KR101728605B1 - Novel microorganisms - Google Patents

Abstract

And to provide a new microorganism useful for the treatment of organic wastes such as urine, urine, sludge and the like.
2) Oxidase test is negative; catalase test is positive and aerobic; 3) 1% of sugar and 2.0% of testosterone are of the genus Frateribacillus ; 1) Gram stain is negative and spores are formed; When D-galactose or sucrose is added to the LB medium (pH 8.0) containing NaCl, acid is not produced, but D-glucose, lactose, D-mannitol, glycerin or N-acetylglucosamine is added In this case, it provides an acid production, and provides Frateribacillus flavoalbus which has the characteristics of the like.

Description

NOVEL MICROORGANISMS

The present invention relates to a novel microorganism of the genus Fratelli Bacillus (genus Frateribacillus ). More particularly, the present invention relates to Frateribacillus flavoalbus having the following characteristics (1) to (10).

1) Gram stain is negative, forming spores

2) oxydase test is negative, catalase test is positive and aerobic

3) When D-galactose or sucrose was added to the LB medium (pH 8.0) containing 1% sugar and 2.0% NaCl, no acid was produced, but D-glucose, lactose, D-mannitol , When glycerin or N-acetylglucosamine is added, acid production is performed

4) Intellectual growth temperature is 45 ℃ ~ 55 ℃. No growth is observed at 35 ℃ or above or above 65 ℃

5) The intellectual pH is 7.0 ~ 8.0, and the proliferation is not confirmed when the pH is lower than 5.7 or above 10.0.

6) Proliferation was confirmed by addition of NaCl to the CYC medium up to a concentration of 7%.

7) The main menaquinone is MK7

8) The amino acid composition of the cell wall peptidoglycan includes alanine, glutamic acid, and a small diamino pimelic acid (meso-DAP).

9) GC content of genomic DNA is 51.2% ~ 52.4%

10) The most important fungal fatty acid is iso-C16: 0

In the conventional composting process, bacteria such as fungi, actinomycetales ( Actinomycetales ) called actinomycetes, bacteria of the thermoactinomycetaceae family such as Thermoactinomyces ( genus Thermoactinomyces ) Bacteria, bacteria of the genus Bacillus (genus Bacillus ) and genus of the genus Geobacillus (genus Geobacillus ) and Bacillaceae ( Bacillaceae ) bacteria decompose and magnetize the organic matter is responsible for composting. These microorganisms have spore-forming ability and can exhibit heat resistance in a spore state in an environment where the temperature is raised above the growth temperature of each individual, and can stably stay in the compost. Therefore, even after decomposing and magnetizing organic wastes at proper temperature for growth, It can be repeatedly used for composting.

In composting, aerobic fermentation is preferable to anaerobic fermentation because decomposition of organic matter is faster and odor can be reduced. In the process of composting by aerobic fermentation, aerobic fermentation by ventilation starts, within 10 days, the inside temperature of the compost becomes higher than 45 ℃, and the high temperature condition is maintained for a long time until the end of composting. In a high-temperature environment in the manure of 45 ℃ ~70 ℃ micro mono sports rajigwa (Micromonosporaceae and), Streptomyces three tajigwa (and Streptomycetaceae), Thermo mono sports rajigwa (and Thermomonosporanceae), streptomycin sports field such as liquid Martino Jia jigwa (Streptosporangiaceae and) Actinomycetes in the mycetellar necks ( Actinomycetales ), and bacterial strains in the thermoactinomycetaceae family, which are thermophilic and in the form of yarn , are responsible for decomposition of organic matter One). In addition, bacteria belonging to the genus Geobacillus (genus Geobacillus ) and of the genus Bacillaceae are also present in the compost under high temperature environment (Non-Patent Document 2 and Non-Patent Document 3). In the compost of high temperature, the bacteria belonging to the limited classification group are mainly responsible for composting. Since the bacteria in each family are composed of bacteria having similar characteristics, The population is low in diversity.

Patent Document 1 describes a method of producing compost using a strain in Anoxybacillus , and Patent Document 2 discloses a method of producing compost using a strain of Bacillus genus. However, in the case of Bacillaceae And bacteria) are used.

On the other hand, organic wastes from waste disposal vary widely in pH, salt concentration, corrosive acid concentration, and C / N ratio depending on the type of waste materials, including organic sludge, food residues and animal manure. Therefore, in order to efficiently compost various organic wastes, it is preferable to grow and grow the compost by adding a different strain group to the existing strain, and to decompose and magnetize the organic matter by various species of bacteria. Further, it is preferable that the added strain is capable of maintaining a dormant state by having a spore forming ability and capable of withstanding environmental changes. The stable spore state is economical because it is easy to store and transport, and can be used repeatedly as recycled compost.

Up to now, the present inventors have searched for new bacteria that can be used for composting, and found a new super thermophile belonging to a new genus that grows in an environment of 80 ° C or higher (Patent Document 3).

Patent Document 1: Japanese Patent Laid-Open Publication No. 2011-24569 Patent Document 2: Japanese Patent Application Laid-Open No. 2008-278890 Patent Document 3: Japanese Patent Application Laid-Open No. 2005-13063

Non-Patent Document 1: Journal of bioscience and bioengineering, Vol.99, p1-11, 2005 Non-Patent Document 2: International Journal of Systematic and Evolutionary Microbiology, Vol. 52, p2251-2255, 2002 Non-Patent Document 3: Systematic and Applied Microbiology, Vol.34, p419-423, 2011

The present invention aims to provide a novel microorganism useful for the treatment of organic wastes such as urine and sludge.

The present inventors have completed the present invention by discovering a novel microorganism, isolating it, and identifying it.

That is, the present invention is as follows.

(1) PRA PRA Terry Terry Bacillus Bacillus Plastic beam albus (Frateribacillus flavoalbus) having a swine, and the characteristics of the following 1) to 10) of (Frateribacillus in).

1) Gram stain is negative bacillus, forming spores

2) oxydase test is negative, catalase test is positive and aerobic

3) When D-galactose or sucrose was added to the LB medium (pH 8.0) containing 1% sugar and 2% NaCl, no acid was produced, but D-glucose, lactose, D-mannitol , When glycerin or N-acetylglucosamine is added, acid production is performed

4) Intellectual growth temperature is 45 ℃ ~ 55 ℃. No growth is observed at 35 ℃ or above or above 65 ℃

5) The intellectual pH is 7.0 ~ 8.0, and the proliferation is not confirmed when the pH is lower than 5.7 or above 10.0.

6) Proliferation was confirmed by addition of NaCl to the CYC medium up to a concentration of 7%.

7) The main menaquinone is MK7

8) The amino acid composition of the cell wall peptidoglycan includes alanine, glutamic acid, and a small diamino pimelic acid (meso-DAP).

9) GC content of genomic DNA is 51.2% ~ 52.4%

10) The most important fungal fatty acid is iso-C16: 0

[2] The bacteriophage according to [1] above, which has a 16S rRNA gene consisting of a nucleotide sequence having a homology of 98.7% or more with respect to the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: Bee albus ( Frateribacillus flavoalbus ).

[3] YM17 strain NITE BP-01764, YM17-2 strain NITE BP-01948, YM17-3 strain NITE BP-01949, YM17-4 strain NITE BP- ( Frateribacillus flavoalbus ) according to the above [1] or [2], wherein a DNA-DNA hybridization homology value with the NITE BP-01925 is 70% or more.

[4] The microbicidal composition according to any one of [1] to [3], wherein the microbial fatty acid comprises any one or more of anteiso-C15: 0, C16: 0, anteiso-C17: 0 and iso-C17: ( Frateribacillus flavoalbus ) described in < / RTI >

[5] Frateribacillus flavoalbus subsp. Flavoalbus containing anteiso-C15: 0, anteiso-C17: 0, iso-C17: 0 in addition to iso-C16: The Frateribacillus flavoalbus according to any one of the above [1] to [4],

[6] Frateribacillus flavoalbus subsp. Fimetarium including anteiso-C15: 0, C16: 0, and anteiso-C17: 0 in addition to iso-C16: , Frateribacillus flavoalbus according to any one of [1] to [4] above.

[7] Frateribacillus flavoalbus according to any one of [1] to [6], which is used for treating organic wastes.

[8] The Frateribacillus flavoalbus according to [7], wherein the organic waste is urine, organic sludge, food remnants , or animal manure.

[9] YM17 strain (NITE BP-01764), NaCl_20131016_04 strain, YM17-2 strain (NITE BP-01948), YM17-3 strain (NITE BP-01949), YM17-4 strain Described in any one of [1] to [8] above, which is any one of the above-mentioned [1] to [8], wherein the YM17-5 strain (NITE BP-01925), NaCl_20131016_24 strain, NaCl_20131016_28 strain, NaCl_20131016_30 strain, NaCl_20131016_34 strain, NaCl_20131016_37 strain, Flavo albus ( Frateribacillus flavoalbus ).

[10] A method for treating organic wastes using the Frateribacillus flavoalbus according to any one of [1] to [9] above.

[11] A method for degrading a protein using the Frateribacillus flavoalbus according to any one of [1] to [9] above.

The novel microorganism obtained by the present invention is a new species of the genus Frateribacillus (genus Frateribacillus ). This new microorganism is useful for treatment of organic wastes such as urine, sludge and the like, decomposition of proteins, and the like, and can be used for various purposes.

Fig. 1 is a view showing a cell photograph of YM17 (Example 1).
Fig. 2 is a diagram showing the number of lines generated by the neighborhood combining method (Example 1). Fig.
Fig. 3 is a diagram showing the phylogenetic tree of 12 weeks 16S rRNA genes isolated from YM17 strain (Example 2).
4 is a view showing the state of bacteria during compost cultivation (Example 2).
Fig. 5 is a diagram showing the results of gelatin self-assemble (Example 2).

Plastic Plastic Terry Bacillus beam albus (Frateribacillus flavoalbus) of the present invention refers to a new quick plastic Terry Bacillus having the characteristics of the following 1) to 10) (in Frateribacillus).

1) Gram stain is negative, forming spores

2) oxydase test is negative, catalase test is positive and aerobic

3) When D-galactose or sucrose was added to the LB medium (pH 8.0) containing 1% sugar and 2.0% NaCl, no acid was produced, but D-glucose, lactose, D-mannitol , When glycerin or N-acetylglucosamine is added, acid production is performed

4) Intellectual growth temperature is 45 ℃ ~ 55 ℃. No growth is observed at 35 ℃ or above or above 65 ℃

5) The intellectual pH is 7.0 ~ 8.0, and the proliferation is not confirmed when the pH is lower than 5.7 or above 10.0.

6) Proliferation was confirmed by addition of NaCl to the CYC medium up to a concentration of 7%.

7) The main menaquinone is MK7

8) The amino acid composition of the cell wall peptidoglycan includes alanine, glutamic acid, and a small diamino pimelic acid (meso-DAP).

9) GC content of genomic DNA is 51.2% ~ 52.4%

10) The most important fungal fatty acid is iso-C16: 0

As the " main menaquinone ", there can be mentioned those containing 81.8% or more of MK7.

As the " major fungal fatty acid ", iso-C16: 0 is contained in an amount of not less than 34.1% and not more than 42.6%.

Further, as the " fungal fatty acid ", one or more of anteiso-C15: 0, C16: 0, anteiso-C17: 0 and iso-C17: 0 may be contained in addition to iso-C16: 0. The Frateribacillus flavoalbus of the present invention preferably contains 86.1% or more of these fatty acids in total.

In addition, the Frateribacillus flavoalbus of the present invention comprises a 16S rRNA gene comprising a nucleotide sequence having a homology of 98.7% or more with respect to the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 8 to 19, .

In addition, the Frateribacillus flavoalbus of the present invention has a homology of 70% with DNA-DNA hybridization with YM17 strain, YM17-2 strain, YM17-3 strain, YM17-4 strain or YM17-5 strain, .

The Frateribacillus flavoalbus of the present invention has the above-mentioned properties 1) to 10), and further has an anteiso-C15: 0, anteiso-C17: Frateribacillus flavoalbus subsp. Flavoalbus , which contains the Fr.beta . 0, iso-C17: 0.

Such a Frateribacillus flavoalbus subsp. Flavoalbus is exemplified by YM17 strain and YM17-5 strain.

The present invention also relates to a Frateribacillus flavoalbus of the present invention which has the above-mentioned properties 1) to 10), and has an anteiso-C15: 0, C16: 0 Frateribacillus flavoalbus subsp. fimetarium , including anteiso-C17: 0, may also be mentioned.

As the Frateribacillus flavoalbus subsp. Fimetarium , there were used NaCl_20131016_04, YM17-2, YM17-3, YM17-4, NaCl_20131016_19, NaCl_20131016_24, NaCl_20131016_28, NaCl_20131016_30, NaCl_20131016_34 weeks, NaCl_20131016_37 weeks and NaCl_20131016_38 weeks.

The YM17 strain as Frateribacillus flavoalbus of the present invention has the above-mentioned characteristics, and the base sequence of 16S rRNA is 100% identical to the nucleotide sequence of SEQ ID NO: 1.

This strain has been deposited internationally on the basis of the Budapest Treaty at the Patent and Trademark Office of the Agency for Product Assessment and Technology (NITE) at the request of the applicant. Hereinafter, the content specifying the deposit is described.

(1) Depositary Institution: Independent administrative corporation Product evaluation Technology-based organization Patented microorganism deposit center

(2) Contact: Postal 292-0818 2-5-8 Matsuri, Kazusaka, Kisaratsu, Chiba Prefecture

Phone number 0438-20-5580

(3) Accession number: NITE BP-01764

(4) Indication for identification: YM17

(5) Date of original deposit: November 29, 2013

In addition, regarding the YM17-2 strain, YM17-3 strain, YM17-4 strain and YM17-5 strain, which are also known as Frateribacillus flavoalbus of the present invention, The Agency for Product Assessment and Technology (NITE) has been deposited with the Patent Microbial Depository Center on the basis of the Budapest Treaty. Details of specifying these deposits are disclosed in Examples and the like.

The Frateribacillus flavoalbus of the present invention also includes a mutant strain of the strain corresponding to this Frateribacillus flavoalbus . In the case of Frateribacillus flavoalbus flavoalbus , it is also possible to use the Frateribacillus flavoalbus subsp. Flavoalbus or the Frateribacillus flavoalbus subsp. Fimetarium Includes mutations.

This mutant in the present invention is a modified version of the physical and chemical properties of the mutant by genetic engineering techniques such as chemical mutagenesis, natural mutation, morphological mutation and transfection. .

The Frateribacillus flavoalbus of the present invention can be used for the treatment of organic wastes such as organic sludge, food remnants , or animal manure. It is also possible to produce compost and the like by adding Frateribacillus flavoalbus to these organic wastes.

In the method of treating organic wastes using the Frateribacillus flavoalbus of the present invention, not only Frateribacillus flavoalbus is added to the organic wastes, but also useful in the treatment of organic wastes Or may include an outside process.

In addition, the Frateribacillus flavoalbus of the present invention can be used for protein degradation.

In the method for decomposing proteins using Frateribacillus flavoalbus according to the present invention, not only Frateribacillus flavoalbus is added to a digestion target containing protein or protein, May include other processes useful in the process of the invention.

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

Example

[Example 1]

Identification and identification of Frateribacillus flavoalbus ( Frateribacillus flavoalbus )

1. Isolation of strain

We isolated YM17 week from compost of composting ground of Sojo, Kagoshima.

That is, 4 mL of a 1/2 nutrient medium (peptone: 0.25%, sodium chloride: 0.25%, yeast extract: 0.1%, meat extract: 0.05%, pH 7.4) containing 50 μg / mL novobiocin After 400 mg of the above compost was added and cultured at 55 ° C for 2 days, this liquid culture was applied to a nutrient agar medium containing 50 μg / mL novobiocin and cultured for 4 days to collect colonies of YM17 Isolated.

2. Taxonomic properties

A. Test Method

The taxonomic properties of the strains can be found in "Classification of Microorganisms", "Classification and Identification of Microorganisms", "Classification and Identification of Microorganisms in Revised Edition" &Quot; Supuringo, Japan "), BERGEY'S MANUAL OF SYSTEMIC BACTERIOLOGY. The classification and identification of the isolates were carried out by comparison with the species described in the International Journal of Systematic and Evolutionary Microbiology.

The morphological, cultural, physiological, growth, and chemical taxonomic properties obtained by this analysis are shown in Tables 2-1 and 2-5. A photograph of a cell cultured in YM17 strain at 55 占 폚 is shown in Fig. In Fig. 1, it was confirmed that YM17 was a bacterium and spores were formed at the end of the cell. The bar in Fig. 1 represents 10 mu m. In Table 2 (Tables 2-1 to 2-5), the taxonomic properties of the 12 strains isolated in Example 2 were also shown.

B. Test media

The pH of each medium is as described in Table 2-1. Each medium except litmus milk was autoclaved at 121 占 폚 for 15 minutes. The Litmus milk was autoclaved at 110 캜 for 10 minutes. The element of the Christensen urea medium, the sugar in the OF test medium, and the acid production from the saccharide were examined by filter sterilization and added to each medium so as to be the indicated concentration. In addition, acid production from sugar was investigated 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 7hydrate 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 7hydrate 0.05%, potassium chloride 0.05% Iron sulfate heptahydrate: 0.001%)

(3) Juicy agar medium (Meat extract: 1%, peptone: 1%, sodium chloride: 0.5%, agar: 1.5%)

(4) Juicy liquid medium (Meat extract: 1%, peptone: 1%, sodium chloride: 0.5%)

(5) Litmus milk (skim milk: 10%, 1% litmus liquid: several drops)

(6) VP medium (yeast extract: 0.1%, tryptone: 0.7%, soytone: 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%, iron citrate: 0.05 g,

(8) LB agar medium (tryptone: 1%, sodium chloride: 0.5%, yeast extract: 0.5%, agar: 1.5%)

(9) SY agar medium (0.2% ammonium sulfate, 0.14% disodium hydrogen phosphate, 0.07% potassium dihydrogen phosphate, 0.02% magnesium sulfate heptahydrate, 0.0005% iron sulfate heptahydrate, 0.0005% iron sulfate heptahydrate %, Glucose: 1%)

(10) Nut agar medium (meat extract: 0.3%, peptone: 0.5%, agar: 1.5%)

(11) Simmons medium (0.1% ammonium dihydrogen phosphate, 0.1% sodium dihydrogen phosphate, 0.5% sodium chloride, 0.2% sodium citrate, 0.02% magnesium sulfate heptahydrate, 1.5% 0.008%)

(12) Cultured yeast extract medium (yeast extract: 0.05%, cysteine hydrochloride: 0.01%, sodium chloride: 0.5%, sodium citrate: 0.3%, glucose: 0.02%, potassium dihydrogenphosphate: 0.1% Phenol red: 0.0012%)

(13) Nitrate medium (glucose: 1.0%, potassium dihydrogenphosphate: 0.1%, magnesium sulfate heptahydrate: 0.05%, potassium chloride: 0.02%, sodium nitrate: 0.1%

(14) Ammonium salt medium (glucose: 1.0%, potassium dihydrogenphosphate: 0.1%, magnesium sulfate monohydrate: 0.05%, potassium chloride: 0.02%, ammonium sulfate: 0.078%)

(15) Kristensen urea medium (peptone: 0.1%, sodium chloride: 0.5%, glucose: 0.1 g, KH2PO4: 0.2%, phenol red: 0.0008%, agar: 1.5%

(16) OF test medium (peptone: 0.2%, sodium chloride: 0.5%, dipotassium hydrogen phosphate: 0.03%, agar: 0.2%, BTB: 0.008%, glucose: 1%

C. Analysis of fatty acids

1) Sample preparation and analysis by gas chromatography

5 L of Erlenmeyer flask, 3 L of CYC liquid medium was placed in the culture medium, and cultured at 52 캜 from the late logarithmic growth phase to the early stage of the steady state, and the cells were recovered by centrifugation. The recovered cells were washed twice with physiological saline. Approximately 500 mg to 700 mg of the wet cells were placed in a centrifuge tube equipped with a screw cap. 1 mL of an alkali saponifying solution was added and kept at 100 占 폚 for 5 minutes in a tight state. After 5 minutes, it was shaken well for 10 seconds and maintained at 100 DEG C for 30 minutes. After the centrifuge tube was cooled, 2 mL of a methanolic reagent of hydrochloric acid was added and kept at 80 DEG C for 10 minutes in a tight state. After cooling the centrifuge tube, 1.25 mL of extraction solvent was added and stirred slowly for 10 minutes. The centrifuge tube was centrifuged at 1000 rpm for 5 minutes. Then, the upper layer was transferred to a centrifuge tube with a separate screw cap, and 3 mL of the washing solution was added. The mixture was stirred again for 5 minutes. The centrifugal tube was centrifuged at 1000 rpm for 5 minutes, and then the solvent layer was taken in a glass ampule and dried with nitrogen gas. The composition of each reagent is shown below. The obtained dried sample was analyzed by gas chromatography on a Technosuruga Labo Co., Ltd. as a fatty acid analysis sample. The obtained results are shown in Table 1.

2) Reagent

(1) Alkali saponification solution (sodium hydroxide: 7.5 g, methanol: 25 mL, ultrapure water: 25 mL)

(2) Hydrochloric acid Methanol reagent (6 N hydrochloric acid: 21.7 mL, methanol: 18.3 mL)

(3) Extraction solvent (hexane: 10 mL, t-butyl methyl ether: 10 mL)

(4) Washing solution (sodium hydroxide: 0.586 g, ultrapure water: 48.8 mL)

D. Cell wall peptidoglycan

1) Sample adjustment

Approximately 2 g of the wet cells were suspended in 6 mL of 50 mM phosphate buffer (pH 7.2) and treated with an ultrasonic disrupter (VP-30S, manufactured by TAITEC) for 30 minutes to disrupt the cells. The disrupted solution was centrifuged at 4 캜 and 3000 rpm for 10 minutes, and the supernatant was recovered. The supernatant was centrifuged at 4000 rpm at 9000 rpm for 60 minutes to remove the supernatant, and the precipitate was transferred to a new centrifuge tube. The precipitate was washed with 6 mL of 50 mM phosphate buffer (pH 7.2), centrifuged at 4000 rpm at 9000 rpm for 60 minutes, and the supernatant was removed. 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 DEG C was further added, stirred, and centrifuged at a temperature of 30 DEG C at 9000 rpm for 60 minutes.

The supernatant was removed, and 5 mL of 50 mM phosphate buffer (pH 7.2) warmed to 65 DEG C was added to the precipitate, followed by washing. The supernatant was removed by centrifugation at 9000 rpm for 30 minutes at 30 DEG C. This cleaning 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 the mixture was maintained at 37 ° C. for 2 hours. 5 mL of 50 mM phosphate buffer solution (pH 7.6) was added to the solution after the proteinase reaction, and the cell wall components were washed, and the supernatant was removed by centrifugation at 9000 rpm for 30 minutes at 4 ° C. This cleaning operation was performed twice.

To the precipitate after washing, 2 mL of 5% trichloroacetic acid aqueous solution was added, and the mixture was maintained at 100 DEG C for 20 minutes. The suspension was transferred to a centrifugal tube equipped with a glass screw cap, washed with 5 mL of 50 mM phosphate buffer (pH 7.2), centrifuged at 1000 rpm for 1 minute at room temperature, and the supernatant was removed. This cleaning operation was performed twice. 3 mL of ethanol was added to the precipitate after washing, centrifugation was performed at room temperature for 1 minute at 1000 rpm, and the supernatant was removed. 3 mL of diethyl ether was added to the precipitate, centrifugation was performed 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

To 5 mg of the cell wall sample, 250 μL of 4 N hydrochloric acid was added and hydrolysis was carried out at 100 ° C. for 16 hours. After the hydrolysis reaction, hydrochloric acid was removed in a desiccator in which potassium hydroxide was placed, and the obtained treatment solution was provided to TLC.

The TLC plate was a TLC cellulose plate No.1.05716.0001 manufactured by Merck. For the detection of constituent amino acids, the first dimension was developed with a developing solvent of isopropanol: acetic acid: ultrapure water = 75: 10: 15 and the second dimension was developed with methanol: pyridine: 10 N hydrochloric acid: ultrapure water = 64: 8: did. After the development, a ninhydrin reagent was sprayed on the TLC plate, and the amino acid contained in the cell wall peptidoglycan was detected. Further, the detection of the isomeric form of diaminopimelic acid was carried out using the same TLC plate and developed with methanol: pyridine: 10 N hydrochloric acid: ultrapure water = 64: 8: 2: 14, . As a result, it was confirmed that the amino acid composition of the cell wall peptidoglycan contained alanine, glutamic acid, and meiamidopimelic acid (meso-DAP).

E. menaquinone

1) Sample adjustment

To a dried cell mass of about 500 mg, 20 mL of chloroform: methanol = 2: 1 was added, and the mixture was slowly stirred overnight in a cold dark place. The suspension was filtered, and the filtrate was concentrated using a rotary evaporator at a bath temperature of 30 ° C. The dry solid was dissolved in 3 mL of acetone and concentrated to about 200 μL with nitrogen gas. HPTLC Silica gel 60 F254 plate No.1.05628.0001 manufactured by Merck Co., Ltd. This concentrate was provided, and developed with toluene as a developing solvent. After development, the TLC plate was dried and the menaquinone band was identified 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 scratched silica gel, and the mixture was stirred slowly, and the silica gel particles were removed by filtration. The obtained supernatant was concentrated to about 200 μL with nitrogen gas, and used as a sample for HPLC.

2) Analysis by HPLC

25 μL of the sample was injected into the HPLC. An ODS column (CAPCELL PAK C18 UG120 S-5 manufactured by SHISEIDO) was used as a column, and methanol: isopropanol = 2: 1 solution was used as a developing solvent. At a moving speed of 1.5 mL / min, at a column temperature of 40 DEG C, and detected by UV (270 nm). The HPLC machine was a SERIES 1100 manufactured by HEWLETT PACKART. This analysis was repeated a plurality of times. Since the content of MK7 was 83.9% at the first time and was 85.2% at the second time, it was confirmed that the main menaquinone was MK7.

F. GC content

1) DNA regulation

Genomic DNA was extracted according to the method of Reference 1.

Namely, 0.9 g to 1.6 g of wet cells were washed with Saline-0.1 M 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 the lysate solution was frozen. An equal volume of Tris-SDS (pH 9.0) was added to the frozen product, which was melted in a hot water bath at 60 캜 and held 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 recovered by centrifugation, and two-fold amount of ethanol was added, and the fibrous DNA was wound up with a glass rod. The wound DNA was washed with 70%, 90%, and 99.5% ethanol stepwise, and the phenol was removed. The DNA was dried and dissolved in an appropriate amount of 0.1XSSC solution, and RNase was added thereto, followed by reaction at 35 DEG C for at least 30 minutes to remove RNA. After ice-cooling, 1/5 amount of water-saturated phenol was added and dissolved by the same shaking, centrifugation, ethanol precipitation, phenol removal, 0.1XSSC solution, and RNase treatment was carried out.

The DNA after the RNase treatment was subjected to phenol treatment, centrifuged, and the DNA-soluble fraction was separately collected. To the DNA-soluble fraction was added 1/10 of the amount of acetate-EDTA and 0.6-fold amount of isopropanol, and the DNA was rewound again. The wound DNA was washed with 70%, 90%, and 99.5% ethanol, dried, and dissolved in a 0.1XSSC solution to a DNA concentration of 500 / / mL or more. When the purity of the DNA was low, removal of the protein by phenol and RNase treatment were repeated.

References 1: Saito, H. & Miura, K. 1963. Preparation of transforming deoxyribonucleic acid by phenol treatment. Biochim. Biophys. Acta 72: 619-629.

2) Reagent

(1) Saline-0.1 M EDTA (NaCl: 8.77 g, EDTA.2Na.2H2O: 37.22 g, pH: 8.0, ultrapure water: total amount = 1000 mL)

(2) Tris-SDS (12.1 g of Tris, 20.0 g of SDS, pH 9.0, and ultrapure water: total amount of 1000 mL)

(3) 20X SSC (175 g of NaCl, 77.4 g of trisodium citrate, and ultrapure water: total amount of 1000 mL) were prepared by diluting 20XSSC with ultra-pure water in 0.1XSSC and 1XSSC)

(4) Acetate-EDTA (solution A: CH3COONa.3H2O: 81.65 g / ultrapure water 200 mL, solution B: CH3COOH: 36.03 g / ultrapure water 200 mL) Solution A and solution B were adjusted to pH 7.0 EDTA.2Na 2H2O: 74.45 mg / 200 mL of the adjusted solution to give acetate-EDTA)

3) Measurement of GC content

The GC content was measured using a DNA GC kit (manufactured by Yamasa Co., Ltd.). 7 ㎍ equivalent of DNA was dissolved in 20 μL of 0.1 × SSC, denatured at 100 ° C. for 10 minutes, and immediately frozen. After ice-cooling, 20 μL of 4 U / mL nuclease P1 solution was added and reacted at 50 ° C. for 1 hour. After nuclease P1 treatment, 20 μL of a 2.4 U / mL alkaline phosphatase solution was added and reacted at 37 ° C. for 1 hour to provide HPLC.

An ODS column (CAPCELL PAK C18 UG120 S-5, manufactured by SHISEIDO) was used as the HPLC column, and 0.02 M NH4H2PO4: acetonitrile = 20: 1 solution was used as the developing solvent. At a moving speed of 1.0 mL / min, at a column temperature of 40 DEG C, and detected by UV (270 nm). The GC content of YM17 was calculated based on the standard attached to the DNA GC kit, and the GC content was 51.4%.

[Table 2-1]

[Table 2-2]

[Table 2-3]

[Table 2-4]

[Table 2-5]

G. Analysis of base sequence of 16S rRNA gene

1) PCR reaction

The 16S rRNA gene of YM17 was amplified by the colony direct PCR method. Tks Gflex (registered trademark) DNA Polymerase (Takara Bio), EU10F primer (SEQ ID NO: 2) and EU1500R primer (SEQ ID NO: 3) were used for the PCR reaction. The composition of the PCR reaction solution was in accordance with the mixing ratios described in the manual of Tks Gflex (registered trademark) DNA Polymerase. The PCR reaction was carried out for one cycle of heat treatment at 94 占 폚 for 1 minute, followed by 30 cycles of reaction at 98 占 폚 for 10 seconds, 55 占 폚 for 15 seconds, and 68 占 폚 for 1 minute and 30 seconds.

2) Sequence response

The PCR reaction product was purified by Wizard (registered trademark) SV Gel and PCR Clean-Up System (Promega). Sequence reaction was performed using the purified product and the EU10F primer, EU520F primer (SEQ ID NO: 4), EU907R primer (SEQ ID NO: 5), or EU1500R primer to determine the base sequence of the 16S rRNA gene of the isolate.

The nucleotide sequence of the 16S rRNA gene of YM17 is shown in SEQ ID NO: 1.

Regarding the 16S rRNA gene sequence of the YM17 strain, the BLAST homology search (http://blast.ncbi.nlm.nih.gov/Blast.cgi) of the National Center for Biotechnology Information, which is a gene database, The species exhibiting the highest homology with the rRNA gene was Mechercharimyces asporophorigenens and the homology was 91%.

Subsequently, the 16S rRNA gene of the relatives was collected from the gene database of The National Center for Biotechnology Information in the YM17 strain, and systematic analysis was performed.

The collected 16S rRNA gene sequence was aligned by the Clustal X 2.0.12 program, and the phylogenetic tree was prepared by the neighborhood binding method. The obtained number of trees is shown in Fig. The numbers in parentheses indicate the liquid session number of GenBank, and YM16 indicates the strain isolated by the present inventors in the same manner as the present invention.

H. Identification and Classification of YM17 State

1) YM17 week

FIG YM17 note as shown in the phylogenic analysis of 2 16S rRNA gene forms a branch that is different from largely separated Basil rajigwa (Bacillaceae and) or inform a cycloalkyl Basil rajigwa (Alicyclobacillaceae with) Thermo liquid Martino my three tajigwa (Thermoactinomycetaceae and).

The form of bacillus and the form of apolipoprotein expressed in YM17 strain are the ones seen in the Bacillaceae and Alicyclobacillaceae, and the thermoactinomycetaceae , which form mycelia of mating , ).

The homology of the 16S rRNA gene sequences of the species belonging to the YM17 strain, the Bacillaceae strain, and the Alicyclobacillaceae strain was compared. The results are shown in Table 3. In all species homology is less than 89%, YM17 is taxonomically very different from the species belonging to Basilagia ( Bacillaceae ), and Alicyclobacillaceae ( Alicyclobacillaceae ). In the phylogenetic tree of FIG. 2, Alcyclobacillus acidocaldarius , Calditerricola satsumensis , Caldalkalibacillus thermarum , CaldicBacillus devilis (which is closely related to the YM17 strain ) Caldibacillus debilis ) and YM17. The results are shown in Table 4.

Data in Table 4 other than the YM17 strain are reported in International Journal of Systematic and Evolutionary Microbiology (2002), 52, 1681-1685, Journal of General Microbiology (1971), 67, 9-15, , 61, 631-636, International Journal of Systematic and Evolutionary Microbiology (2006), 56, 1217-1221, and International Journal of Systematic and Evolutionary Microbiology (2004), 54, 2197-2201.

Alicyclobacillus genus ( Alicyclobacillus genus) differs from YM17 strain in that it has ω-cyclohexane-C17: 0, ω-cyclohexane-C19: 0 as a major fatty acid.

Calditoricola (genus Calditerricola ) is a highly thermophilic bacterium that can grow above 75 ℃ and is different from YM17 strain.

The genus Caldarkalibacillus (genus Caldalkalibacillus ) is gram-positive, and the base composition of DNA is 45% different from that of YM17.

The genus Caldibacillus (the genus Caldibacillus ) grows above 65 ° C and differs from the YM17 strain because no acid production from glucose is seen.

From the above, the YM17 strain was judged to be a new strain of the genus (Praterella basilagia) and swift (genus Praterella), and YM17 strain was identified as Frateribacillus flavoalbus (hereinafter simply referred to as Pratery Bacillus flavobalbus ) ).

[Example 2]

Isolation of Frateribacillus flavoalbus species

According to the above-described Example 1, it was found that the species of Fraser Bacillus flavobus differs from that of the known plants, genus and species. Therefore, in order to clarify the characteristics of Pratery Bacillus flavobus species, isolates belonging to the species of Pratery Bacillus flavobus other than YM17 strain were isolated.

1. Isolation of Prateri Bacillus flavobus species

We tried to separate the strain of Pratery Bacillus flavobus species from the compost of Kagoshima-shi city and compost of Kagoshima-shi composting ground as the separation source.

With reference to the culture characteristics of YM17 in Example 1, the culture broth was prepared by adding 7% or 10% of sodium chloride and 50 / / mL of ampicillin to CYC agar medium or no addition thereof to the culture medium at a culture temperature of 55 캜 After culturing for 6 days and isolating the bacteria, colonies of numerous colonies of the mushroom bacteria and bacteria of the liver balance appeared on the separation medium.

Among these, colonies of bacteria in the liver balance were separated and subjected to the determination by specific PCR for detecting the species of Prateri Bacillus flavus albus shown in the following 2. As shown in Table 5, a total of 12 strains Prater Bacillus Flavobacterium was determined to be a species.

In particular, the frequency of isolation in CYC agar medium supplemented with 7% sodium chloride and 50 μg / mL ampicillin was high, and it was possible to efficiently isolate Prateria Bacillus flavobus species by culturing at 55 ° C. using this medium .

The 12 isolated strains were analyzed for NaCl_20131016_04, YM17-2, YM17-3, YM17-4, NaCl_20131016_19, YM17-5, NaCl_20131016_24, NaCl_20131016_28, NaCl_20131016_30, NaCl_20131016_34, NaCl_20131016_37, or NaCl_20131016_37 Mainly.

Of these, YM17-2 strain, YM17-3 strain, YM17-4 strain and YM17-5 strain strain were tested in accordance with the application of the present applicant by Budapest Institute for Product Assessment Technology (NITE) It has been internationally deposited on the basis of a treaty. Hereinafter, the content specifying the deposit is described.

YM17-2 week

(1) Depositary Institution: Independent administrative corporation Product evaluation Technology-based organization Patented microorganism deposit center

(2) Contact: Postal 292-0818 2-5-8 Matsuri, Kazusaka, Kisaratsu, Chiba Prefecture

Phone number 0438-20-5580

(3) Accession number: NITE BP-01948

(4) Indication for identification: YM17-2

(5) Date of donation: October 6, 2014

YM17-3 week

(1) Depositary Institution: Independent administrative corporation Product evaluation Technology-based organization Patented microorganism deposit center

(2) Contact: Postal 292-0818 2-5-8 Matsuri, Kazusaka, Kisaratsu, Chiba Prefecture

Phone number 0438-20-5580

(3) Accession number: NITE BP-01949

(4) Indication for identification: YM17-3

(5) Date of donation: October 6, 2014

YM17-4 week

(1) Depositary Institution: Independent administrative corporation Product evaluation Technology-based organization Patented microorganism deposit center

(2) Contact: Postal 292-0818 2-5-8 Matsuri, Kazusaka, Kisaratsu, Chiba Prefecture

Phone number 0438-20-5580

(3) Accession number: NITE BP-01950

(4) Indication for identification: YM17-4

(5) Date of donation: October 6, 2014

YM17-5 week

(1) Depositary Institution: Independent administrative corporation Product evaluation Technology-based organization Patented microorganism deposit center

(2) Contact: Postal 292-0818 2-5-8 Matsuri, Kazusaka, Kisaratsu, Chiba Prefecture

Phone number 0438-20-5580

(3) Accession number: NITE BP-01925

(4) Indication for identification: YM17-5

(5) Date of donation: August 28, 2014

2. Specific PCR for detection of Prateri Bacillus flavobus species

Specific PCR for detecting Prateri Bacillus flavobus species was carried out as follows. First, TIP155F1 primer (5'-ATACCGGATAAGAGGCTTTT-3 ') (SEQ ID NO: 6) and TIP155R1 primer (5'-TGGTACCGTCACGCTAAGA-3') (sequence of SEQ ID NO: 6) were prepared using the base sequence specific to 16S rRNA gene of YM17 Number 7).

For the PCR reaction, Tks Gflex (R) DNA Polymerase, TIP155F1 primer, TIP155R1 primer and DNA of the isolate were used. The composition of the PCR reaction solution was in accordance with the mixing ratios described in the manual of Tks Gflex (registered trademark) DNA Polymerase. The PCR reaction was carried out for 1 cycle of heat treatment at 94 캜 for 1 minute, followed by 25 cycles of reaction at 98 캜 for 10 seconds, 55 캜 for 15 seconds, and 68 캜 for 1 minute, and amplification products The identified strain was judged to be Prateri Bacillus flavobus species.

3. Taxonomic analysis of isolated strains

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

4. Analysis of the base sequence of the 16S rRNA gene of the isolate

The nucleotide sequences of the 16S rRNA genes of the 12 strains isolated by 1. and 2. above were examined in the same manner as in Example 1 regarding the YM17 strain. The nucleotide sequences of the 16S rRNA genes of each state are shown in SEQ ID NOs: 8 to 19, respectively. SEQ ID NO: 8 is NaCl_20131016_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_20131016_19, SEQ ID NO: 13 is YM17-5, SEQ ID NO: 14 corresponds to NaCl_20131016_24 week, SEQ ID NO: 15 corresponds to NaCl_20131016_28 week, SEQ ID NO: 16 corresponds to NaCl_20131016_30 week, SEQ ID NO: 17 corresponds to NaCl_20131016_34 week, SEQ ID NO: 18 corresponds to NaCl_20131016_37 week and SEQ ID NO: 19 corresponds to NaCl_20131016_38 week.

The nucleotide sequences of the 16S rRNA gene of YM17 and the 16S rRNA gene of 12 of these strains are shown in Table 6. As shown in Table 6, the nucleotide sequence of the 16S rRNA gene of 12 strains isolated was 98.7% or more homologous with the nucleotide sequence of the 16S rRNA gene of YM17.

5. DNA-DNA hybridization

DNA-DNA hybridization tests were carried out using 12 strains isolated by 1. and 2. above and YM17 genomic DNA. In addition, as a negative control, the DNA of Calditerricola satsumensis YMO81 was used as a near continuous line. Adjustment of the DNA was carried out by the same method as in the method of adjusting the GC content of [Example 1].

All of the obtained genomic DNAs had a concentration of at least 500 / / mL, an OD260 / OD280 value of at least 1.8, and an OD260 / OD230 value of at least 2.0, which were high in quality that could be used for DNA-DNA hybridization tests .

The DNA-DNA hybridization test was performed according to the method described in Reference 2 and the classification and identification experiment of microorganisms (Supringing, Japan).

First, a DNA plate was prepared. The genomic DNA solution was diluted with 0.1 X SSC to 100 쨉 g / mL. The diluted DNA solution was heated at 100 DEG C for 5 minutes to form a single strand, and immediately frozen. The thermally denatured DNA solution was diluted 10-fold with PBS-Mg solution to 10 쨉 g / mL. 10 μg / mL was added to each well of a microplate in an amount of 100 μL. The microplate was sealed and allowed to stand at 30 ° C. for 3 hours or more. Thereafter, the DNA solution in the wells was discarded and used as a test DNA plate. The background control used salmon sperm DNA.

In parallel with the preparation of the DNA plate, photobiotin labeling of the DNA was carried out. 30 μL of a 500 μg / mL DNA solution was prepared, treated with an ultrasonic cleaner for 10 minutes, and the DNA was cut. 25 μL of 1 mg / mL photo biotin solution was added to the cleaved DNA solution, and the DNA was completely dissolved. Then, the DNA was irradiated with a low pressure mercury lamp of 250 W for 30 minutes while being ice-cooled, and the DNA was labeled with photobiotin. To remove excess photobiotin, 540 μL of 0.1 M Tris-HCl buffer (pH 9.0) was added to the irradiated DNA solution, and then 600 μL of 2-butanol was added and mixed. Centrifuged at 15,000 rpm for 5 seconds, and the supernatant containing excess photobiotin was discarded. The operation of adding 2-butanol and removing excess photobiotin was performed twice in total. The photo-biotin-labeled DNA solution was heated at 100 ° C for 5 minutes, immediately frozen, and used for hybridization.

Next, a hybridization operation was performed. 200 μL of the prehybridization mixture was injected into each well of the DNA plate and allowed to stand at 37 ° C for 15 minutes. 300 μL of thermo-denatured photobiotin-labeled DNA solution was mixed with 6 mL of a hybridization mixture. The prehybridization mixture was removed from each well of the DNA plate, and 100 .mu.l of the hybridization mixture containing the photo-biotin-labeled DNA was dividedly injected, and the DNA plate was sealed. This DNA plate was allowed to stand at 46 DEG C for 2 hours or more, and hybridization treatment was carried out. After the hybridization treatment, the hybridization mixture containing the photobiotin-labeled DNA was discarded and the wells were washed three times with 300 μL of 1 × SSC solution.

100 μL of Streptavidin-enzyme solution was injected into each well after washing. After standing at 37 DEG C for 30 minutes, the streptavidin-enzyme solution was discarded. After washing each well with 100 μL of 1 × SSC solution, each well was washed with 300 μL of 1 × SSC solution three times in total. 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). Kept at 37 캜, and the fluorescence intensity was measured every minute. The fluorescence intensity of each combination of DNA was calculated by calculating an average value of four central values in six wells and subtracting the mean fluorescence intensity value of the well to which salmon sperm DNA was attached as a background value. Homologs were expressed as a percentage of homozygous numbers using the same strain of DNA on wells and covers.

References 2: Ezaki, T., Hashimoto, Y. & Yabuuchi, 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 / 100 mL of PBS)

(2) 0.1 M Tris-HCl buffer (Tris: 12.1 g, EDTA 占 Na: 0.4 g, pH 9.0, ultrapure water:

(3) Prepare hybridization mixture (20XSSC: 1 mL, 50XDenhardt solution: 1 mL, 10 mg / mL denatured salmon DNA: 0.1 mL, formamide: 5 mL, and ultrapure water: 2.9 mL)

(4) Hybridization mixture (1 mL of 20XSSC, 1 mL of 50XDenhardt solution, 0.1 mL of 10 mg / mL denatured salmon DNA, 5 mL of formamide, 0.25 g of dextran sulfate, 2.8 mL of ultrapure water)

(5) Streptavidin-enzyme solution (10 μL of streptavidin-β-D-galactosidase conjugate, 0.5% bovine serum albumin / PBS solution: 10 mL)

(6) Fluorescent substrate solution (100 μL of 10 mg / mL 4-methylumbelliferyl-β-D-galactopyranoside solution, 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 of the mutants of YM-17 strain, YM17-2 strain, YM17-3 strain, YM17-4 strain, YM17-5 strain, NaCl_20131016_37 strain and NaCl_20131016_38 strain was more than 70%.

As described in Reference 3, in the microbial taxonomy, when the DNA-DNA homology value is 70% or more, it is regarded as the same species.

Reference 3: Stackebrandt, E. & Ebers, J., 2006. Taxonomic parameters revisited: tarnished gold standards. Microbiol. Today Nov. : p152-155

6. Properties of Pratery Bacillus Flavo Albus

By the analysis and test described above, it was confirmed that Frateribacillus flavoalbus was a species possessing the following characteristics (1) to (10) as properties.

1) Gram stain is negative, forming spores

2) oxydase test is negative, catalase test is positive and aerobic

3) When D-galactose or sucrose was added to the LB medium (pH 8.0) containing 1% sugar and 2.0% NaCl, no acid was produced, but D-glucose, lactose, D-mannitol , When glycerin or N-acetylglucosamine is added, acid production is performed

4) Intellectual growth temperature is 45 ℃ ~ 55 ℃. No growth is observed at 35 ℃ or above or above 65 ℃

5) The intellectual pH is 7.0 ~ 8.0, and the proliferation is not confirmed when the pH is lower than 5.7 or above 10.0.

6) Proliferation was confirmed by addition of NaCl to the CYC medium up to a concentration of 7%.

7) The main menaquinone is MK7

8) The amino acid composition of the cell wall peptidoglycan includes alanine, glutamic acid, and a small diamino pimelic acid (meso-DAP).

9) GC content of genomic DNA is 51.2% ~ 52.4%

10) The most important fungal fatty acid is iso C16: 0

7. Classification of subspecies in Prateri Bacillus flavobus species

(NaCl_20131016_4 week, YM17-2 week, YM17-3 week, YM17-4 week, NaCl_20131016_19 week, YM17-5 week, NaCl_20131016_24 week, NaCl_20131016_28 week, NaCl_20131016_30 week, NaCl_20131016_30 week, NaCl_20131016_34 week, NaCl-20131016_37 Figure 3 shows the phylogenetic tree by the 16S rRNA gene of the main and NaCl_20131016_38 weeks. As shown in FIG. 3, the 16S rRNA gene of YM17 strain and YM17-5 strain diverged from other strains.

As shown in Table 8, the four major iso-C16: 0, anteiso-C17: 0, anteiso-C15: 0 and iso-C17: 0 of the four major fatty acids of YM17 and YM17-5, 2 weeks, YM17-3 week, YM17-4 week, NaCl_20131016_37 week and NaCl_20131016_38 weeks were different by iso-C16: 0, C16: 0, anteiso-C17: 0, anteiso-C15: For this reason, the YM17 strain and YM17-5 strain and the other strains were the same species as the Fusarium albicans, but were different subspecies.

From the above results, two strains of YM17 strain and YM17-5 strain were named as Frateribacillus flavoalbus subsp. Flavoalbus , and strains of NaCl_20131016_04 strain , YM17-2 strain, YM17-3 strain, YM17- 4 weeks, NaCl_20131016_19 weeks, NaCl_20131016_24 weeks, NaCl_20131016_28 weeks, NaCl_20131016_30 weeks, NaCl_20131016_34 weeks, NaCl_20131016_37 weeks and NaCl_20131016_38 weeks were named as Frateribacillus flavoalbus subsp. Fimetarium .

That is to say, the Frateribacillus flavoalbus belongs to the genus Frateribacillus flavoalbus subsp. Flavoalbus and the Frateribacillus flavoalbus subsp. Fimetarium . It was swift and new which consisted of two subspecies.

8. Proteolytic activity of Pratery Bacillus flavobus

Next, the proteolytic activity of Frateribacillus flavoalbus was examined. 2.0% of gelatin or 0.4% of casein was added to the basic medium (2.0% of sodium chloride, 0.1% of yeast extract), the pH was adjusted to 8.0, 1.5% of agar was added and the autoclave And sterilized to prepare a test medium.

The test medium was supplemented with YM17, NaCl_20131016_04, YM17-2, YM17-3, YM17-4, NaCl_20131016_19, YM17-5, NaCl_20131016_24, NaCl_20131016_28, NaCl_20131016_30, NaCl_20131016_34, NaCl_20131016_37, NaCl_20131016_38, And cultured at 55 ° C for 7 days. After the incubation, the hydrochloric acid-mercuric chloride solution (15 g of mercuric chloride, 20 mL of concentrated hydrochloric acid, 100 mL of distilled water) was added dropwise to the test medium, and the degradation activity of the protein was examined by halo formation. As a result of the test, degradation activity of gelatin and casein was observed in all 13 strains.

9. Growth and decomposition of organic matter in the compost environment of Pratery Bacillus flavobus

The growth and organic degradation activity of Frateribacillus flavoalbus in compost was investigated.

1 ml of a 10% yeast extract solution sterilized by filtration, 3 ml of a 1% casein solution or sterilized water, and 2 ml of an LB medium (containing 2% NaCl) were added to 10 g of compost obtained by autoclaving sterilization at 120 ° C for 30 minutes pH 8.0), 1 ml of the culture solution of the bacteria cultured at 55 占 폚 for 1 day was added, and the mixture was sterilized so that the water spread evenly throughout the compost. The mixture was sealed in a state of sufficient vapor 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 2% NaCl sterilized in place of the bacterial culture solution. As test organisms, YM17 strain, YM17-2 strain, YM17-3 strain, YM17-4 strain, YM17-5 strain, NaCl_20131016_37 strain and NaCl_20131016_38 strain were used.

The cultures after 3 days of compost cultivation were observed under a microscope. In the test group supplemented with YM17 strain, YM17-2 strain, YM17-3 strain, YM17-4 strain, YM17-5 strain, NaCl_20131016_37 strain or NaCl_20131016_38 strain, The growth of good bacteria was confirmed. On the other hand, no cells were observed in the negative control to which the culture of the bacteria was not added. Fig. 4 shows a photomicrograph of the YM17 main or negative control after 3 days of casein-added compost cultivation.

As indicated by the circle A in FIG. 4, good growth of the cells was confirmed with respect to the YM17 strain. On the other hand, as shown in Fig. 4B, only the solid matter of the compost was observed in the negative control, and the cells were not observed.

Proteins were extracted from the cultures after 3 days of compost cultivation. First, 30 mL of Tris-HCl (pH 8.0) was added to the culture to make a suspension, and the mixture was allowed to stand at room temperature for 3 hours. The suspension after filtration was filtered (filter paper: Adovan Tech Co., model No. 5B, 110 mm), and the filtrate was recovered. The remaining residue was washed with Tris-HCl (pH 8.0), and the total amount of the filtrate was adjusted to 30 mL. To 30 mL of the filtrate, 14.2 g of ammonium sulfate was added, and the mixture was gradually stirred to dissolve the ammonium sulfate. After dissolving ammonium sulfate, it was allowed to stand overnight on ice. After the standing, the solution was centrifuged at 10,000 째 C for 30 minutes at 4 째 C, the supernatant was removed, and the precipitate was recovered. The precipitate was dissolved in 1 mL of Tris-HCl (pH 8.0) and the lysate was placed in a dialysis membrane (Spectrum Laboratories, Inc., Spectra / Pore TM 1, MW 6,000-8,000) ) And desalted by dialysis. After desalting, the volume of the solution was adjusted to 2 mL with Tris-HCl (pH 8.0), and the sample was used as a sample providing a gammagram.

Gel electrophoresis gel for gel electrophoresis was prepared by adding 0.1% gelatin as a substrate. 15 μL of protein solution after desalting and 5 μL of Loading Buffer were mixed, and 20 μL of the mixture was supplied to the mammography.

The gel after electrophoresis was placed in Tris-HCl (pH 8.0) containing 0.2% TritonX for 10 minutes, and the buffer was discarded and washed twice with Tris-HCl (pH 8.0) containing no TritonX. The washed gel was immersed in 20 mL of Tris-HCl (pH 8.0) containing 2 mM of CaCl 2 and 2 mM of MgSO 4, allowed to stand at 55 ° C for 16 hours, and then subjected to 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 min, discolored after Coomassie staining, I investigated.

As a result of the zymography, the protein extracted from the compost culture with no casein showed a gelatinolytic activity, but the protein extracted from the compost culture with casein showed a stronger activity. Further, in the negative control to which the culture medium of the bacteria was not added, the protein extracted from the compost culture showed no gelatin decomposition activity regardless of whether casein was added or not.

In the case of the addition of the culture medium of the microorganism, the decomposition activity of the protein was also shown in the case of the compost cultivation with no casein added because the protein remained in the compost as a base and the activity was induced. In addition, when the casein was added and cultivation was carried out, the activity induction was more strongly appeared.

From the above results, Frateribacillus flavoalbus species such as YM17 strain , YM17-2 strain , YM17-3 strain , YM17-4 strain , YM17-5 strain , NaCl_20131016_37 strain or NaCl_20131016_38 strain were grown in compost , And has proteolytic activity. Fig. 5 shows the results of the gelatinization. Fig. 5A shows the result of the glycation using the protein extracted from the casein-free compost culture, and Fig. 5B shows the result of the glycation using the protein extracted from the casein-added compost culture.

reagent

(1) Tris-HCl (pH 8.0) (Tris: 6.1 g, adjusted to pH 8.0 with concentrated hydrochloric acid, ultrapure water: added to a total volume of 1000 mL)

(2) Electrophoresis gel for zymogram

Separate layer (30% Acrylamide: 2000 μL, 1.5 μM Tris-HCl (pH 8.5): 1500 μL, Ultrapure Water: 1800 μL, 10% SDS: 60 μL, 10% APS: 50 μL, TEMED: Gelatin: 600 [mu] L)

Stacking layer (750 μL of 30% acrylamide, 750 μL of 0.5 M Tris-HCl (pH 6.5), 1700 μL of ultrapure water, 30 μL of 10% SDS, 50 μL of 10% APS, 5 μL of TEMED)

After the separate layer is solidified, the stacking layer is layered and solidified.

(3) Electrophoresis buffer (Tris: 30.3 g, glycine: 144 g, ultrapure water: 1000 mL total amount) was added to a 10X genomogram. In the electrophoresis of zymogram, the electrophoresis buffer for 10 × Zymogram was diluted with ultrapure water to 1 × buffer, and SDS was added so that the final concentration was 0.1%.

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

[Access number]

[Reference to Deposited Biomaterials]

(1) Depositary Institution: Independent administrative corporation Product evaluation Technology-based organization Patented microorganism deposit center

(2) Contact: Postal 292-0818 2-5-8 Matsuri, Kazusaka, Kisaratsu, Chiba Prefecture

Phone number 0438-20-5580

(3) Accession number: NITE BP-01764

(4) Indication for identification: YM17

(5) Date of original deposit: November 29, 2013

[Reference to Deposited Biomaterials]

(1) Depositary Institution: Independent administrative corporation Product evaluation Technology-based organization Patented microorganism deposit center

(2) Contact: Postal 292-0818 2-5-8 Matsuri, Kazusaka, Kisaratsu, Chiba Prefecture

Phone number 0438-20-5580

(3) Accession number: NITE BP-01948

(4) Indication for identification: YM17-2

(5) Date of donation: October 6, 2014

[Reference to Deposited Biomaterials]

(1) Depositary Institution: Independent administrative corporation Product evaluation Technology-based organization Patented microorganism deposit center

(2) Contact: Postal 292-0818 2-5-8 Matsuri, Kazusaka, Kisaratsu, Chiba Prefecture

Phone number 0438-20-5580

(3) Accession number: NITE BP-01949

(4) Indication for identification: YM17-3

(5) Date of donation: October 6, 2014

[Reference to Deposited Biomaterials]

(1) Depositary Institution: Independent administrative corporation Product evaluation Technology-based organization Patented microorganism deposit center

(2) Contact: Postal 292-0818 2-5-8 Matsuri, Kazusaka, Kisaratsu, Chiba Prefecture

Phone number 0438-20-5580

(3) Accession number: NITE BP-01950

(4) Indication for identification: YM17-4

(5) Date of donation: October 6, 2014

[Reference to Deposited Biomaterials]

(1) Depositary Institution: Independent administrative corporation Product evaluation Technology-based organization Patented microorganism deposit center

(2) Contact: Postal 292-0818 2-5-8 Matsuri, Kazusaka, Kisaratsu, Chiba Prefecture

Phone number 0438-20-5580

(3) Accession number: NITE BP-01925

(4) Indication for identification: YM17-5

(5) Date of donation: August 28, 2014

                         SEQUENCE LISTING <110> SANYU CO., LTD.   <120> Novel Bacterium <130> SANYU-130 <160> 19 <170> PatentIn version 3.5 <210> 1 <211> 1416 <212> DNA <213> Frateribacillus flavoalbus <400> 1 gt gcggcggacg ggtgagtaac acgtgggcaa cctgcctgca agaccgggat aactgcggga 120 aaccggagct aataccggat aagaggcttt yccgcatggg ggagccttaa aaggcggcgy 180 aagtcgtcac ttgcagatgg gcccgcggcg cattagctcg ttggtgaggt aagggctcac 240 caaggcgacg atgcgtagcc gacctgagag ggtgaccggc cacactggga ctgagacacg 300 gcccagactc ctacgggagg cagcagtagg gaattttcgg caatgggggg aaccctgacc 360 gagcaacgcc gcgtgagcga ggacggcctt cgggttgtaa agctctgtca tgtgggaaga 420 acggctagga gagggcatgc tcttagcgtg acggtaccac aggaggaagc cccggctaac 480 tacgtgccag cagccgcggt aacacgtagg gggcgagcgt tgtccggaat tattgggcgt 540 aaagggcgcg caggcggtct cttaagtctg atgtgaaagg ccacggctca accgtgggat 600 ggcattggaa actgggggac ttgagtgcag gagaggggag cggaattccc ggtgtagcgg 660 tgaaatgcgt agagatcggg aggaacacca gtggcgaagg cggctctctg gcctgtaact 720 gacgctgagg cgcgaaagcg tggggagcga acaggattag ataccctggt agtccacgcc 780 gtaaacgatg agtgctaggt gttaggggyg tcasrycctt agtgccgaag tgaacacatt 840 aagcactccg cctggggagt acggccgcaa ggctgaaact caaaggaatt gacgggggcc 900 cgcacaagcg gtggagcatg tggtttaatt cgaagcaacg cgaagaacct taccagggct 960 tgacatcctc tgacccctct agagatagag gtttcccttc ggggacagag agacaggtgg 1020 tgcatggttg tcgtcagctc gtgtcgtgag atgttgggtt aagtcccgca acgagcgcaa 1080 cccttgagct tagttgccag cattgagttg ggcactctaa gctgactgcc agtgacaagc 1140 tggaggaagg tggggatgac gtcaaatcat catgcccctt atgtcctggg cgacacacgt 1200 gctacaatgg ctggtacaga gggcagcgaa gcggtgacgc ggagccaatc ccagaaagcc 1260 agtctcagtt cggatcgcag gctgcaactc gcctgcgtga aggaggaatc gctagtaatc 1320 gcggatcagc atgccgcggt gaatacgttc ccgggccttg tacacaccgc ccgtcacacc 1380 acgagagtct gtaacacccg aagtcggtga ggcaac 1416 <210> 2 <211> 19 <212> DNA <213> Artificial sequence <220> <223> primer <400> 2 agagtttgat cctggctca 19 <210> 3 <211> 19 <212> DNA <213> Artificial sequence <220> <223> primer <400> 3 ggttaccttg ttacgactt 19 <210> 4 <211> 16 <212> DNA <213> Artificial sequence <220> <223> primer <400> 4 gtgccagcag ccgcgg 16 <210> 5 <211> 20 <212> DNA <213> Artificial sequence <220> <223> primer <400> 5 ccgtcaattc atttgagttt 20 <210> 6 <211> 20 <212> DNA <213> Artificial sequence <220> <223> primer <400> 6 ataccggata agaggctttt 20 <210> 7 <211> 19 <212> DNA <213> Artificial sequence <220> <223> primer <400> 7 tggtaccgtc acgctaaga 19 <210> 8 <211> 1406 <212> DNA <213> Frateribacillus flavoalbus <400> 8 gtcgtgcggg tgaagccgac caccccttcg ggggcggatg gtggaaccag cggcggacgg 60 gtgagtaaca cgtgggcaac ctgcctgcaa gaccgggata actgcgggaa accggagcta 120 ataccggata agaggctttt ccgcatgggg gagccttaaa aggcggcgga agtcgtcrct 180 tgcagatggg cycgcggcgc attagctagt tggtgaggta agggctcacc aaggcgacga 240 tgcgtagccg acctgagagg gtgaccggcc acactgggac tgagacacgg cccagactcc 300 tacgggaggc agcagtaggg aattttcggc aatgggggga accctgaccg agcaacgccg 360 cgtgagcgag gacggccttc gggttgtaaa gctctgtcat gtgggaagaa cggctaggag 420 agggcatgct cttagcgtga cggtaccaca ggaggaagcc ccggctaact acgtgccagc 480 agccgcggta acacgtaggg ggcgagcgtt gtccggaatt attgggcgta aagggcgcgc 540 aggcggtctc ttaagtctga tgtgaaaggc cacggctcaa ccgtgggatg gcattggaaa 600 ctgggggact tgagtgcagg agaggggagc ggaattcccg gtgtagcggt gaaatgcgta 660 gagatcggga ggaacaccag tggcgaaggc ggctctctgg cctgtaactg acgctgaggc 720 gcgaaagcgt ggggagcgaa caggattaga taccctggta gtccacgccg taaacgatga 780 gtgctaggtg ttaggggcgt cacgccctta gtgccgaagt aaacacatta agcactccgc 840 ctggggagta cggccgcaag gctgaaactc aaaggaattg acgggggccc gcacaagcgg 900 tggagcatgt ggtttaattc gaagcaacgc gaagaacctt accagggctt gacatcctct 960 gacccctcta gagatagagg tttcccttcg gggacagaga gacaggtggt gcatggttgt 1020 cgtcagctcg tgtcgtgaga tgttgggtta agtcccgcaa cgagcgcaac ccttgagctt 1080 agttgccagc attgagttgg gcactctaag ctgactgcca gtgacaagct ggaggaaggt 1140 ggggatgacg tcaaatcatc atgcccctta tgtcctgggc gacacacgtg ctacaatggc 1200 tggtacagag ggcagcgaag cggtgacgcg gagccaatcc ctaaaaagcc agtctcagtt 1260 cggatcgcag gctgcaactc gcctgcgtga aggaggaatc gctagtaatc gcggatcagc 1320 atgccgcggt gaatacgttc ccgggccttg tacacaccgc ccgtcacacc acgagagtct 1380 gtaacacccg aagtcggtga ggcaac 1406 <210> 9 <211> 1406 <212> DNA <213> Frateribacillus flavoalbus <400> 9 gtcgtgcggg tgaagccgac caccccttcg ggggcggatg gtggaaccag cggcggacgg 60 gtgagtaaca cgtgggcaac ctgcctgcaa gaccgggata actgcgggaa accggagcta 120 ataccggata agaggctttt ccgcatgggg gagccttaaa aggcggcgga agtcgtcact 180 tgcagatggg cccgcggcgc attagctagt tggtgaggga agggctcacc aaggcgacga 240 tgcgtagccg acctgagagg gtgaccggcc acactgggac tgagacacgg cccagactcc 300 tacgggaggc agcagtaggg aattttcggc aatgggggga accctgaccg agcaacgccg 360 cgtgagcgag gacggccttc gggttgtaaa gctctgtcat gtgggaagaa cggctaggag 420 agggcatgct cttagcgtga cggtaccaca ggaggaagcc ccggctaact acgtgccagc 480 agccgcggta acacgtaggg ggcgagcgtt gtccggaatt attgggcgta aagggcgcgc 540 aggcggtctc ttaagtctga tgtgaaaggc cacggctcaa ccgtgggatg gcattggaaa 600 ctgggggact tgagtgcagg agaggggagc ggaattcccg gtgtagcggt gaaatgcgta 660 gagatcggga ggaacaccag tggcgaaggc ggctctctgg cctgtaactg acgctgaggc 720 gcgaaagcgt ggggagcgaa caggattaga taccctggta gtccacgccg taaacgatga 780 gtgctaggtg ttaggggcgt cacgccctta gtgccgaagt aaacacatta agcactccgc 840 ctggggagta cggccgcaag gctgaaactc aaaggaattg acgggggccc gcacaagcgg 900 tggagcatgt ggtttaattc gaagcaacgc gaagaacctt accagggctt gacatcctct 960 gacccctcta gagatagagg tttcccttcg gggacagaga gacaggtggt gcatggttgt 1020 cgtcagctcg tgtcgtgaga tgttgggtta agtcccgcaa cgagcgcaac ccttgagctt 1080 agttgccagc attgagttgg gcactctaag ctgactgcca gtgacaagct ggaggaaggt 1140 ggggatgacg tcaaatcatc atgcccctta tgtcctgggc gacacacgtg ctacaatggc 1200 tggtacagag ggcagcgaag cggtgacgcg gagccaatcc ctaaaaagcc agtctcagtt 1260 cggatcgcag gctgcaactc gcctgcgtga aggaggaatc gctagtaatc gcggatcagc 1320 atgccgcggt gaatacgttc ccgggccttg tacacaccgc ccgtcacacc acgagagtct 1380 gtaacacccg aagtcggtga ggcaac 1406 <210> 10 <211> 1406 <212> DNA <213> Frateribacillus flavoalbus <400> 10 gtcgtgcggg tgaagccgac caccccttcg ggggcggatg gcggaaccag cggcggacgg 60 gtgagtaaca cgtgggcaac ctgcctgcaa gaccgggata actgcgggaa accggagcta 120 ataccggata agaggctttt ccgcatgggg gagccttaaa aggcggcgga agtcgtcact 180 tgcagatggg cccgcggcgc attagctagt tggtgaggga agggctcacc aaggcgacga 240 tgcgtagccg acctgagagg gtgaccggcc acactgggac tgagacacgg cccagactcc 300 tacgggaggc agcagtaggg aattttcggc aatgggggga accctgaccg agcaacgccg 360 cgtgagcgag gacggccttc gggttgtaaa gctctgtcat gtgggaagaa cggctaggag 420 agggcatgct cttagcgtga cggtaccaca ggaggaagcc ccggctaact acgtgccagc 480 agccgcggta acacgtaggg ggcgagcgtt gtccggaatt attgggcgta aagggcgcgc 540 aggcggtctc ttaagtctga tgtgaaaggc cacggctcaa ccgtgggatg gcattggaaa 600 ctgggggact tgagtgcagg agaggggagc ggaattcccg gtgtagcggt gaaatgcgta 660 gagatcggga ggaacaccag tggcgaaggc ggctctctgg cctgtaactg acgctgaggc 720 gcgaaagcgt ggggagcgaa caggattaga taccctggta gtccacgccg taaacgatga 780 gtgctaggtg ttaggggcgt cacgccctta gtgccgaagt aaacacatta agcactccgc 840 ctggggagta cggccgcaag gctgaaactc aaaggaattg acgggggccc gcacaagcgg 900 tggagcatgt ggtttaattc gaagcaacgc gaagaacctt accagggctt gacatcctct 960 gacccctcta gagatagagg tttcccttcg gggacagaga gacaggtggt gcatggttgt 1020 cgtcagctcg tgtcgtgaga tgttgggtta agtcccgcaa cgagcgcaac ccttgagctt 1080 agttgccagc attgagttgg gcactctaag ctgactgcca gtgacaagct ggaggaaggt 1140 ggggatgacg tcaaatcatc atgcccctta tgtcctgggc gacacacgtg ctacaatggc 1200 tggtacagag ggcagcgaag cggtgacgcg gagccaatcc ctaaaaagcc agtctcagtt 1260 cggatcgcag gctgcaactc gcctgcgtga aggaggaatc gctagtaatc gcggatcagc 1320 atgccgcggt gaatacgttc ccgggccttg tacacaccgc ccgtcacacc acgagagtct 1380 gtaacacccg aagtcggtga ggcaac 1406 <210> 11 <211> 1406 <212> DNA <213> Frateribacillus flavoalbus <400> 11 gtcgtgcggg tgaagccgac caccccttcg ggggcggatg gtggaaccag cggcggacgg 60 gtgagtaaca cgtgggcaac ctgcctgcaa gaccgggata actgcgggaa accggagcta 120 ataccggata agaggctttt ccgcatgggg gagccttaaa aggcggcgga agtcgtcact 180 tgcagatggg cccgcggcgc attagctagt tggtgaggga agggctcacc aaggcgacga 240 tgcgtagccg acctgagagg gtgaccggcc acactgggac tgagacacgg cccagactcc 300 tacgggaggc agcagtaggg aattttcggc aatgggggga accctgaccg agcaacgccg 360 cgtgagcgag gacggccttc gggttgtaaa gctctgtcat gtgggaagaa cggctaggag 420 agggcatgct cttagcgtga cggtaccaca ggaggaagcc ccggctaact acgtgccagc 480 agccgcggta acacgtaggg ggcgagcgtt gtccggaatt attgggcgta aagggcgcgc 540 aggcggtctc ttaagtctga tgtgaaaggc cacggctcaa ccgtgggatg gcattggaaa 600 ctgggggact tgagtgcagg agaggggagc ggaattcccg gtgtagcggt gaaatgcgta 660 gagatcggga ggaacaccag tggcgaaggc ggctctctgg cctgtaactg acgctgaggc 720 gcgaaagcgt ggggagcgaa caggattaga taccctggta gtccacgccg taaacgatga 780 gtgctaggtg ttaggggcgt cacgccctta gtgccgaagt aaacacatta agcactccgc 840 ctggggagta cggccgcaag gctgaaactc aaaggaattg acgggggccc gcacaagcgg 900 tggagcatgt ggtttaattc gaagcaacgc gaagaacctt accagggctt gacatcctct 960 gacccctcta gagatagagg tttcccttcg gggacagaga gacaggtggt gcatggttgt 1020 cgtcagctcg tgtcgtgaga tgttgggtta agtcccgcaa cgagcgcaac ccttgagctt 1080 agttgccagc attgagttgg gcactctaag ctgactgcca gtgacaagct ggaggaaggt 1140 ggggatgacg tcaaatcatc atgcccctta tgtcctgggc gacacacgtg ctacaatggc 1200 tggtacagag ggcagcgaag cggtgacgcg gagccaatcc ctaaaaagcc agtctcagtt 1260 cggatcgcag gctgcaactc gcctgcgtga aggaggaatc gctagtaatc gcggatcagc 1320 atgccgcggt gaatacgttc ccgggccttg tacacaccgc ccgtcacacc acgagagtct 1380 gtaacacccg aagtcggtga ggcaac 1406 <210> 12 <211> 1406 <212> DNA <213> Frateribacillus flavoalbus <400> 12 gtcgtgcggg tgaagccgac caccccttcg ggggcggatg gcggaaccag cggcggacgg 60 gtgagtaaca cgtgggcaac ctgcctgcaa gaccgggata actgcgggaa accggagcta 120 ataccggata agaggctttt ccgcatgggg gagccttaaa aggcggcgga agtcgtcact 180 tgcagatggg cccgcggcgc attagctagt tggtgaggka agggctcacc aaggcgacga 240 tgcgtagccg acctgagagg gtgaccggcc acactgggac tgagacacgg cccagactcc 300 tacgggaggc agcagtaggg aattttcggc aatgggggga accctgaccg agcaacgccg 360 cgtgagcgag gacggccttc gggttgtaaa gctctgtcat gtgggaagaa cggctaggag 420 agggcatgct cttagcgtga cggtaccaca ggaggaagcc ccggctaact acgtgccagc 480 agccgcggta acacgtaggg ggcgagcgtt gtccggaatt attgggcgta aagggcgcgc 540 aggcggtctc ttaagtctga tgtgaaaggc cacggctcaa ccgtgggatg gcattggaaa 600 ctgggggact tgagtgcagg agaggggagc ggaattcccg gtgtagcggt gaaatgcgta 660 gagatcggga ggaacaccag tggcgaaggc ggctctctgg cctgtaactg acgctgaggc 720 gcgaaagcgt ggggagcgaa caggattaga taccctggta gtccacgccg taaacgatga 780 gtgctaggtg ttaggggcgt cacgccctta gtgccgaagt aaacacatta agcactccgc 840 ctggggagta cggccgcaag gctgaaactc aaaggaattg acgggggccc gcacaagcgg 900 tggagcatgt ggtttaattc gaagcaacgc gaagaacctt accagggctt gacatcctct 960 gacccctcta gagatagagg tttcccttcg gggacagaga gacaggtggt gcatggttgt 1020 cgtcagctcg tgtcgtgaga tgttgggtta agtcccgcaa cgagcgcaac ccttgagctt 1080 agttgccagc attgagttgg gcactctaag ctgactgcca gtgacaagct ggaggaaggt 1140 ggggatgacg tcaaatcatc atgcccctta tgtcctgggc gacacacgtg ctacaatggc 1200 tggtacagag ggcagcgaag cggtgacgcg gagccaatcc ctaaaaagcc agtctcagtt 1260 cggatcgcag gctgcaactc gcctgcgtga aggaggaatc gctagtaatc gcggatcagc 1320 atgccgcggt gaatacgttc ccgggccttg tacacaccgc ccgtcacacc acgagagtct 1380 gtaacacccg aagtcggtga ggcaac 1406 <210> 13 <211> 1405 <212> DNA <213> Frateribacillus flavoalbus <400> 13 gtcgtgcggg agaagccgac cgccccttcg ggggaggatg gcggaaccag cggcggacgg 60 gtgagtaaca cgtgggcaac ctgcctgcaa gaccgggata actgcgggaa accggagcta 120 ataccggata agaggctttt ccgcatgggg gagccttaaa aggcggcggg agtcgtcact 180 tgcagatggg cccgcggcgc attagctagt tggtgaggga agggctcacc aaggcgacga 240 tgcgtagccg acctgagagg gtgaccggcc acactgggac tgagacacgg cccagactcc 300 tacgggaggc agcagtaggg aattttcggc aatgggggga accctgaccg agcaacgccg 360 cgtgagcgag gacggccttc gggttgtaaa gctctgtcat gtgggaagaa cggctaggag 420 agggcatgct cttagcgtga cggtaccaca ggaggaagcc ccggctaact acgtgccagc 480 agccgcggta acacgtaggg ggcgagcgtt gtccggaatt attgggcgta aagggcgcgc 540 aggcggtctc ttaagtctga tgtgaaaggc cacggctcaa ccgtgggatg gcattggaaa 600 ctgggggact tgagtgcagg agaggggagc ggaattcccg gtgtagcggt gaaatgcgta 660 gagatcggga ggaacaccag tggcgaaggc ggctctctgg cctgtaactg acgctgaggc 720 gcgaaagcgt ggggagcgaa caggattaga taccctggta gtccacgccg taaacgatga 780 gtgctaggtg ttaggggcgt cacgccctta gtgccgcagt gaacacatta agcactccgc 840 ctggggagta cggccgcaag gctgaaactc aaaggaattg acgggggccc gcacaagcgg 900 tggagcatgt ggtttaattc gaagcaacgc gaagaacctt accagggctt gacatcctct 960 gacccctcta gagatagagg tttcccttcg gggacagaga gacaggtggt gcatggttgt 1020 cgtcagctcg tgtcgtgaga tgttgggtta agtcccgcaa cgagcgcaac ccttgagctt 1080 agttgccagc attaagttgg gcactctaag ctgactgcca gtgacaagct ggaggaaggt 1140 ggggatgacg tcaaatcatc atgcccctta tgtcctgggc gacacacgtg ctacaatggc 1200 tggtacagag ggcagcgaag cggtgacgcg gagccaatcc cagaaagcca gtctcagttc 1260 ggatcgcagg ctgcaactcg cctgcgtgaa ggaggaatcg ctagtaatcg cggatcagca 1320 tgccgcggtg aatacgttcc cgggccttgt acacaccgcc cgtcacacca cgagagtctg 1380 taacacccga agtcggtgag gcaac 1405 <210> 14 <211> 1406 <212> DNA <213> Frateribacillus flavoalbus <400> 14 gtcgtgcggg tgaagccgac caccccttcg ggggcggatg gtggaaccag cggcggacgg 60 gtgagtaaca cgtgggcaac ctgcctgcaa gaccgggata actgcgggaa accggagcta 120 ataccggata agaggctttt ccgcatgggg gagccttaaa aggcggcgga agtcgtcact 180 tgcagatggg cccgcggcgc attagctagt tggtgaggga agggctcacc aaggcgacga 240 tgcgtagccg acctgagagg gtgaccggcc acactgggac tgagacacgg cccagactcc 300 tacgggaggc agcagtaggg aattttcggc aatgggggga accctgaccg agcaacgccg 360 cgtgagcgag gacggccttc gggttgtaaa gctctgtcat gtgggaagaa cggctaggag 420 agggcatgct cttagcgtga cggtaccaca ggaggaagcc ccggctaact acgtgccagc 480 agccgcggta acacgtaggg ggcgagcgtt gtccggaatt attgggcgta aagggcgcgc 540 aggcggtctc ttaagtctga tgtgaaaggc cacggctcaa ccgtgggatg gcattggaaa 600 ctgggggact tgagtgcagg agaggggagc ggaattcccg gtgtagcggt gaaatgcgta 660 gagatcggga ggaacaccag tggcgaaggc ggctctctgg cctgtaactg acgctgaggc 720 gcgaaagcgt ggggagcgaa caggattaga taccctggta gtccacgccg taaacgatga 780 gtgctaggtg ttaggggcgt cacgccctta gtgccgaagt aaacacatta agcactccgc 840 ctggggagta cggccgcaag gctgaaactc aaaggaattg acgggggccc gcacaagcgg 900 tggagcatgt ggtttaattc gaagcaacgc gaagaacctt accagggctt gacatcctct 960 gacccctcta gagatagagg tttcccttcg gggacagaga gacaggtggt gcatggttgt 1020 cgtcagctcg tgtcgtgaga tgttgggtta agtcccgcaa cgagcgcaac ccttgagctt 1080 agttgccagc attgagttgg gcactctaag ctgactgcca gtgacaagct ggaggaaggt 1140 ggggatgacg tcaaatcatc atgcccctta tgtcctgggc gacacacgtg ctacaatggc 1200 tggtacagag ggcagcgaag cggtgacgcg gagccaatcc ctaaaaagcc agtctcagtt 1260 cggatcgcag gctgcaactc gcctgcgtga aggaggaatc gctagtaatc gcggatcagc 1320 atgccgcggt gaatacgttc ccgggccttg tacacaccgc ccgtcacacc acgagagtct 1380 gtaacacccg aagtcggtga ggcaac 1406 <210> 15 <211> 1406 <212> DNA <213> Frateribacillus flavoalbus <400> 15 gtcgtgcggg tgaagccgac cgccccttcg ggggcggatg gtggaaccag cggcggacgg 60 gtgagtaaca cgtgggcaac ctgcctgcaa gaccgggata actgcgggaa accggagcta 120 ataccggata agaggctttt ccgcatgggg gagccttaaa aggcggcgga agtcgtcact 180 tgcagatggg cccgcggcgc attagctagt tggtgaggga agggctcacc aaggcgacga 240 tgcgtagccg acctgagagg gtgaccggcc acactgggac tgagacacgg cccagactcc 300 tacgggaggc agcagtaggg aattttcggc aatgggggga accctgaccg agcaacgccg 360 cgtgagcgag gacggccttc gggttgtaaa gctctgtcat gtgggaagaa cggctaggag 420 agggcatgct cttagcgtga cggtaccaca ggaggaagcc ccggctaact acgtgccagc 480 agccgcggta acacgtaggg ggcgagcgtt gtccggaatt attgggcgta aagggcgcgc 540 aggcggtctc ttaagtctga tgtgaaaggc cacggctcaa ccgtgggatg gcattggaaa 600 ctgggggact tgagtgcagg agaggggagc ggaattcccg gtgtagcggt gaaatgcgta 660 gagatcggga ggaacaccag tggcgaaggc ggctctctgg cctgtaactg acgctgaggc 720 gcgaaagcgt ggggagcgaa caggattaga taccctggta gtccacgccg taaacgatga 780 gtgctaggtg ttaggggcgt cacgccctta gtgccgaagt aaacacatta agcactccgc 840 ctggggagta cggccgcaag gctgaaactc aaaggaattg acgggggccc gcacaagcgg 900 tggagcatgt ggtttaattc gaagcaacgc gaagaacctt accagggctt gacatcctct 960 gacccctcta gagatagagg tttcccttcg gggacagaga gacaggtggt gcatggttgt 1020 cgtcagctcg tgtcgtgaga tgttgggtta agtcccgcaa cgagcgcaac ccttgagctt 1080 agttgccagc attgagttgg gcactctaag ctgactgcca gtgacaagct ggaggaaggt 1140 ggggatgacg tcaaatcatc atgcccctta tgtcctgggc gacacacgtg ctacaatggc 1200 tggtacagag ggcagcgaag cggtgacgcg gagccaatcc ctaaaaagcc agtctcagtt 1260 cggatcgcag gctgcaactc gcctgcgtga aggaggaatc gctagtaatc gcggatcagc 1320 atgccgcggt gaatacgttc ccgggccttg tacacaccgc ccgtcacacc acgagagtct 1380 gtaacacccg aagtcggtga ggcaac 1406 <210> 16 <211> 1406 <212> DNA <213> Frateribacillus flavoalbus <400> 16 gtcgtgcggg tgaagccgac caccccttcg ggggcggatg gtggaaccag cggcggacgg 60 gtgagtaaca cgtgggcaac ctgcctgcaa gaccgggata actgcgggaa accggagcta 120 ataccggata agaggctttt ccgcatgggg gagccttaaa aggcggcgga agtcgtcact 180 tgcagatggg cccgcggcgc attagctagt tggtgaggta agggctcacc aaggcgacga 240 tgcgtagccg acctgagagg gtgaccggcc acactgggac tgagacacgg cccagactcc 300 tacgggaggc agcagtaggg aattttcggc aatgggggga accctgaccg agcaacgccg 360 cgtgagcgag gacggccttc gggttgtaaa gctctgtcat gtgggaagaa cggctaggag 420 agggcatgct cttagcgtga cggtaccaca ggaggaagcc ccggctaact acgtgccagc 480 agccgcggta acacgtaggg ggcgagcgtt gtccggaatt attgggcgta aagggcgcgc 540 aggcggtctc ttaagtctga tgtgaaaggc cacggctcaa ccgtgggatg gcattggaaa 600 ctgggggact tgagtgcagg agaggggagc ggaattcccg gtgtagcggt gaaatgcgta 660 gagatcggga ggaacaccag tggcgaaggc ggctctctgg cctgtaactg acgctgaggc 720 gcgaaagcgt ggggagcgaa caggattaga taccctggta gtccacgccg taaacgatga 780 gtgctaggtg ttaggggcgt cacgccctta gtgccgaagt aaacacatta agcactccgc 840 ctggggagta cggccgcaag gctgaaactc aaaggaattg acgggggccc gcacaagcgg 900 tggagcatgt ggtttaattc gaagcaacgc gaagaacctt accagggctt gacatcctct 960 gacccctcta gagatagagg tttcccttcg gggacagaga gacaggtggt gcatggttgt 1020 cgtcagctcg tgtcgtgaga tgttgggtta agtcccgcaa cgagcgcaac ccttgagctt 1080 agttgccagc attgagttgg gcactctaag ctgactgcca gtgacaagct ggaggaaggt 1140 ggggatgacg tcaaatcatc atgcccctta tgtcctgggc gacacacgtg ctacaatggc 1200 tggtacagag ggcagcgaag cggtgacgcg gagccaatcc ctaaaaagcc agtctcagtt 1260 cggatcgcag gctgcaactc gcctgcgtga aggaggaatc gctagtaatc gcggatcagc 1320 atgccgcggt gaatacgttc ccgggccttg tacacaccgc ccgtcacacc acgagagtct 1380 gtaacacccg aagtcggtga ggcaac 1406 <210> 17 <211> 1406 <212> DNA <213> Frateribacillus flavoalbus <400> 17 gtcgtgcggg tgaagccgac caccccttcg ggggcggatg gtggaaccag cggcggacgg 60 gtgagtaaca cgtgggcaac ctgcctgcaa gaccgggata actgcgggaa accggagcta 120 ataccggata agaggctttt ccgcatgggg gagccttaaa aggcggcgga agtcgtcact 180 tgcagatggg cccgcggcgc attagctagt tggtgaggga agggctcacc aaggcgacga 240 tgcgtagccg acctgagagg gtgaccggcc acactgggac tgagacacgg cccagactcc 300 tacgggaggc agcagtaggg aattttcggc aatgggggga accctgaccg agcaacgccg 360 cgtgagcgag gacggccttc gggttgtaaa gctctgtcat gtgggaagaa cggctaggag 420 agggcatgct cttagcgtga cggtaccaca ggaggaagcc ccggctaact acgtgccagc 480 agccgcggta acacgtaggg ggcgagcgtt gtccggaatt attgggcgta aagggcgcgc 540 aggcggtctc ttaagtctga tgtgaaaggc cacggctcaa ccgtgggatg gcattggaaa 600 ctgggggact tgagtgcagg agaggggagc ggaattcccg gtgtagcggt gaaatgcgta 660 gagatcggga ggaacaccag tggcgaaggc ggctctctgg cctgtaactg acgctgaggc 720 gcgaaagcgt ggggagcgaa caggattaga taccctggta gtccacgccg taaacgatga 780 gtgctaggtg ttaggggcgt cacgccctta gtgccgaagt aaacacatta agcactccgc 840 ctggggagta cggccgcaag gctgaaactc aaaggaattg acgggggccc gcacaagcgg 900 tggagcatgt ggtttaattc gaagcaacgc gaagaacctt accagggctt gacatcctct 960 gacccctcta gagatagagg tttcccttcg gggacagaga gacaggtggt gcatggttgt 1020 cgtcagctcg tgtcgtgaga tgttgggtta agtcccgcaa cgagcgcaac ccttgagctt 1080 agttgccagc attgagttgg gcactctaag ctgactgcca gtgacaagct ggaggaaggt 1140 ggggatgacg tcaaatcatc atgcccctta tgtcctgggc gacacacgtg ctacaatggc 1200 tggtacagag ggcagcgaag cggtgacgcg gagccaatcc ctaaaaagcc agtctcagtt 1260 cggatcgcag gctgcaactc gcctgcgtga aggaggaatc gctagtaatc gcggatcagc 1320 atgccgcggt gaatacgttc ccgggccttg tacacaccgc ccgtcacacc acgagagtct 1380 gtaacacccg aagtcggtga ggcaac 1406 <210> 18 <211> 1406 <212> DNA <213> Frateribacillus flavoalbus <400> 18 gtcgtgcggg tgaagccgac caccccttcg ggggcggatg gcggaaccag cggcggacgg 60 gtgagtaaca cgtgggcaac ctgcctgcaa gaccgggata actgcgggaa accggagcta 120 ataccggata agaggctttt ccgcatgggg gagccttaaa aggcggcgga agtcgtcact 180 tgcagatggg cccgcggcgc attagctagt tggtgaggga agggctcacc aaggcgacga 240 tgcgtagccg acctgagagg gtgaccggcc acactgggac tgagacacgg cccagactcc 300 tacgggaggc agcagtaggg aattttcggc aatgggggga accctgaccg agcaacgccg 360 cgtgagcgag gacggccttc gggttgtaaa gctctgtcat gtgggaagaa cggctaggag 420 agggcatgct cttagcgtga cggtaccaca ggaggaagcc ccggctaact acgtgccagc 480 agccgcggta acacgtaggg ggcgagcgtt gtccggaatt attgggcgta aagggcgcgc 540 aggcggtctc ttaagtctga tgtgaaaggc cacggctcaa ccgtgggatg gcattggaaa 600 ctgggggact tgagtgcagg agaggggagc ggaattcccg gtgtagcggt gaaatgcgta 660 gagatcggga ggaacaccag tggcgaaggc ggctctctgg cctgtaactg acgctgaggc 720 gcgaaagcgt ggggagcgaa caggattaga taccctggta gtccacgccg taaacgatga 780 gtgctaggtg ttaggggcgt cacgccctta gtgccgaagt aaacacatta agcactccgc 840 ctggggagta cggccgcaag gctgaaactc aaaggaattg acgggggccc gcacaagcgg 900 tggagcatgt ggtttaattc gaagcaacgc gaagaacctt accagggctt gacatcctct 960 gacccctcta gagatagagg tttcccttcg gggacagaga gacaggtggt gcatggttgt 1020 cgtcagctcg tgtcgtgaga tgttgggtta agtcccgcaa cgagcgcaac ccttgagctt 1080 agttgccagc attgagttgg gcactctaag ctgactgcca gtgacaagct ggaggaaggt 1140 ggggatgacg tcaaatcatc atgcccctta tgtcctgggc gacacacgtg ctacaatggc 1200 tggtacagag ggcagcgaag cggtgacgcg gagccaatcc ctaaaaagcc agtctcagtt 1260 cggatcgcag gctgcaactc gcctgcgtga aggaggaatc gctagtaatc gcggatcagc 1320 atgccgcggt gaatacgttc ccgggccttg tacacaccgc ccgtcacacc acgagagtct 1380 gtaacacccg aagtcggtga ggcaac 1406 <210> 19 <211> 1406 <212> DNA <213> Frateribacillus flavoalbus <400> 19 gtcgtgcggg tgaagccgac caccccttcg ggggcggatg gcggaaccag cggcggacgg 60 gtgagtaaca cgtgggcaac ctgcctgcaa gaccgggata actgcgggaa accggagcta 120 ataccggata agaggctttt ccgcatgggg gagccttaaa aggcggcgga agtcgtcact 180 tgcagatggg cccgcggcgc attagctagt tggtgaggta agggctcacc aaggcgacga 240 tgcgtagccg acctgagagg gtgaccggcc acactgggac tgagacacgg cccagactcc 300 tacgggaggc agcagtaggg aattttcggc aatgggggga accctgaccg agcaacgccg 360 cgtgagcgag gacggccttc gggttgtaaa gctctgtcat gtgggaagaa cggctaggag 420 agggcatgct cttagcgtga cggtaccaca ggaggaagcc ccggctaact acgtgccagc 480 agccgcggta acacgtaggg ggcgagcgtt gtccggaatt attgggcgta aagggcgcgc 540 aggcggtctc ttaagtctga tgtgaaaggc cacggctcaa ccgtgggatg gcattggaaa 600 ctgggggact tgagtgcagg agaggggagc ggaattcccg gtgtagcggt gaaatgcgta 660 gagatcggga ggaacaccag tggcgaaggc ggctctctgg cctgtaactg acgctgaggc 720 gcgaaagcgt ggggagcgaa caggattaga taccctggta gtccacgccg taaacgatga 780 gtgctaggtg ttaggggcgt cacgccctta gtgccgaagt aaacacatta agcactccgc 840 ctggggagta cggccgcaag gctgaaactc aaaggaattg acgggggccc gcacaagcgg 900 tggagcatgt ggtttaattc gaagcaacgc gaagaacctt accagggctt gacatcctct 960 gacccctcta gagatagagg tttcccttcg gggacagaga gacaggtggt gcatggttgt 1020 cgtcagctcg tgtcgtgaga tgttgggtta agtcccgcaa cgagcgcaac ccttgagctt 1080 agttgccagc attgagttgg gcactctaag ctgactgcca gtgacaagct ggaggaaggt 1140 ggggatgacg tcaaatcatc atgcccctta tgtcctgggc gacacacgtg ctacaatggc 1200 tggtacagag ggcagcgaag cggtgacgcg gagccaatcc ctagaaagcc agtctcagtt 1260 cggatcgcag gctgcaactc gcctgcgtga aggaggaatc gctagtaatc gcggatcagc 1320 atgccgcggt gaatacgttc ccgggccttg tacacaccgc ccgtcacacc acgagagtct 1380 gtaacacccg aagtcggtga ggcaac 1406

Claims (11)

The present invention relates to a novel 16S rRNA gene comprising a nucleotide sequence selected from SEQ ID NOs: 1 and 8 to 19, which is a novel type of the genus Frateribacillus (genus Frateribacillus ), has a proteolytic activity and also has the following characteristics 1) to 10) (NITE BP-01964), YM17-2 strain (NITE BP-01964), YM17-2 strain (NITE BP- -01925), Frateribacillus flavoalbu s.
1) Gram stain is negative bacillus, forming spores
2) oxydase test is negative, catalase test is positive and aerobic
3) When D-galactose or sucrose was added to the LB medium (pH 8.0) containing 1% sugar and 2.0% NaCl, no acid was produced, but D-glucose, lactose, D-mannitol , When glycerin or N-acetylglucosamine is added, acid production is performed
4) Intellectual growth temperature is 45 ℃ ~ 55 ℃. No growth is observed at 35 ℃ or above or above 65 ℃
5) The intellectual pH is 7.0 ~ 8.0, and the proliferation is not confirmed when the pH is lower than 5.7 or above 10.0.
6) Proliferation was confirmed even when NaCl was added to the CYC medium to a final concentration of 7% w / v.
7) The main menaquinone is MK7
8) The amino acid composition of the cell wall peptidoglycan includes alanine, glutamic acid, and a small diamino pimelic acid (meso-DAP).
9) GC content of genomic DNA is 51.2% ~ 52.4%
10) The major fungal fatty acid is iso-C16: 0 delete delete delete delete delete delete A method for treating organic wastes using the Frateribacillus flavoalbus according to claim 1. A method for degrading a protein using the Frateribacillus flavoalbus according to claim 1. delete delete

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