using LB medium (pH 8.0) containing 1% sugar and 2% NaCl.

(1) CYC agar medium (sucrose: 3%, casamino acid: 0.6%, sodium nitrate: 0.3%, yeast extract: 0.2%, dipotassium hydrogen phosphate: 0.1%, magnesium sulfate seven (Hydrate: 0.05%, Potassium chloride: 0.05%, Iron sulfate heptahydrate: 0.001%, Agar: 1.5%)
(2) CYC liquid medium (sucrose: 3%, casamino acid: 0.6%, sodium nitrate: 0.3%, yeast extract: 0.2%, dipotassium hydrogen phosphate: 0.1%, magnesium sulfate seven Hydrate: 0.05%, potassium chloride: 0.05%, iron sulfate heptahydrate: 0.001%)
(3) Meat juice agar medium (meat extract: 1%, peptone: 1%, sodium chloride: 0.5%, agar: 1.5%)
(4) Meat broth liquid medium (meat extract: 1%, peptone: 1%, sodium chloride: 0.5%)
(5) Litmus milk (skimmed milk: 10%, 1% litmus liquid: several drops)
(6) VP medium (yeast extract: 0.1%, tryptone: 0.7%, soyton: 0.5%, glucose: 1%, sodium chloride: 0.5%, agar: 0.3%)
(7) SIM medium (meat extract: 0.3%, peptone: 3%, sodium thiosulfate: 0.005%, cysteine hydrochloride: 0.02%, ammonium iron citrate: 0.05 g, agar: 0.5 %)
(8) LB agar medium (tryptone: 1%, sodium chloride: 0.5%, yeast extract: 0.5%, agar: 1.5%)
(9) SY agar medium (ammonium sulfate: 0.2%, disodium hydrogen phosphate: 0.14%, potassium dihydrogen phosphate: 0.07%, magnesium sulfate heptahydrate: 0.02%, iron sulfate (Heptahydrate: 0.0005%, manganese sulfate pentahydrate: 0.0005%, glucose: 1%)
(10) Nutrient agar medium (meat extract: 0.3%, peptone: 0.5%, agar: 1.5%)
(11) Simmons medium (ammonium dihydrogen phosphate: 0.1%, dipotassium hydrogen phosphate: 0.1%, sodium chloride: 0.5%, sodium citrate: 0.2%, magnesium sulfate heptahydrate Material: 0.02%, Agar: 1.5%, BTB: 0.008%)
(12) Christensen medium (yeast extract: 0.05%, cysteine hydrochloride: 0.01%, sodium chloride: 0.5%, sodium citrate: 0.3%, glucose: 0.02%, diphosphate) (Potassium hydrogen: 0.1%, agar: 1.5%, phenol red: 0.0012%)
(13) Nitrate medium (glucose: 1.0%, potassium dihydrogen phosphate: 0.1%, magnesium sulfate heptahydrate: 0.05%, potassium chloride: 0.02%, sodium nitrate: 0.1 %)
(14) Ammonium salt medium (glucose: 1.0%, potassium dihydrogen phosphate: 0.1%, magnesium sulfate heptahydrate: 0.05%, potassium chloride: 0.02%, ammonium sulfate: 0.078 %)
(15) Christensen urea medium (peptone: 0.1%, sodium chloride: 0.5%, glucose: 0.1 g, KH2PO4: 0.2%, phenol red: 0.0008%, agar: 1.5%, (Urea: 2%)
(16) OF test medium (peptone: 0.2%, sodium chloride: 0.5%, dipotassium hydrogen phosphate: 0.03%, agar: 0.2%, BTB: 0.008%, sugar: 1 %)

C. Analysis of fatty acids 1) Sample preparation and analysis by gas chromatography Inoculate the seed cultures into the main culture medium containing 3L CYC liquid medium in a 5L Erlenmeyer flask, and at 52 ° C from the late logarithmic growth phase to the stationary phase The cells were cultured until the beginning, and the cells were collected by centrifugation. The collected cells were washed twice with physiological saline. About 500 mg to 700 mg of wet cells were placed in a centrifuge tube with a screw cap. 1 mL of alkaline saponification solution was added, sealed and kept at 100 ° C. for 5 minutes. After 5 minutes, it was shaken well for 10 seconds and further kept at 100 ° C. for 30 minutes. After the centrifuge tube was cooled, 2 mL of methanolic hydrochloric acid reagent was added, sealed, and kept at 80 ° C. for 10 minutes. After cooling the centrifuge tube, 1.25 mL of extraction solvent was added and stirred slowly for 10 minutes. After centrifuging the centrifuge tube at 1000 rpm for 5 minutes, the upper layer was transferred to another centrifuge tube with a screw cap, and 3 mL of washing solution was added. Sealed again and stirred for 5 minutes. After centrifuging the centrifuge tube at 1000 rpm for 5 minutes, the solvent layer was taken in a glass ampoule and dried with nitrogen gas. The composition of each reagent is shown below. The obtained dried sample was analyzed by gas chromatography at Techno Suruga Lab Co., Ltd. as a fatty acid analysis sample. The obtained results are shown in Table 1.

2) Reagent (1) Alkaline saponification solution (sodium hydroxide: 7.5 g, methanol: 25 mL, ultrapure water: 25 mL)
(2) Hydrochloric acid methanol reagent (6N hydrochloric acid: 21.7 mL, methanol: 18.3 mL)
(3) Extraction solvent (hexane: 10 mL, t-butyl methyl ether: 10 mL)
(4) Cleaning solution (sodium hydroxide: 0.586 g, ultrapure water: 48.8 mL)

D. Cell wall peptidoglycan 1) Preparation of sample Approximately 2 g of wet cells were suspended in 6 mL of 50 mM phosphate buffer (pH 7.2), and the cells were disrupted by treatment with an ultrasonic disrupter (VP-30S manufactured by TAITEC) for 30 minutes. did. The crushed liquid was centrifuged at 3000 rpm for 10 minutes at a temperature of 4 ° C., and the supernatant was collected. The supernatant was centrifuged at 9000 rpm for 60 minutes at a temperature of 4 ° C., the supernatant was removed, and the precipitate was transferred to a new centrifuge tube. The precipitate was washed by adding 6 mL of 50 mM phosphate buffer (pH 7.2), and then centrifuged at 9000 rpm for 60 minutes at a temperature of 4 ° C. to remove the supernatant. 2 mL of 50 mM phosphate buffer (pH 7.2) and 400 μL of 25% SDS were added to the precipitate and kept at 100 ° C. for 40 minutes. 10 mL of 50 mM phosphate buffer (pH 7.2) warmed to 65 ° C. was further added and stirred, and then centrifuged at 9000 rpm for 60 minutes at a temperature of 30 ° C.

After removing the supernatant and washing the precipitate with 5 mL of 50 mM phosphate buffer (pH 7.2) warmed to 65 ° C., the supernatant was centrifuged at 9000 rpm for 30 minutes at 30 ° C. Removed. This washing operation was performed twice. After washing, 2 mL of 50 mM phosphate buffer (pH 7.6) and 100 μL of 1 mg / mL Pronase solution were added to the precipitate and kept at 37 ° C. for 2 hours. 5 mL of 50 mM phosphate buffer (pH 7.6) was added to the solution after this Pronase reaction to wash the cell wall components, and then centrifuged at 9000 rpm for 30 minutes at a temperature of 4 ° C. to remove the supernatant. This washing operation was performed twice.
2 mL of 5% trichloroacetic acid aqueous solution was added to the washed precipitate, and kept at 100 ° C. for 20 minutes. This suspension was transferred to a glass screw cap centrifuge tube, washed with 5 mL of 50 mM phosphate buffer (pH 7.2), and then centrifuged at 1000 rpm for 1 minute at room temperature. Removed. This washing operation was performed twice. 3 mL of ethanol was added to the washed precipitate, followed by centrifugation at 1000 rpm for 1 minute at room temperature, and the supernatant was removed. 3 mL of diethyl ether was added to the precipitate, centrifuged at 1000 rpm for 1 minute at room temperature, the supernatant was removed, and the precipitate was dried to obtain a cell wall sample.

2) Analysis by TLC 250 μL of 4N hydrochloric acid was added to a 5 mg cell wall sample, and a hydrolysis reaction was performed at 100 ° C. for 16 hours. After the hydrolysis reaction, hydrochloric acid was removed in a desiccator where potassium hydroxide was placed, and the resulting treatment solution was subjected to TLC.
The TLC plate is TLC cellulose plate No. manufactured by Merck. 1.05716.0001 was used. In detection of constituent amino acids, the first dimension is developed with a developing solvent of isopropanol: acetic acid: ultra pure water = 75: 10: 15, and the second dimension is methanol: pyridine: 10N hydrochloric acid: ultra pure water = 64: 8: 2: 14 developed. After the development, the ninhydrin reagent was sprayed on the TLC plate, and amino acids contained in the cell wall peptidoglycan were detected. In addition, the detection of the isomeric form of diaminopimelic acid was carried out using the same TLC plate with methanol: pyridine: 10N hydrochloric acid: ultra pure water = 64: 8: 2: 14, and identified by comparing the mobility with a standard product. . As a result, it was confirmed that the amino acid composition of the cell wall peptidoglycan contains alanine, glutamic acid and meso-type diaminopimelic acid (meso-DAP).

E. Menaquinone 1) Sample preparation 20 mL of chloroform: methanol = 2: 1 solution was added to about 500 mg of dried cells and gently stirred overnight in a cool and dark place. This suspension was filtered, and the filtrate was concentrated and dried by a rotary evaporator having a hot water bath temperature of 30 ° C. The dried solid was dissolved in 3 mL of acetone and concentrated to about 200 μL with nitrogen gas. HPLC LC Silica Gel 60 F254 Plate No. manufactured by Merck This concentrated solution was tested at 1.0562.0001 and developed using toluene as a developing solvent. After the development, the TLC plate was dried, the menaquinone band was confirmed under a UV lamp (254 nm), and the silica gel in that portion was scraped off with a spatula and placed in a test tube. 4 mL of acetone was added to the scraped silica gel, and the mixture was gently stirred and filtered to remove silica gel particles. The obtained supernatant was concentrated to about 200 μL with nitrogen gas, and used as a sample for HPLC.

2) Analysis by HPLC A 25 μL sample was injected into the HPLC. The column used was an ODS column (CAPISEL PAK C18 UG120 S-5 manufactured by SHISEIDO), and methanol: isopropanol = 2: 1 was used as the developing solvent. Development was carried out at a moving speed of 1.5 mL / min and a column temperature of 40 ° C., and detection was performed with UV (270 nm). As the HPLC machine, SERIES 1100 manufactured by HEWLETT PACKART was used. When this analysis was performed a plurality of times, the content of MK7 was 83.9% for the first time and 85.2% for the second time, confirming that the main menaquinone was MK7.

F. GC content 1) DNA preparation Genomic DNA was extracted according to the method of Reference 1.
Specifically, 0.9 to 1.6 g of wet cells were washed with Saline-0.1M EDTA (pH 8.0) and suspended in 5 mL of the same solution. Egg white lysozyme was added at a final concentration of 1 mg / mL and lysed at 35 ° C., and then the lysate was frozen. An equal amount of Tris-SDS (pH 9.0) was added to the frozen product, thawed in a 60 ° C. hot water bath, and kept for 10 minutes. Thereafter, the mixture was ice-cooled, an equal amount of water-saturated phenol was added, and the mixture was shaken for 10 minutes. The supernatant was collected by centrifugation, twice the amount of ethanol was added, and the fibrous DNA was wound up with a glass rod. The wound DNA was washed stepwise with 70%, 90%, and 99.5% ethanol to remove phenol. After the DNA was dried, it was dissolved in an appropriate amount of 0.1XSSC solution, RNase was added, and the mixture was reacted at 35 ° C. for 30 minutes or more to remove RNA. After ice cooling, 1/5 amount of water-saturated phenol was added, and similarly, shaking, centrifugation, ethanol precipitation, phenol removal, dissolution with 0.1XSSC solution, and RNase treatment were performed.
The DNA after RNase treatment is treated with phenol, centrifuged to collect the DNA-lysed fraction, and 1/10 amount of Acetate-EDTA and 0.6 times the amount of isopropanol are added to the DNA-lysed fraction, and the DNA is wound up again. It was. The wound DNA was washed with 70%, 90%, and 99.5% ethanol, dried, and then dissolved in a 0.1XSSC solution so that the DNA concentration was 500 μg / mL or more. When the purity of DNA was low, protein removal with phenol and RNase treatment were repeated.
Reference 1: Saito, H .; & Miura, K .; 1963. Preparation of transforming deoxyribonucleic acid by phenol treatment. Biochim. Biophys. Acta 72: 619-629.

2) Reagent (1) Saline-0.1M EDTA (NaCl: 8.77 g, EDTA · 2Na · 2H 2 O: 37.22 g, pH: 8.0, ultrapure water: added so that the total liquid volume becomes 1000 mL )
(2) Tris-SDS (Tris: 12.1 g, SDS: 20.0 g, pH 9.0, ultrapure water: added so that the total liquid volume becomes 1000 mL)
(3) 20XSSC (NaCl: 175 g, Trisodium citrate: 77.4 g, ultrapure water: added so that the total liquid volume becomes 1000 mL. 0.1XSSC and 1XSSC were prepared by diluting 20XSSC with ultrapure water)
(4) Acetate-EDTA (A liquid: CH3COONa.3H2O: 81.65 g / 200 mL of ultrapure water, B liquid: CH3COOH: 36.03 g / 200 mL of ultrapure water. A liquid and B liquid are mixed to pH 7.0. EDTA · 2Na · 2H 2 O: 74.45 mg / acetate-EDTA as adjustment solution 200 mL)

3) Measurement of GC content The GC content was measured using DNA GC kit (manufactured by Yamasa Shoyu Co., Ltd.). 7 μg of DNA was dissolved in 20 μL of 0.1XSSC, heat-denatured at 100 ° C. for 10 minutes, and immediately cooled on ice. After cooling with ice, 20 μL of 4 U / mL nuclease P1 solution was added and reacted at 50 ° C. for 1 hour. After treatment with nuclease P1, 20 μL of 2.4 U / mL alkaline phosphatase solution was added and reacted at 37 ° C. for 1 hour for use in HPLC.
As an HPLC column, an ODS column (CAPISEL PAK C18 UG120 S-5 manufactured by SHISEIDO) was used, and 0.02 M NH4H2PO4: acetonitrile = 20: 1 solution was used as a developing solvent. Development was performed at a moving speed of 1.0 mL / min and a column temperature of 40 ° C., and detection was performed with UV (270 nm). When the GC content of the YM17 strain was calculated based on the standard attached to the DNA GC kit, the GC content was 51.4%.

G. Analysis of nucleotide sequence of 16S rRNA gene 1) PCR reaction The 16S rRNA gene of the YM17 strain was amplified by colony direct PCR. For the PCR reaction, Tks Gflex (registered trademark) DNA Polymerase (Takara Bio Inc.), EU10F primer (SEQ ID NO: 2) and EU1500R primer (SEQ ID NO: 3) were used. The composition of the PCR reaction solution was in accordance with the mixing ratio described in the instruction manual of Tks Gflex (registered trademark) DNA Polymerase. In the PCR reaction, 1 cycle of heat treatment at 94 ° C. was performed for 1 cycle, and then 30 cycles of reaction at 98 ° C. for 10 seconds, 55 ° C. for 15 seconds, and 68 ° C. for 1 minute 30 seconds were performed.

2) Sequence reaction The obtained PCR reaction product was purified by Wizard (registered trademark) SV Gel and PCR Clean-Up System (Promega). A sequence reaction was performed using the purified product and EU10F primer, EU520F primer (SEQ ID NO: 4), EU907R primer (SEQ ID NO: 5), or EU1500R primer, and the base sequence of the 16S rRNA gene of the isolated strain was determined. .
The nucleotide sequence of 16S rRNA gene of YM17 strain is shown in SEQ ID NO: 1 in the sequence listing.

BLAST homology search (http://blast.ncbi.nlm.nih.gov/Blast.cgi) of the YS17 strain 16S rRNA gene base sequence of The National Center for Biotechnology Information (http://blast.ncbi.nlm.nih.gov/Blast.cgi), 17 results. The species exhibiting the highest homology with the 16S rRNA gene was Mechacharimyces aspophorigenens with a homology of 91%.
Subsequently, a 16S rRNA gene related to the YM17 strain was collected from the gene database of The National Center for Biotechnology Information, and phylogenetic analysis was performed.
The collected 16S rRNA gene base sequences were aligned by the Clustal X 2.0.12 program, and a phylogenetic tree was created by the neighborhood joining method. The obtained phylogenetic tree is shown in FIG. The numbers in parentheses indicate GenBank accession numbers, and YM16 indicates a strain isolated by the present inventors in the same manner as the present invention.

H. Identification and Classification of YM17 Strain 1) YM17 Strain As shown in the phylogenetic analysis of FIG. 2, the 16S rRNA gene of the YM17 strain is a moth family (Bacillaceae family), alicyclobatillaceae family, Thermoactinomyceta family (Themoactinaceae family) A branch that is largely divided is formed.
The form of gonococci and the form of spore formation in the cells indicated by the YM17 strain are the properties found in the Bacillusae family (Bacylaceae family) and Alicyclobacteridae family (Alicylobacillaceae family), and the thermoactinomycetes that form filamentous mycelia It is clearly different from the family (Themoactinomycetaceae family).
The homology of 16S rRNA gene sequences of species belonging to the YM17 strain and the species belonging to the family Bacillaceae (family Bacillaceae) and the family Alicyclobacilaceae (family Alicyclobacillaceae) was compared. The results are shown in Table 3. In all genera, the homology is 89% or less, and the YM17 strain is greatly taxonomically different from the species belonging to the family Bacillusae (Bacilliaceae) and Alicyclobacilaeae (Alicylobacillaceae). In the phylogenetic tree of FIG. 2, the alicyclobatillas acid caldarius (Alycyclobacillus acidocaldarius), Calditericula satsumensis (Calditericula satsumensis), Caldarbali calsium calbalis caldalbalis calmarbium (Caldalkalibalis calma) The taxonomic properties of the strains were compared. The results are shown in Table 4.

  The data other than YM17 strain in Table 4 are International Journal of Systemic and Evolutionary Microbiology (2002), 52, 1681-1685, Journal of General Microbiology (1971), 67 ), 61, 631-636, International Journal of Systemic and Evolutionary Microbiology (2006), 56, 1217-1221, and International Journal. l of Systematic and Evolutionary Microbiology (2004), 54, 2197-2201.

The genus Alicyclobacillus (genus Alicyclobacillus) is characterized by having ω-Cyclohexane-C17: 0 and ω-Cyclohexane-C19: 0 as main fatty acids, which is different from the YM17 strain.
The genus Calditericola (genus Calditericola) is a highly thermophilic bacterium that can grow at 75 ° C. or higher and is different from the YM17 strain.
Unlike the YM17 strain, the genus Caldalkalibibacillus is positive for Gram staining and the base composition of DNA is 45%.
The genus Caldivibacillus is different from the YM17 strain because it grows even at 65 ° C. or higher and acid production from glucose is not observed.
Based on the above, the YM17 strain is determined as a new species of the new family (Flateribatiridae) and a new genus (Flateribatillas genus), and the YM17 strain is referred to as Frateribacillus flavalbus (hereinafter sometimes simply referred to as Frateribacillus flavalbus). Named.

[Example 2]
Separation of Frateribacillus flavalbus species According to Example 1 above, it was shown that Frateribacillus flavalbus species differ from known families, genera, and species. Therefore, in order to clarify the characteristics of Frateribacillus flavoarubus species, strains belonging to Frateribacillus flavoarubus species other than the YM17 strain were isolated.

1. Separation of Frateribacillus flavoarubus species We tried to isolate a strain of Frateribacillus flavoarubus species using the composting of the composting plant in Kaoshima Prefecture and Kagoshima City as the separation source.
With reference to the culture characteristics of the YM17 strain in Example 1 above, a culture medium with CYC agar added with or without 7% or 10% sodium chloride and 50 μg / mL ampicillin as a separation medium, and a culture temperature of 55 ° C. When the bacteria were separated by culturing for 6 days, numerous colonies of filamentous bacteria and colonies of gonococcal bacteria appeared on the separation medium.
From these, a colony of gonococcal bacteria is isolated and the following 2. As shown in Table 5, a total of 12 strains were determined to be Frateribacillus flav albus species as shown in Table 5.
In particular, the separation frequency is high in a medium in which 7% sodium chloride and 50 μg / mL ampicillin are added to a CYC agar medium. By culturing at 55 ° C. using this medium, it is possible to efficiently isolate the Frateribacillus flav albus species. Met.

For the 12 isolated strains, NaCl_2013016_04 strain, YM17-2 strain, YM17-3 strain, YM17-4 strain, NaCl_2013016_19 strain, YM17-5 strain, NaCl_20131016_24 strain, NaCl_20131016_28 strain, NaCl_20131016_30 strain, NaCl_201316_34_ strain_Na_201616_16_____________ It was.
Of these, the YM17-2, YM17-3, YM17-4 and YM17-5 strains were submitted to the Budapest Treaty by the National Institute of Technology and Evaluation (NITE) Patent Microorganism Depositary upon application of the applicant. Based on international deposits. The contents specifying the deposit are described below.

YM17-2 strain (1) Depositary institution: National Institute of Technology and Evaluation, Patent Microorganism Depositary Center (2) Contact: 2-5-8 Kazusa Kamashichi, Kisarazu City, Chiba Prefecture 292-0818
Phone number 0438-20-5580
(3) Accession number: NITE BP-01948
(4) Display for identification: YM17-2
(5) Original deposit date: October 6, 2014

YM17-3 strain (1) Depositary institution: National Institute of Technology and Evaluation, Patent Microorganisms Deposit Center (2) Contact: 2-5-8 Kazusa Kamashichi, Kisarazu City, Chiba Prefecture 292-0818
Phone number 0438-20-5580
(3) Accession number: NITE BP-01949
(4) Display for identification: YM17-3
(5) Original deposit date: October 6, 2014

YM17-4 shares (1) Depositary institution: National Institute of Technology and Evaluation, Patent Microorganisms Deposit Center (2) Contact: 2-5-8 Kazusa Kamashichi, Kisarazu City, Chiba Prefecture 292-0818
Phone number 0438-20-5580
(3) Accession number: NITE BP-01950
(4) Display for identification: YM17-4
(5) Original deposit date: October 6, 2014

YM17-5 shares (1) Depositary institution: National Institute of Technology and Evaluation, Patent Microorganisms Deposit Center (2) Contact: 2-5-8 Kazusa Kamashichi, Kisarazu City, Chiba Prefecture 292-0818
Phone number 0438-20-5580
(3) Accession number: NITE BP-012925
(4) Display for identification: YM17-5
(5) Original deposit date: August 28, 2014

2. Specific PCR for the detection of Frateribacillus flavo albus species
Specific PCR for detecting Frateribacillus flav albus species was performed as follows. First, using a base sequence specific for the 16S rRNA gene of YM17 strain, TIP155F1 primer (5′-ATACCGGATATAGAGGCTTTT-3 ′) (SEQ ID NO: 6) and TIP155R1 primer (5′-TGGTACCGTCACGCTGAGA-3 ′) (arrangement) Column table SEQ ID NO: 7) was prepared.
In the PCR reaction, Tks Gflex (registered trademark) DNA Polymerase, TIP155F1 primer, TIP155R1 primer, and DNA of the isolated strain were used. The composition of the PCR reaction solution was in accordance with the mixing ratio described in the instruction manual of Tks Gflex (registered trademark) DNA Polymerase. The PCR reaction was carried out for 1 cycle at 94 ° C for 1 minute, followed by 25 cycles of reaction at 98 ° C for 10 seconds, 55 ° C for 15 seconds and 68 ° C for 1 minute, and the amplification product was found around 328 bp by electrophoresis. The confirmed strain was determined to be Frateribacillus flavobas species.

3. Taxonomic analysis of isolates 1. And 2. The taxonomic properties of 12 strains isolated by the above were examined by the same method as in Example 1 for the YM17 strain, and the results are shown in Tables 2-1 to 2-5.

4). Analysis of base sequence of 16S rRNA gene of isolated isolate. And 2. The base sequences of 16S rRNA genes of 12 strains isolated by the above were examined in the same manner as in Example 1 for the YM17 strain. The nucleotide sequences of 16S rRNA genes of each strain are shown in SEQ ID NOs: 8 to 19, respectively. SEQ ID NO: 8 is NaCl_2013016_04, SEQ ID NO: 9 is YM17-2, SEQ ID NO: 10 is YM17-3, SEQ ID NO: 11 is YM17-4, SEQ ID NO: 12 is NaCl_2013016_19, SEQ ID NO: 13 is YM17-5, SEQ ID NO: 14 corresponds to the NaCl_20130316_24 strain, SEQ ID NO: 15 corresponds to the NaCl_20131016_28 strain, SEQ ID NO: 16 corresponds to the NaCl_2013016_30 strain, SEQ ID NO: 17 corresponds to the NaCl_2013010_34 strain, SEQ ID NO: 18 corresponds to the NaCl_2013016_37 strain, and SEQ ID NO: 19 corresponds to the NaCl_20131016_38 strain.
Table 6 shows the homology between the 16S rRNA gene of the YM17 strain and the base sequence of the 16S rRNA genes of these 12 strains. As shown in Table 6, the nucleotide sequences of the 16S rRNA genes of the 12 isolated strains had a homology of 98.7% or more with the nucleotide sequence of the 16S rRNA gene of the YM17 strain.

5. DNA-DNA hybridization And 2. A DNA-DNA hybridization test was performed using the genomic DNA of 12 strains and YM17 strain isolated from each other. Moreover, as a negative control, DNA of Calditericola satsumensy YMO81 strain, which is a related genus, was used. The DNA was prepared by the same method as that prepared by the GC content measurement in [Example 1].

All of the obtained genomic DNAs have a concentration of 500 μg / mL or more, an OD260 / OD280 value of 1.8 or more, an OD260 / OD230 value of 2.0 or more and no RNA contamination, and should be used for a DNA-DNA hybridization test. High quality that can be.
The DNA-DNA hybridization test was carried out in accordance with the method described in Reference 2 and microorganism classification / identification experiment method (issued by Springer Japan).

First, a DNA plate was prepared. The genomic DNA solution was diluted with 0.1XSSC to make 100 μg / mL. The diluted DNA solution was heated to 100 ° C. for 5 minutes to form a single strand, and then immediately cooled on ice. The thermally denatured DNA solution was diluted 10-fold with PBS-Mg solution to 10 μg / mL. 100 μL of the DNA solution adjusted to 10 μg / mL was dispensed into each microplate well, sealed on the microplate, and allowed to stand at 30 ° C. for 3 hours or more. Thereafter, the DNA solution in the well was discarded to obtain a test DNA plate. Salmon sperm DNA was used for background control.
In parallel with the preparation of the DNA plate, DNA was labeled with photobiotin. 30 μL of a 500 μg / mL DNA solution was prepared and treated with an ultrasonic cleaner for 10 minutes to cleave the DNA. 25 μL of a 1 mg / mL photobiotin solution was added to the cleaved DNA solution to completely dissolve the DNA, and then irradiated with a 250 W low-pressure mercury lamp for 30 minutes with ice cooling to label the DNA with photobiotin. In order to remove excess photobiotin, 540 μL of 0.1 M Tris-HCl buffer (pH 9.0) was added to the DNA solution after irradiation, and then 600 μL of 2-butanol was added and mixed. After centrifugation at 15,000 rpm for 5 seconds, the supernatant containing excess photobiotin was discarded. The operation of adding 2-butanol to remove excess photobiotin was performed twice in total. The photobiotin-labeled DNA solution was heated at 100 ° C. for 5 minutes, immediately cooled on ice, and used for hybridization.

  Next, a hybridization operation was performed. 200 μL of Prehybridization mixture was dispensed into each well of the DNA plate and allowed to stand at 37 ° C. for 15 minutes. 300 μL of the heat-denatured photobiotin-labeled DNA solution was mixed with 6 mL of hybridization mixture. Prehybridization mixture was removed from each well of the DNA plate, and 100 μL of hybridization mixture containing photobiotin-labeled DNA was dispensed, and the DNA plate was sealed. The DNA plate was allowed to stand at 46 ° C. for 2 hours or longer to perform a hybridization treatment. After the hybridization treatment, the hybridization mixture containing photobiotin-labeled DNA was discarded and each well was washed three times with 300 μL of 1XSSC solution.

100 μL of Streptavidin-enzyme solution was dispensed into each well after washing. After standing at 37 ° C. for 30 minutes, the Streptavidin-enzyme solution was discarded. After washing each well with 100 μL of 1XSSC solution, each well was further washed three times with 300 μL of 1XSSC solution. 100 μL of the fluorescent substrate solution was added to each well after washing, and the fluorescence intensity was measured with a microplate reader (excitation wavelength 360 nm, measurement wavelength 450 nm). The temperature was kept at 37 ° C., and the fluorescence intensity was measured every minute. The fluorescence intensity of each combination of DNA was calculated by calculating the average value of the median 4 wells from among the 6-well replicates, and subtracting the average fluorescence intensity value of the wells attached with Salmon sperm DNA as the background value. Homology values are expressed as a percentage of the homozygous value used for DNA of the same strain for wells and labeling.
Reference 2: Ezaki, T .; , Hashimoto, Y .; & Yabuchi, E .; 1989. Fluorometric deoxyribonucleic acid-deoxyribonucleic acid hybridization in microdilution wells as an alternative to membrane filter hybridization in which radioisotopes are used to determine genetic relatedness among bacterial strains. International journal of systematic bacteriology. 39: p224-229

Reagent (1) PBS-Mg solution (MgCl2: 0.95 g / PBS 100 mL)
(2) 0.1 M Tris-HCl buffer (Tris: 12.1 g, EDTA · 2Na: 0.4 g, pH 9.0, ultrapure water: added so that the total liquid volume becomes 1000 mL)
(3) Prehybridization mixture (20XSSC: 1 mL, 50X Denhardt solution: 1 mL, 10 mg / mL denatured Salmon DNA: 0.1 mL, formamide: 5 mL, ultrapure water: 2.9 mL)
(4) Hybridization mix (20XSSC: 1 mL, 50X Denhardt solution: 1 mL, 10 mg / mL denatured Salmon DNA: 0.1 mL, formamide: 5 mL, dextran sulfate: 0.25 g, ultrapure water: 2.8 mL)
(5) Streptavidin-enzyme solution (Streptavidin-β-D-galactosidase conjugate: 10 μL, 0.5% bovine serum albumin (fraction V) / PBS solution: 10 mL)
(6) Fluorescent substrate solution (10 mg / mL 4-methylbelliferyl-β-D-galactopyranoside solution: 100 μL, PBS + 1 mmol MgCl 2 solution: 10 mL)

The results of the DNA-DNA hybridization test are shown in Table 7.
As shown in Table 7, the DNA-DNA homology values of the YM-17 strain, YM17-2 strain, YM17-3 strain, YM17-4 strain, YM17-5 strain, NaCl_2013016_37 strain and NaCl_2013016_38 strain are all 70%. That was all.
As shown in Reference Document 3, it is considered that these strains are of the same species because the microbe taxonomy is considered to be the same species if the DNA-DNA homology value is 70% or more.
Reference 3: Stackebrandt, E .; & Ebers, J .; , 2006. Taxonic parameters reviewed: tarnished gold standards. Microbiol. Today Nov. : P152-155

6). Properties of Frateribacillus Flavoalbus From the above analyzes and tests, it was confirmed that Frateribacillus flavalbus is a species having the following characteristics 1) to 10) as properties.
1) Gram staining is negative, spore formation 2) Oxidase test is negative, catalase test is positive and aerobic 3) LB medium (pH 8.0) containing 1% sugar and 2.0% NaCl ), When D-galactose or sucrose is added as the sugar, acid generation is not performed, but when D-glucose, lactose, D-mannitol, glycerin or N-acetylglucosamine is added, acid generation is not performed. 4) Optimal growth temperature is 45 ° C. to 55 ° C., and growth is not confirmed at 35 ° C. or lower or 65 ° C. or higher 5) Optimal pH is 7.0 to 8.0, pH 5.7 or lower, or pH 10 Growth is not confirmed at more than 0. 6) Growth is confirmed even when NaCl is added to CYC medium to a final concentration of 7%. 7) The main menaquinone is MK7. The amino acid composition of tidoglycan includes alanine, glutamic acid, and meso-type diaminopimelic acid (meso-DAP) 9) The genomic DNA has a GC content of 51.2% to 52.4% 10) The most important cell fatty acid is iso C16: 0

7). Subspecies classification of Frateribacillus flavoabus species 12 strains isolated from YM17 strain (NaCl_2013016_04 strain, YM17-2 strain, YM17-3 strain, YM17-4 strain, NaCl_20131016_19 strain, YM17-5 strain, NaCl_20131016_24 strain, NaCl_20131016_28 strain, NaCl3011 The phylogenetic tree of 16S rRNA genes of the strains, NaCl_20131016_34 strain, NaCl_2013016_37 strain and NaCl_20131016_38 strain) is shown in FIG. As shown in FIG. 3, the 16S rRNA genes of YM17 strain and YM17-5 strain were branched from other strains.

  In addition, as shown in Table 8, the top four major cell fatty acids of YM17 strain and YM17-5 strain are iso-C16: 0, antiiso-C17: 0, antiiso-C15: 0, iso-C17: 0. In contrast, YM17-2, YM17-3, YM17-4, NaCl_2013016_37 and NaCl_2013016_38 are iso-C16: 0, C16: 0, antiiso-C17: 0, antiiso-C15: 0. It was different. For this reason, the YM17 strain / YM17-5 strain and other strains were the same species in Frateribacillus flavoarubus, but were different subspecies.

From the above results, two strains, YM17 strain and YM17-5 strain, were named Frateribacillus flavalbus flavalbus (Frateribacillus flavalbus subsp. Flavalbus), NaCl_2013016_04, YM17-3, YM17-3, YM17-3, 11 strains of NaCl_20130316_19, NaCl_2013016_24, NaCl_2013016_28, NaCl_2013016_30, NaCl_2013016_34, NaCl_2013016_37, and NaCl_20131016_38 strains etarium) was named.
In other words, Frateribacillus flavalbus is a new species of Frateribacillus flavalbus subspecies um, and a new species of genus flavus s. Met.

8). Proteolytic activity of Frateribacillus flavalbus Next, the proteolytic activity of Frateribacillus flavalbus was examined. After adjusting pH to 8.0 by adding 2.0% gelatin or 0.4% casein to basic medium (sodium chloride: 2.0%, yeast extract: 0.1%), 1.5% Of agar was added and autoclaved at 120 ° C. for 20 minutes to obtain a test medium.
YM17 strain, NaCl_2013016_04 strain, YM17-2 strain, YM17-3 strain, YM17-4 strain, NaCl_20131016_19 strain, YM17-5 strain, NaCl_201301016_24 strain, NaCl_2013016_28 strain, NaCl_20131016_30 strain, NaCl_201316_38 strain, It was applied and cultured at 55 ° C. for 7 days. After the culture, a hydrochloric acid-mercuric chloride solution (mercuric chloride 15 g, concentrated hydrochloric acid 20 mL, distilled water 100 mL) was added dropwise to the test medium, and the protein degradation activity was examined by halo formation. As a result of the test, gelatin and casein degradation activity was observed in all 13 strains.

9. Growth and organic matter decomposition activity of Frateribacillus flavo albus in compost environment Growth and organic matter decomposition activity of Frateribacillus flavo albus in compost were investigated.
LB medium (pH 8.0) containing 10% yeast extract solution 1 mL, 1% casein solution or 3 mL of sterilized water 2% NaCl, and 10% of 10% compost sterilized by autoclaving at 120 ° C. for 30 minutes twice 1 mL of a culture solution of bacteria cultivated at 55 ° C. for 1 day was added, and mixed with a sterilized medicine spoon so that the water could reach the entire compost. The mixture was sealed in a state where there was a sufficient gas phase, and cultured at 55 ° C. for 3 days (hereinafter referred to as compost culture). A negative control was prepared by adding LB medium (pH 8.0) containing sterilized 2% NaCl in place of the bacterial culture. As a test bacterium, YM17 strain, YM17-2 strain, YM17-3 strain, YM17-4 strain, YM17-5 strain, NaCl_2013016_37 strain and NaCl_2013016_38 strain were used.

When the culture after 3 days of compost culture was observed with a microscope, the culture solution of YM17 strain, YM17-2 strain, YM17-3 strain, YM17-4 strain, YM17-5 strain, NaCl_2013016_37 strain or NaCl_2013016_38 strain was added. In all, good fungus growth was confirmed. On the other hand, no bacterial cells were found in the negative control to which no bacterial culture was added. FIG. 4 shows a photomicrograph after 3 days of compost culture of casein-added compost of YM17 strain or negative control.
As shown by the circle in FIG. 4A, good growth of the bacterial cells was confirmed for the YM17 strain. On the other hand, as shown in FIGS. 4 and B, only the solid matter of compost was observed in the negative control, and no cells were observed.

  Protein was extracted from the culture after 3 days of compost culture. First, 30 mL of Tris-HCl (pH 8.0) was added to the culture to form a suspension, which was allowed to stand at room temperature for 3 hours. The suspension after standing was filtered (filter paper: Advantech Toyo, model No. 5B, 110 mm), and the filtrate was recovered. The remaining residue was washed with Tris-HCl (pH 8.0), and the filtrate was adjusted to a total volume of 30 mL. 14.2 g of ammonium sulfate was added to 30 mL of the filtrate, and the mixture was gently stirred to dissolve the ammonium sulfate. After dissolution of ammonium sulfate, the solution was allowed to stand overnight on ice. The liquid after standing was centrifuged at 10,000 g for 30 minutes at 4 ° C., and the supernatant was removed to collect the precipitate. The precipitate was dissolved with 1 mL of Tris-HCl (pH 8.0), and the lysate was placed in a dialysis membrane (Spectrum Laboratories, Inc., Spectra / Pore® 1, MW 6,000-8,000). Desalting was performed by dialysis in Tris-HCl (pH 8.0). After desalting, it was adjusted with Tris-HCl (pH 8.0) to make the liquid volume 2 mL, and used as a test sample of zymogram.

Gelatin having a final concentration of 0.1% was added as a substrate to the electrophoresis gel for zymography. 15 μL of protein solution after desalting and 5 μL of Loading Buffer were mixed, and 20 μL of the mixed solution was used for zymography.
The gel after electrophoresis was placed in Tris-HCl (pH 8.0) containing 0.2% Triton X for 10 minutes, and then this buffer was discarded and washed twice with Tris-HCl (pH 8.0) not containing Triton X. did. The washed gel was immersed in 20 mL of Tris-HCl (pH 8.0) containing 2 mM CaCl 2 and 2 mM MgSO 4 and allowed to stand at 55 ° C. for 16 hours for active culture. After the active culture, 20 mL of Tris-HCl (pH 8.0) containing 2 mM CaCl 2 and 2 mM MgSO 4 was discarded, the gel was stained with Coomassie for 30 minutes, and decolorized after Coomassie staining to examine the protein degradation activity.

As a result of zymography, proteins extracted from compost cultures without addition of casein also showed gelatinolytic activity, whereas proteins extracted from compost cultures with addition of casein showed stronger activities. In addition, in the negative control to which no fungal culture solution was added, the protein extracted from the compost culture showed no gelatinolytic activity regardless of the presence or absence of casein addition.
When the fungal culture solution was added, protein degradation activity was observed even in the case of compost culture without addition of casein because the protein remained in the compost used as the base material and the activity was induced. Met. Moreover, activity induction appeared more strongly by composting with the addition of casein.
From the above results, Frateribacillus flavalbus species such as YM17 strain, YM17-2 strain, YM17-3 strain, YM17-4 strain, YM17-5 strain, NaCl_2013016_37 strain or NaCl_2013016_38 strain grow in compost, It was shown to have proteolytic activity. FIG. 5 shows the results of gelatin zymography. FIGS. 5A and 5B show the results of zymography using proteins extracted from compost culture without addition of casein, and FIGS. 5 and B show the results of zymography using proteins extracted from compost culture with addition of casein.

Reagent (1) Tris-HCl (pH 8.0) (Tris: 6.1 g, adjusted to pH 8.0 with concentrated hydrochloric acid, ultrapure water: added so that the total liquid volume becomes 1000 mL)
(2) Zymogram electrophoresis gel separate layer (30% Acrylamide: 2000 μL, 1.5 M Tris-HCl (pH 8.5): 1500 μL, ultrapure water: 1800 μL, 10% SDS: 60 μL, 10% APS: 50 μL, TEMED : 5 μL, 1% gelatin: 600 μL)
Stacking layer (30% Acrylamide: 450 μL, 0.5 M Tris-HCl (pH 6.5): 750 μL, ultrapure water: 1700 μL, 10% SDS: 30 μL, 10% APS: 50 μL, TEMED: 5 μL)
After the separate layer is solidified, the stacking layer is overlaid and solidified.
(3) 10X zymogram electrophoresis buffer (Tris: 30.3 g, Glycine: 144 g, ultrapure water: added so that the total liquid volume becomes 1000 mL). In the electrophoresis of zymogram, 10 × zymogram electrophoresis buffer was diluted with ultrapure water to make 1 × buffer, and SDS was added to a final concentration of 0.1%.

  The novel microorganism obtained by the present invention is useful as a new species of the new genus Frateribacillus, for the treatment of organic waste such as human waste and sludge, and can be used for various applications.

[Reference to deposited biological materials]
(1) Name of depositary institution: National Institute of Technology and Evaluation, Patent Microorganisms Deposit Center (2) Contact: 2-5-8 Kazusa Kamashi, Kisarazu City, Chiba Prefecture 292-0818
Phone number 0438-20-5580
(3) Accession number: NITE BP-0176
(4) Display for identification: YM17
(5) Original deposit date: November 29, 2013

[Reference to deposited biological materials]
(1) Name of depositary institution: National Institute of Technology and Evaluation, Patent Microorganisms Deposit Center (2) Contact: 2-5-8 Kazusa Kamashi, Kisarazu City, Chiba Prefecture 292-0818
Phone number 0438-20-5580
(3) Accession number: NITE BP-01948
(4) Display for identification: YM17-2
(5) Original deposit date: October 6, 2014

[Reference to deposited biological materials]
(1) Name of depositary institution: National Institute of Technology and Evaluation, Patent Microorganisms Deposit Center (2) Contact: 2-5-8 Kazusa Kamashi, Kisarazu City, Chiba Prefecture 292-0818
Phone number 0438-20-5580
(3) Accession number: NITE BP-01949
(4) Display for identification: YM17-3
(5) Original deposit date: October 6, 2014

[Reference to deposited biological materials]
(1) Name of depositary institution: National Institute of Technology and Evaluation, Patent Microorganisms Deposit Center (2) Contact: 2-5-8 Kazusa Kamashi, Kisarazu City, Chiba Prefecture 292-0818
Phone number 0438-20-5580
(3) Accession number: NITE BP-01950
(4) Display for identification: YM17-4
(5) Original deposit date: October 6, 2014

[Reference to deposited biological materials]
(1) Name of depositary institution: National Institute of Technology and Evaluation, Patent Microorganisms Deposit Center (2) Contact: 2-5-8 Kazusa Kamashi, Kisarazu City, Chiba Prefecture 292-0818
Phone number 0438-20-5580
(3) Accession number: NITE BP-01925
(4) Display for identification: YM17-5
(5) Original deposit date: August 28, 2014

Claims (9)

16S rRNA gene which is a new species of the genus Frateribacillus and has a base sequence having a homology of 98.7% or more with respect to the base sequence shown in SEQ ID NO: 1 or SEQ ID NO: 8 to SEQ ID NO: 19. YM17 strain (NITE BP-0176), YM17-2 strain (NITE BP-01948), YM17-3 strain (NITE BP-01949), YM17-4 strain (NITE BP-01950) or YM17-5 strain Frateribacillus flavalbus having a DNA-DNA hybridization homology value of (NITE BP-01925) of 70% or more and the following characteristics 1) to 10).
1) Neisseria gonorrhoeae with negative Gram staining, forming spores 2) Negative oxidase test, positive and aerobic catalase test 3) LB medium (pH 8) containing 1% sugar and 2.0% NaCl 0.0), when D-galactose or sucrose is added as the sugar, acid generation is not carried out, but when D-glucose, lactose, D-mannitol, glycerin or N-acetylglucosamine is added, acid is not generated. 4) Optimal growth temperature is 45 ° C to 55 ° C, and growth is not confirmed at 35 ° C or lower or 65 ° C or higher 5) Optimal pH is 7.0 to 8.0, pH 5.7 or lower Or, growth is not confirmed at pH 10.0 or higher. 6) Growth is confirmed even when NaCl is added to CYC medium to a final concentration of 7%. 7) The main menaquinone is MK7. The amino acid composition of wall peptidoglycan includes alanine, glutamic acid, and meso-type diaminopimelic acid (meso-DAP) 9) The genomic DNA has a GC content of 51.2% to 52.4% 10) The most important cell fatty acids are iso-C16: 0. Furthermore, as bacteria fatty, anteiso-C15: 0, C16 : 0, anteiso-C17: 0, iso-C17: 0 either containing one or more, Furateribachirasu flavonol albus (Frateribacillus flavoalbus) of claim 1. It is Frateribacillus flavalbus subspecies flavalbus (Ferraibacillus flavalbus subsp. Flavalbus) containing anteiso-C15: 0, anteiso-C17: 0, and iso-C17: 0 in addition to iso-C16: 0 as a cell fatty acid. Item 3. A Frateribacillus flavalbus according to Item 1 or 2 . It is Frateribacillus flavalbus subspecies fimetalium containing anteiso-C15: 0, C16: 0, anteiso-C17: 0 in addition to iso-C16: 0 as a microbial fatty acid. 1 according to any one of 3 Furateribachirasu Flavobacterium albus (Frateribacillus flavoalbus). The Frateribacillus flavalbus according to any one of claims 1 to 4 , which is used for the treatment of organic waste. 6. The Frateribacillus flavalbus according to claim 5 , wherein the organic waste is human waste, organic sludge, food residue, or animal manure. YM17 strain (NITE BP-0176) , YM17-2 strain (NITE BP-01948), YM17-3 strain (NITE BP-01949), YM17-4 strain (NITE BP-01950) or YM17-5 strain (NITE BP) -01925) is either, according to any of claims 1-6 Furateribachirasu Flavobacterium albus (Frateribacillus flavoalbus). The method of treating organic waste using Furateribachirasu Flavobacterium albus (Frateribacillus flavoalbus) according to any one of claims 1-7. A method for degrading a protein using the Frateribacillus flavalbus according to any one of claims 1 to 7 .