KR20160051572A - Novel microorganisms - Google Patents

Novel microorganisms Download PDF

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KR20160051572A
KR20160051572A KR1020150110508A KR20150110508A KR20160051572A KR 20160051572 A KR20160051572 A KR 20160051572A KR 1020150110508 A KR1020150110508 A KR 1020150110508A KR 20150110508 A KR20150110508 A KR 20150110508A KR 20160051572 A KR20160051572 A KR 20160051572A
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frateribacillus
flavoalbus
nacl
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dna
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KR101728605B1 (en
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요시미치 오카무라
마사이치 야마무라
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가부시끼가이샤 산유
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    • BPERFORMING OPERATIONS; TRANSPORTING
    • B09DISPOSAL OF SOLID WASTE; RECLAMATION OF CONTAMINATED SOIL
    • B09BDISPOSAL OF SOLID WASTE NOT OTHERWISE PROVIDED FOR
    • B09B3/00Destroying solid waste or transforming solid waste into something useful or harmless
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    • C02TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02FTREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F1/00Treatment of water, waste water, or sewage
    • CCHEMISTRY; METALLURGY
    • C02TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02FTREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F11/00Treatment of sludge; Devices therefor
    • C02F11/02Biological treatment
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    • C05FORGANIC FERTILISERS NOT COVERED BY SUBCLASSES C05B, C05C, e.g. FERTILISERS FROM WASTE OR REFUSE
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    • C12N1/00Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N1/00Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20Bacteria; Culture media therefor
    • YGENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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    • Y02ATECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A40/00Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
    • Y02A40/10Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
    • Y02A40/20Fertilizers of biological origin, e.g. guano or fertilizers made from animal corpses
    • YGENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02WCLIMATE CHANGE MITIGATION TECHNOLOGIES RELATED TO WASTEWATER TREATMENT OR WASTE MANAGEMENT
    • Y02W10/00Technologies for wastewater treatment
    • Y02W10/20Sludge processing

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Abstract

The present invention provides Frateribacillus flavoalbus, which is a novel Frateribacillus genus microorganism usefully used in treating organic waste such as excrement and sewage sludge. 1) Gram coloring is negative, and spores are formed. 2) An oxidase test is negative, a catalase test is positive, and the microorganism is aerobic. 3) Acid is not generated when D-galactose or sucrose is added as sugar to an LB culture medium (pH 8.0) including 1% of the sugar and 2.0% of NaCl, but acid is generated when D-glucose, lactose, D-mannitol, glycerin, or N-acetylglucosamine is added.

Description

신규 미생물{NOVEL MICROORGANISMS}NOVEL MICROORGANISMS

본 발명은, 신속(新屬) 프라테리바실러스속(Frateribacillus 속)의 신종이 되는 신규 미생물에 관한 것이다. 더욱 자세하게는, 다음 1)∼10)의 특성을 갖는 프라테리바실러스 플라보알버스(Frateribacillus flavoalbus)에 관한 것이다.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) 그램 염색이 음성이고, 포자를 형성함1) Gram stain is negative, forming spores

2) 옥시다아제 테스트는 음성, 카탈라아제 테스트는 양성이고 호기성임2) oxydase test is negative, catalase test is positive and aerobic

3) 1%의 당 및 2.0%의 NaCl을 포함하는 LB 배지(pH 8.0)에, 상기 당으로서 D-갈락토오스 또는 수크로오스를 첨가한 경우에는 산 생성을 하지 않지만, D-글루코오스, 락토오스, D-만니톨, 글리세린 또는 N-아세틸글루코사민을 첨가한 경우에는 산 생성을 함3) 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) 지적 생육 온도는 45℃∼55℃이고, 35℃ 이하 또는 65℃ 이상에서는 증식이 확인되지 않음4) Intellectual growth temperature is 45 ℃ ~ 55 ℃. No growth is observed at 35 ℃ or above or above 65 ℃

5) 지적 pH는 7.0∼8.0이고, pH 5.7 이하 또는 pH 10.0 이상에서는 증식이 확인되지 않음5) 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) CYC 배지에 NaCl을 종농도 7%까지 첨가해도 증식이 확인됨6) Proliferation was confirmed by addition of NaCl to the CYC medium up to a concentration of 7%.

7) 주요한 메나퀴논은 MK7임7) The main menaquinone is MK7

8) 세포벽 펩티도글리칸의 아미노산 조성으로서 알라닌, 글루타민산, 및 메소형 디아미노피멜산(meso-DAP)을 포함함8) The amino acid composition of the cell wall peptidoglycan includes alanine, glutamic acid, and a small diamino pimelic acid (meso-DAP).

9) 게놈 DNA의 GC 함량은 51.2%∼52.4%임9) GC content of genomic DNA is 51.2% ~ 52.4%

10) 가장 주요한 균체 지방산은 iso-C16:0임10) The most important fungal fatty acid is iso-C16: 0

종래의 퇴비화의 과정에서는, 곰팡이 등의 진균, 방선균이라고 불리는 액티노마이세테일즈목(Actinomycetales 목)의 세균, 서모액티노마이세스속(Thermoactinomyces 속) 등 서모액티노마이세타지과(Thermoactinomycetaceae 과)의 세균, 바실러스속(Bacillus 속)이나 게오바실러스속(Geobacillus 속) 등 바실라지과(Bacillaceae 과)의 세균이 유기물을 분해·자화하여, 퇴비화를 담당하고 있다. 이들 미생물은 포자 형성능을 갖고, 각 개체의 생육 온도 이상으로 승온한 환경에서는 포자 상태에서 내열성을 나타내어 안정적으로 퇴비 내에 머물 수 있기 때문에, 생육 적온에 있어서 유기 폐기물을 분해·자화한 후에도, 재활용 퇴비로서 반복적으로 퇴비화에 이용할 수 있다.In the process of the conventional composting, bacterial, thermo-fluid Martino My process in (Thermoactinomyces genus), such as thermo-fluid Martino my three tajigwa (Thermoactinomycetaceae with) the fungi, called Streptomyces liquid Martino my three Tales neck (Actinomycetales neck) of the mold and 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.

퇴비화에서는, 혐기적 발효보다 호기적 발효 쪽이 유기물의 분해가 빠르고, 악취도 저감할 수 있기 때문에 바람직하다. 호기적 발효에 의한 퇴비화의 과정에서는, 통기에 의한 호기적인 발효를 개시하여 10일 이내에 퇴비 내부의 온도가 45℃를 초과하는 고온이 되고, 고온 상태는 퇴비화 종료까지 장기적으로 유지된다. 45℃∼70℃의 퇴비 내의 고온 환경에서는 미크로모노스포라지과(Micromonosporaceae 과), 스트렙토마이세타지과(Streptomycetaceae 과), 서모모노스포라지과(Thermomonosporanceae 과), 스트렙토스포란지아지과(Streptosporangiaceae 과) 등 액티노마이세테일즈목(Actinomycetales 목)의 방선균군, 호열성이고 사상성(絲狀性)의 형태를 갖는 서모액티노마이세타지과(Thermoactinomycetaceae 과)의 세균군이 유기물의 분해를 담당하고 있다(비특허문헌 1). 또한, 게오바실러스속(Geobacillus 속)이 속하는 바실라지과(Bacillaceae 과)의 세균군도 고온 환경하의 퇴비 내에 존재하고 있다(비특허문헌 2, 비특허문헌 3). 고온 상태의 퇴비 내부에서는 이들의 한정된 분류학군 내에 속한 균군이 주로 퇴비화를 담당하고 있는데, 각 과(family) 내의 균종은 성상이 유사한 균으로 구성되어 있기 때문에, 일반적으로 고온으로 된 이후의 퇴비 내부의 균군은 다양성이 낮은 상태이다.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.

특허문헌 1에는 아녹시바실러스(Anoxybacillus)속의 균주를 이용하여 퇴비를 제조하는 방법, 특허문헌 2에는 바실러스(Bacillus)속의 균주를 이용하여 퇴비를 제조하는 방법이 기재되어 있지만, 어느 것이나 바실라지과(Bacillaceae 과)의 세균을 이용하고 있다.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.

한편, 폐기물 처리에서 나오는 유기 폐기물은 유기성 오니, 식품 잔사물, 동물 분뇨 등 다방면에 걸쳐, 폐기물 원료의 종류에 따라 퇴비 내의 pH나 염 농도, 부식산 농도, C/N 비는 크게 다르다. 이 때문에, 다양한 유기 폐기물을 효율적으로 퇴비화 처리하기 위해서는, 기존의 균군과 상이한 균군을 첨가하여 퇴비 중에서 생육시켜, 다양한 균종에 의해 유기물을 분해·자화하는 것이 바람직하다. 또한, 첨가하는 균군은 포자 형성능을 가짐으로써 휴면 상태를 유지할 수 있고, 환경의 변화에 견딜 수 있는 것이 바람직하다. 안정된 포자의 상태는 보관, 운반이 용이하고, 또한, 재활용 퇴비로서 반복적으로 사용할 수 있기 때문에 경제적이다.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.

지금까지 본 발명자들은, 퇴비화에 이용할 수 있는 신규한 세균을 탐색하여, 80℃ 이상의 환경하에서 생육하는 신규한 속에 속하는 초호열균(超好熱菌)을 발견하였다(특허문헌 3).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).

특허문헌 1 : 일본 특허 공개 제2011-24569호 공보Patent Document 1: Japanese Patent Laid-Open Publication No. 2011-24569 특허문헌 2 : 일본 특허 공개 제2008-278890호 공보Patent Document 2: Japanese Patent Application Laid-Open No. 2008-278890 특허문헌 3 : 일본 특허 공개 제2005-13063호 공보Patent Document 3: Japanese Patent Application Laid-Open No. 2005-13063

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

본 발명은, 시뇨, 오니 등의 유기 폐기물의 처리 등에 유용한 신규 미생물의 제공을 과제로 한다.The present invention aims to provide a novel microorganism useful for the treatment of organic wastes such as urine and sludge.

본 발명자들은, 신규 미생물을 발견하고, 이것을 단리, 동정함으로써, 본 발명을 완성하기에 이르렀다.The present inventors have completed the present invention by discovering a novel microorganism, isolating it, and identifying it.

즉, 본 발명은 이하와 같은 것이다.That is, the present invention is as follows.

[1] 프라테리바실러스속(Frateribacillus 속)의 신종이고, 다음 1)∼10)의 특성을 갖는 프라테리바실러스 플라보알버스(Frateribacillus flavoalbus).(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) 그램 염색이 음성인 간균이고, 포자를 형성함1) Gram stain is negative bacillus, forming spores

2) 옥시다아제 테스트는 음성, 카탈라아제 테스트는 양성이고 호기성임2) oxydase test is negative, catalase test is positive and aerobic

3) 1%의 당 및 2%의 NaCl을 포함하는 LB 배지(pH 8.0)에, 상기 당으로서 D-갈락토오스 또는 수크로오스를 첨가한 경우에는 산 생성을 하지 않지만, D-글루코오스, 락토오스, D-만니톨, 글리세린 또는 N-아세틸글루코사민을 첨가한 경우에는 산 생성을 함3) 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) 지적 생육 온도는 45℃∼55℃이고, 35℃ 이하 또는 65℃ 이상에서는 증식이 확인되지 않음4) Intellectual growth temperature is 45 ℃ ~ 55 ℃. No growth is observed at 35 ℃ or above or above 65 ℃

5) 지적 pH는 7.0∼8.0이고, pH 5.7 이하 또는 pH 10.0 이상에서는 증식이 확인되지 않음5) 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) CYC 배지에 NaCl을 종농도 7%까지 첨가해도 증식이 확인됨6) Proliferation was confirmed by addition of NaCl to the CYC medium up to a concentration of 7%.

7) 주요한 메나퀴논은 MK7임7) The main menaquinone is MK7

8) 세포벽 펩티도글리칸의 아미노산 조성으로서 알라닌, 글루타민산, 및 메소형 디아미노피멜산(meso-DAP)을 포함함8) The amino acid composition of the cell wall peptidoglycan includes alanine, glutamic acid, and a small diamino pimelic acid (meso-DAP).

9) 게놈 DNA의 GC 함량은 51.2%∼52.4%임9) GC content of genomic DNA is 51.2% ~ 52.4%

10) 가장 주요한 균체 지방산은 iso-C16:0임10) The most important fungal fatty acid is iso-C16: 0

[2] 서열번호 1, 또는 서열번호 8∼서열번호 19 중 어느 것에 나타낸 염기 서열에 대하여 98.7% 이상의 상동성을 갖는 염기 서열로 이루어지는 16S rRNA 유전자를 갖는, 상기 [1]에 기재된 프라테리바실러스 플라보알버스(Frateribacillus flavoalbus).[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] YM17주(NITE BP-01764), YM17-2주(NITE BP-01948), YM17-3주(NITE BP-01949), YM17-4주(NITE BP-01950) 또는 YM17-5주(NITE BP-01925)와의 DNA-DNA 하이브리다이제이션 상동치가 70% 이상인, 상기 [1] 또는 [2]에 기재된 프라테리바실러스 플라보알버스(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] 또한, 균체 지방산으로서, anteiso-C15:0, C16:0, anteiso-C17:0, iso-C17:0 중 어느 1종 이상을 포함하는, 상기 [1]∼[3] 중 어느 하나에 기재된 프라테리바실러스 플라보알버스(Frateribacillus flavoalbus).[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] 균체 지방산으로서 iso-C16:0에 더하여, anteiso-C15:0, anteiso-C17:0, iso-C17:0을 포함하는 프라테리바실러스 플라보알버스 아종 플라보알버스(Frateribacillus flavoalbus subsp. flavoalbus)인, 상기 [1]∼[4] 중 어느 하나에 기재된 프라테리바실러스 플라보알버스(Frateribacillus flavoalbus).[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] 균체 지방산으로서 iso-C16:0에 더하여, anteiso-C15:0, C16:0, anteiso-C17:0을 포함하는 프라테리바실러스 플라보알버스 아종 피메타리움(Frateribacillus flavoalbus subsp. fimetarium)인, 상기 [1]∼[4] 중 어느 하나에 기재된 프라테리바실러스 플라보알버스(Frateribacillus flavoalbus).[6] Frateribacillus flavoalbus subsp. Fimetarium containing 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] 유기 폐기물의 처리에 사용하는, 상기 [1]∼[6] 중 어느 하나에 기재된 프라테리바실러스 플라보알버스(Frateribacillus flavoalbus).[7] Frateribacillus flavoalbus according to any one of [1] to [6], which is used for treating organic wastes.

[8] 유기 폐기물이 시뇨, 유기성 오니, 식품 잔사물, 또는 동물 분뇨인, 상기 [7]에 기재된 프라테리바실러스 플라보알버스(Frateribacillus flavoalbus).[8] The Frateribacillus flavoalbus according to [7], wherein the organic waste is urine, organic sludge, food remnants , or animal manure.

[9] YM17주(NITE BP-01764), NaCl_20131016_04주, YM17-2주(NITE BP-01948), YM17-3주(NITE BP-01949), YM17-4주(NITE BP-01950), NaCl_20131016_19주, YM17-5주(NITE BP-01925), NaCl_20131016_24주, NaCl_20131016_28주, NaCl_20131016_30주, NaCl_20131016_34주, NaCl_20131016_37주 또는 NaCl_20131016_38주 중 어느 것인, 상기 [1]∼[8] 중 어느 하나에 기재된 프라테리바실러스 플라보알버스(Frateribacillus flavoalbus).[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] 상기 [1]∼[9] 중 어느 하나에 기재된 프라테리바실러스 플라보알버스(Frateribacillus flavoalbus)를 사용하는, 유기 폐기물의 처리 방법.[10] A method for treating organic wastes using the Frateribacillus flavoalbus according to any one of [1] to [9] above.

[11] 상기 [1]∼[9] 중 어느 하나에 기재된 프라테리바실러스 플라보알버스(Frateribacillus flavoalbus)를 사용하는, 단백질의 분해 방법.[11] A method for degrading a protein using the Frateribacillus flavoalbus according to any one of [1] to [9] above.

본 발명에 의해 얻어진 신규 미생물은, 신속 프라테리바실러스속(Frateribacillus 속)의 신종이다. 이 신규 미생물은 시뇨, 오니 등의 유기 폐기물의 처리, 단백질의 분해 등에 유용하고, 또한, 여러가지 용도에 사용할 수 있다.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.

도 1은, YM17주의 세포 사진을 나타낸 도면이다(실시예 1).
도 2는, 근린 결합법에 의해 작성된 계통수를 나타낸 도면이다(실시예 1).
도 3은, YM17주와 분리한 12주의 16S rRNA 유전자에 의한 계통수를 나타낸 도면이다(실시예 2).
도 4는, 퇴비 배양 중의 균의 상태를 나타낸 도면이다(실시예 2).
도 5는, 젤라틴 자이모그래피의 결과를 나타낸 도면이다(실시예 2).
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).

본 발명의 프라테리바실러스 플라보알버스(Frateribacillus flavoalbus)란, 다음 1)∼10)의 특성을 갖는 신속 프라테리바실러스속(Frateribacillus 속)의 신종을 말한다.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) 그램 염색이 음성이고, 포자를 형성함1) Gram stain is negative, forming spores

2) 옥시다아제 테스트는 음성, 카탈라아제 테스트는 양성이고 호기성임2) oxydase test is negative, catalase test is positive and aerobic

3) 1%의 당 및 2.0%의 NaCl을 포함하는 LB 배지(pH 8.0)에, 상기 당으로서 D-갈락토오스 또는 수크로오스를 첨가한 경우에는 산 생성을 하지 않지만, D-글루코오스, 락토오스, D-만니톨, 글리세린 또는 N-아세틸글루코사민을 첨가한 경우에는 산 생성을 함3) 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) 지적 생육 온도는 45℃∼55℃이고, 35℃ 이하 또는 65℃ 이상에서는 증식이 확인되지 않음4) Intellectual growth temperature is 45 ℃ ~ 55 ℃. No growth is observed at 35 ℃ or above or above 65 ℃

5) 지적 pH는 7.0∼8.0이고, pH 5.7 이하 또는 pH 10.0 이상에서는 증식이 확인되지 않음5) 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) CYC 배지에 NaCl을 종농도 7%까지 첨가해도 증식이 확인됨6) Proliferation was confirmed by addition of NaCl to the CYC medium up to a concentration of 7%.

7) 주요한 메나퀴논은 MK7임7) The main menaquinone is MK7

8) 세포벽 펩티도글리칸의 아미노산 조성으로서 알라닌, 글루타민산, 및 메소형 디아미노피멜산(meso-DAP)을 포함함8) The amino acid composition of the cell wall peptidoglycan includes alanine, glutamic acid, and a small diamino pimelic acid (meso-DAP).

9) 게놈 DNA의 GC 함량은 51.2%∼52.4%임9) GC content of genomic DNA is 51.2% ~ 52.4%

10) 가장 주요한 균체 지방산은 iso-C16:0임10) The most important fungal fatty acid is iso-C16: 0

이 「주요한 메나퀴논」으로서 MK7을 81.8% 이상 포함하는 것을 들 수 있다.As the " main menaquinone ", there can be mentioned those containing 81.8% or more of MK7.

또한, 「주요한 균체 지방산」으로서, iso-C16:0을 34.1% 이상 42.6% 이하 포함하는 것을 들 수 있다.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%.

또한 「균체 지방산」으로서, iso-C16:0에 더하여, anteiso-C15:0, C16:0, anteiso-C17:0, iso-C17:0 중 어느 1종 이상을 포함하는 것이어도 좋다. 본 발명의 프라테리바실러스 플라보알버스(Frateribacillus flavoalbus)는 이들 지방산을 합계하여 86.1% 이상 포함하는 것이 바람직하다.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.

또한, 본 발명의 프라테리바실러스 플라보알버스(Frateribacillus flavoalbus)는, 서열번호 1, 또는 서열번호 8∼19 중 어느 것에 나타낸 염기 서열에 대하여 98.7% 이상의 상동성을 갖는 염기 서열로 이루어지는 16S rRNA 유전자를 갖는 것이 바람직하다.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, .

또한, 본 발명의 프라테리바실러스 플라보알버스(Frateribacillus flavoalbus)는 YM17주, YM17-2주, YM17-3주, YM17-4주 또는 YM17-5주와 DNA-DNA 하이브리다이제이션의 상동치가 70%인 것이 바람직하다.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, .

이러한 본 발명의 프라테리바실러스 플라보알버스(Frateribacillus flavoalbus)로서, 상기 1)∼10)의 성질을 갖고, 또한, 균체 지방산으로서 iso-C16:0에 더하여, anteiso-C15:0, anteiso-C17:0, iso-C17:0을 포함하는 프라테리바실러스 플라보알버스 아종 플라보알버스(Frateribacillus flavoalbus subsp. flavoalbus)를 들 수 있다.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.

이러한 프라테리바실러스 플라보알버스 아종 플라보알버스(Frateribacillus flavoalbus subsp. flavoalbus)로서, YM17주, YM17-5주 등을 들 수 있다.Such a Frateribacillus flavoalbus subsp. Flavoalbus is exemplified by YM17 strain and YM17-5 strain.

또한, 본 발명의 프라테리바실러스 플라보알버스(Frateribacillus flavoalbus)로서, 상기 1)∼10)의 성질을 갖고, 또한, 균체 지방산으로서 iso-C16:0에 더하여, anteiso-C15:0, C16:0, anteiso-C17:0을 포함하는, 프라테리바실러스 플라보알버스 아종 피메타리움(Frateribacillus flavoalbus subsp. fimetarium)도 들 수 있다.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.

이러한 프라테리바실러스 플라보알버스 아종 피메타리움(Frateribacillus flavoalbus subsp. fimetarium)으로서, NaCl_20131016_04주, YM17-2주, YM17-3주, YM17-4주, NaCl_20131016_19주, NaCl_20131016_24주, NaCl_20131016_28주, NaCl_20131016_30주, NaCl_20131016_34주, NaCl_20131016_37주 및 NaCl_20131016_38주 등을 들 수 있다.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.

본 발명의 프라테리바실러스 플라보알버스(Frateribacillus flavoalbus)로서 들 수 있는 YM17주는, 상기한 특성을 가짐과 동시에, 16S rRNA의 염기 서열이 서열표 서열번호 1에 기재된 염기 서열과 100% 동일하다.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.

본 균주는 본 출원인의 신청에 의해, 독립행정법인제품평가기술기반기구(NITE) 특허 미생물 기탁 센터에 부다페스트 조약에 기초하여 국제 기탁되어 있다. 이하에, 기탁을 특정하는 내용을 기재한다.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) 기탁기관명 : 독립행정법인제품평가기술기반기구 특허 미생물 기탁 센터(1) Depositary Institution: Independent administrative corporation Product evaluation Technology-based organization Patented microorganism deposit center

(2) 연락처 : 우편 292-0818 치바현 키사라츠시 카즈사 카마타리 2-5-8(2) Contact: Postal 292-0818 2-5-8 Matsuri, Kazusaka, Kisaratsu, Chiba Prefecture

전화 번호 0438-20-5580Phone number 0438-20-5580

(3) 수탁 번호 : NITE BP-01764(3) Accession number: NITE BP-01764

(4) 식별을 위한 표시 : YM17(4) Indication for identification: YM17

(5) 원기탁일 : 2013년 11월 29일 (5) Date of original deposit: November 29, 2013

또한, 마찬가지로 본 발명의 프라테리바실러스 플라보알버스(Frateribacillus flavoalbus)로서 들 수 있는 YM17-2주, YM17-3주, YM17-4주 및 YM17-5주에 관해서도, 본 출원인의 신청에 의해, 독립행정법인제품평가기술기반기구(NITE) 특허 미생물 기탁 센터에 부다페스트 조약에 기초하여 국제 기탁되어 있다. 이들 기탁을 특정하는 내용은 실시예 등에 개시했다.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.

본 발명의 프라테리바실러스 플라보알버스(Frateribacillus flavoalbus)에는, 이 프라테리바실러스 플라보알버스(Frateribacillus flavoalbus)에 해당하는 균주의 변이주도 포함된다. 또한, 프라테리바실러스 플라보알버스(Frateribacillus flavoalbus) 중, 프라테리바실러스 플라보알버스 아종 플라보알버스(Frateribacillus flavoalbus subsp. flavoalbus)나 프라테리바실러스 플라보알버스 아종 피메타리움(Frateribacillus flavoalbus subsp. fimetarium)의 변이주도 포함된다.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.

본 발명에서의 이 변이주란 화학적 변이 도입, 자연 돌연 변이, 형태 변이, 트랜스펙션 등의 유전자 공학적 수법에 의해, 그 물리적 특성이나 화학적 특성이 개변된 것으로, 상기 1)∼10)의 모든 특성을 갖는 것을 말한다.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. .

본 발명의 프라테리바실러스 플라보알버스(Frateribacillus flavoalbus)는, 유기성 오니, 식품 잔사물, 또는 동물 분뇨 등의 유기 폐기물의 처리에 사용할 수 있다. 이들 유기 폐기물에, 프라테리바실러스 플라보알버스(Frateribacillus flavoalbus)를 첨가함으로써, 퇴비 등을 제조하는 것도 가능하다. 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.

본 발명의 프라테리바실러스 플라보알버스(Frateribacillus flavoalbus)를 사용하는 유기 폐기물의 처리 방법에서는, 유기 폐기물에 프라테리바실러스 플라보알버스(Frateribacillus flavoalbus)를 첨가할 뿐만 아니라, 유기 폐기물의 처리에 있어서 유용한 그 밖의 공정을 포함하고 있어도 좋다.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.

또한, 본 발명의 프라테리바실러스 플라보알버스(Frateribacillus flavoalbus)는, 단백질의 분해에 사용할 수 있다.In addition, the Frateribacillus flavoalbus of the present invention can be used for protein degradation.

본 발명의 프라테리바실러스 플라보알버스(Frateribacillus flavoalbus)를 사용하는 단백질의 분해 방법에서는, 단백질이나 단백질을 포함하는 분해 대상에 프라테리바실러스 플라보알버스(Frateribacillus flavoalbus)를 첨가할 뿐만 아니라, 단백질의 분해에 있어서 유용한 그 밖의 공정을 포함하고 있어도 좋다.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.

이하에 실시예를 들어 본 발명을 보다 구체적으로 설명하지만, 본 발명이 이들에 한정되지 않는 것은 물론이다.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.

실시예Example

[실시예 1][Example 1]

프라테리바실러스 플라보알버스(Frateribacillus flavoalbus)의 단리·동정Identification and identification of Frateribacillus flavoalbus ( Frateribacillus flavoalbus )

1. 균주의 단리1. Isolation of strain

가고시마현 소오시의 퇴비화장의 퇴비로부터 YM17주를 단리했다.We isolated YM17 week from compost of composting ground of Sojo, Kagoshima.

즉, 50 ㎍/mL 노보바이오신(Novobiocin)을 포함하는 4 mL의 1/2 영양 배지(펩톤 : 0.25%, 염화나트륨 : 0.25%, 효모 엑기스 : 0.1%, 미트 엑기스 : 0.05%, pH 7.4)에 상기 퇴비 400 mg을 첨가하고, 55℃에서 2일간 배양한 후, 이 액체 배양액을 50 ㎍/mL 노보바이오신을 포함하는 1/2 영양(Nutrient) 한천 배지에 도포하고 4일간 배양하여 YM17주의 콜로니를 단리했다.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. 분류학적 성질 2. Taxonomic properties

A. 시험 방법A. Test Method

균주의 분류학적 성질은 「신생화학 실험 강좌 제17권 미생물 실험법(동경 화학 동인 발행)」, 「개정판 미생물의 분류와 동정 <하>(학회 출판 센터 발행)」, 「미생물의 분류·동정 실험법(슈프링가·재팬 발행)」, BERGEY'S MANUAL OF Systematic Bacteriology에 기재된 방법에 따라 분석했다. 단리 균주의 분류와 동정은 International Journal of Systematic and Evolutionary Microbiology 지에 기재되어 있는 종과의 비교에 의해 행했다.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 &quot;), 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.

이 해석에 의해 얻어진 형태적 성질, 배양적 성질, 생리학적 성질, 생육 범위, 및 화학 분류학적 성질을 표 2-1, 표 2-5에 나타냈다. 또한, YM17주를 55℃에서 배양한 세포 사진을 도 1에 나타냈다. 도 1에 있어서, YM17주는 간균이고, 세포의 말단에 포자를 형성하는 것을 확인할 수 있었다. 도 1의 바는 10 ㎛를 나타낸다. 또, 표 2(표 2-1∼표 2-5)에는, 실시예 2에 있어서 분리한 12 균주의 분류학적 성질도 아울러 나타냈다.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. 시험 배지B. Test media

각 배지의 pH는 표 2-1 중에 기재한 바와 같다. 리트머스 밀크를 제외한 각 배지는 121℃에서 15분간 오토클레이브 멸균을 행했다. 리트머스 밀크는 110℃에서 10분간 오토클레이브 멸균을 행했다. 크리스텐센 요소 배지의 요소, OF 테스트 배지의 당, 당으로부터의 산 생성을 조사한 당류는 필터 멸균을 행하고, 기재한 종농도가 되도록 각 배지에 첨가했다. 또, 당으로부터의 산 생성은 1%의 당과 2%의 NaCl을 포함하는 LB 배지(pH 8.0)를 이용하여 조사했다.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 한천 배지(수크로오스 : 3%, 카사미노산 : 0.6%, 질산나트륨 : 0.3%, 효모 엑기스 : 0.2%, 인산수소이칼륨 : 0.1%, 황산마그네슘 7수화물 : 0.05%, 염화칼륨 : 0.05%, 황산철 7수화물 : 0.001%, 한천 : 1.5%)(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 액체 배지(수크로오스 : 3%, 카사미노산 : 0.6%, 질산나트륨 : 0.3%, 효모 엑기스 : 0.2%, 인산수소이칼륨 : 0.1%, 황산마그네슘 7수화물 : 0.05%, 염화칼륨 : 0.05%, 황산철 7수화물 : 0.001%)(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) 육즙 한천 배지(미트 엑기스 : 1%, 펩톤 : 1%, 염화나트륨 : 0.5%, 한천 : 1.5%)(3) Juicy agar medium (Meat extract: 1%, peptone: 1%, sodium chloride: 0.5%, agar: 1.5%)

(4) 육즙 액체 배지(미트 엑기스 : 1%, 펩톤 : 1%, 염화나트륨 : 0.5%)(4) Juicy liquid medium (Meat extract: 1%, peptone: 1%, sodium chloride: 0.5%)

(5) 리트머스 밀크(스킴 밀크 : 10%, 1% 리트머스액 : 여러 방울)(5) Litmus milk (skim milk: 10%, 1% litmus liquid: several drops)

(6) VP 배지(효모 엑기스 : 0.1%, 트립톤 : 0.7%, 소이톤 : 0.5%, 글루코오스 : 1%, 염화나트륨 : 0.5%, 한천 : 0.3%)(6) VP medium (yeast extract: 0.1%, tryptone: 0.7%, soytone: 0.5%, glucose: 1%, sodium chloride: 0.5%, agar: 0.3%

(7) SIM 배지(미트 엑기스 : 0.3%, 펩톤 : 3%, 티오황산나트륨 : 0.005%, 염산시스테인 : 0.02%, 시트르산철암모늄 : 0.05 g, 한천 : 0.5%)(7) SIM medium (meat extract: 0.3%, peptone: 3%, sodium thiosulfate: 0.005%, cysteine hydrochloride: 0.02%, iron citrate: 0.05 g,

(8) LB 한천 배지(트립톤 : 1%, 염화나트륨 : 0.5%, 효모 엑기스 : 0.5%, 한천 : 1.5%)(8) LB agar medium (tryptone: 1%, sodium chloride: 0.5%, yeast extract: 0.5%, agar: 1.5%)

(9) SY 한천 배지(황산암모늄 : 0.2%, 인산수소이나트륨 : 0.14%, 인산이수소칼륨 : 0.07%, 황산마그네슘 7수화물 : 0.02%, 황산철 7수화물 : 0.0005%, 황산망간 5수화물 : 0.0005%, 글루코오스 : 1%)(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) 영양 한천 배지(미트 엑기스 : 0.3%, 펩톤 : 0.5%, 한천 : 1.5%)(10) Nut agar medium (meat extract: 0.3%, peptone: 0.5%, agar: 1.5%)

(11) 시몬스(Simmons) 배지(인산이수소암모늄 : 0.1%, 인산수소이칼륨 : 0.1%, 염화나트륨 : 0.5%, 시트르산나트륨 : 0.2%, 황산마그네슘 7수화물 : 0.02%, 한천 : 1.5%, BTB : 0.008%)(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) 크리스텐센(Christensen) 배지(효모 엑기스 : 0.05%, 시스테인염산염 : 0.01%, 염화나트륨 : 0.5%, 시트르산나트륨 : 0.3%, 글루코오스 : 0.02%, 인산이수소칼륨 : 0.1%, 한천 : 1.5%, 페놀 레드 : 0.0012%)(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) 질산염 배지(글루코오스 : 1.0%, 인산이수소칼륨 : 0.1%, 황산마그네슘 7수화물 : 0.05%, 염화칼륨 : 0.02%, 질산나트륨 : 0.1%)(13) Nitrate medium (glucose: 1.0%, potassium dihydrogenphosphate: 0.1%, magnesium sulfate heptahydrate: 0.05%, potassium chloride: 0.02%, sodium nitrate: 0.1%

(14) 암모늄염 배지(글루코오스 : 1.0%, 인산이수소칼륨 : 0.1%, 황산마그네슘 7수화물 : 0.05%, 염화칼륨 : 0.02%, 황산암모늄 : 0.078%)(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) 크리스텐센 요소 배지(펩톤 : 0.1%, 염화나트륨 : 0.5%, 글루코오스 : 0.1 g, KH2PO4 : 0.2%, 페놀 레드 : 0.0008%, 한천 : 1.5%, 요소 : 2%)(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 테스트 배지(펩톤 : 0.2%, 염화나트륨 : 0.5%, 인산수소이칼륨 : 0.03%, 한천 : 0.2%, BTB : 0.008%, 당 : 1%)(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. 지방산의 분석C. Analysis of fatty acids

1) 시료 조정과 가스 크로마토그래피에 의한 분석1) Sample preparation and analysis by gas chromatography

5 L의 삼각 플라스크에 3 L의 CYC 액체 배지를 넣은 본배양 배지에, 종배양한 균을 식균하고, 52℃에서 대수 증식기 후기부터 정상기 초기까지 배양하고, 원심에 의해 균체를 회수했다. 회수한 균체는 생리 식염수로 2회 세정했다. 약 500 mg∼700 mg의 습균체를 스크루 캡이 부착된 원심관에 넣었다. 1 mL의 알칼리 비누화액을 첨가하고, 밀전하여 100℃에서 5분간 유지했다. 5분 후, 10초간 잘 흔들고, 다시 100℃에서 30분간 유지했다. 원심관을 냉각한 후, 2 mL의 염산메탄올 시약을 첨가하고, 밀전하여, 80℃에서 10분간 유지했다. 원심관을 냉각한 후, 1.25 mL의 추출용 용매를 첨가하고, 10분간 천천히 교반했다. 원심관을 1000 rpm으로 5분간 원심한 후, 상층을 별도의 스크루 캡이 부착된 원심관에 옮기고, 3 mL의 세정액을 첨가했다. 다시 밀전하여 5분간 교반했다. 이 원심관을 1000 rpm으로 5분간 원심한 후, 용매층을 유리 앰플에 취하여, 질소 가스로 건조시켰다. 각 시약의 조성은 하기에 나타냈다. 얻어진 건조 시료를 지방산 분석 시료로 하여, 주식회사 테크노스루가·라보에서 가스 크로마토그래피에 의해 분석했다. 얻어진 결과를 표 1에 나타냈다.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) 시약2) Reagent

(1) 알칼리 비누화액(수산화나트륨 : 7.5 g, 메탄올 : 25 mL, 초순수 25 mL)(1) Alkali saponification solution (sodium hydroxide: 7.5 g, methanol: 25 mL, ultrapure water: 25 mL)

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

(3) 추출용 용매(헥산 : 10 mL, t-부틸메틸에테르 : 10 mL)(3) Extraction solvent (hexane: 10 mL, t-butyl methyl ether: 10 mL)

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

Figure pat00001
Figure pat00001

D. 세포벽 펩티도글리칸 D. Cell wall peptidoglycan

1) 시료 조정1) Sample adjustment

약 2 g의 습균체를 6 mL의 50 mM 인산 완충액(pH 7.2)에 현탁하고, 초음파 파쇄기(TAITEC사 제조의 VP-30S)에 의해 30분간 처리하여 세포를 파쇄했다. 파쇄액을 온도 4℃에서 3000 rpm으로 10분간 원심을 하여, 상청을 회수했다. 상청을 온도 4℃에서 9000 rpm으로 60분간 원심을 하여, 상청을 제거하고, 침전물을 새로운 원심관에 옮겼다. 침전물에 6 mL의 50 mM 인산 완충액(pH 7.2)을 첨가하여 세정한 후, 온도 4℃에서 9000 rpm으로 60분간 원심을 하여, 상청을 제거했다. 침전물에 2 mL의 50 mM 인산 완충액(pH 7.2)과 400 μL의 25% SDS를 첨가하고, 100℃에서 40분간 유지했다. 65℃로 데워 놓은 10 mL의 50 mM 인산 완충액(pH 7.2)을 더욱 첨가하고, 교반한 후, 온도 30℃에서 9000 rpm으로 60분간 원심했다.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.

상청을 제거하고, 침전물에 65℃로 데워 놓은 5 mL의 50 mM 인산 완충액(pH 7.2)을 첨가하여 세정한 후, 온도 30℃에서 9000 rpm으로 30분간 원심을 하여 상청을 제거했다. 이 세정 작업을 2회 행했다. 세정 후, 침전물에 2 mL의 50 mM 인산 완충액(pH 7.6)과 100 μL의 1 mg/mL 프로나제(Pronase)액을 첨가하고, 37℃에서 2시간 유지했다. 이 프로나제 반응 후의 액에 5 mL의 50 mM 인산 완충액(pH 7.6)을 첨가하고, 세포벽 성분을 세정한 후, 온도 4℃에서 9000 rpm으로 30분간 원심을 하여 상청을 제거했다. 이 세정 작업을 2회 행했다.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.

세정 후의 침전물에 2 mL의 5% 트리클로로아세트산 수용액을 첨가하고, 100℃에서 20분간 유지했다. 이 현탁액을 유리제의 스크루 캡이 부착된 원심관에 옮기고, 5 mL의 50 mM 인산 완충액(pH 7.2)을 첨가하여 세정한 후, 실온에서 1000 rpm으로 1분간 원심을 하여, 상청을 제거했다. 이 세정 작업을 2회 행했다. 세정 후의 침전물에 3 mL의 에탄올을 첨가하고, 실온에서 1000 rpm으로 1분간 원심을 하여, 상청을 제거했다. 침전물에 3 mL의 디에틸에테르를 첨가하고, 실온에서 1000 rpm으로 1분간 원심을 하여, 상청을 제거하고, 침전물을 건조시켜 세포벽 시료로 했다.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) TLC에 의한 분석2) Analysis by TLC

5 mg의 세포벽 시료에 250 μL의 4 N 염산을 첨가하고, 100℃에서 16시간 가수 분해 반응을 행했다. 가수 분해 반응 후, 수산화칼륨을 놓은 데시케이터 내에서 염산을 제거하고, 얻어진 처리액을 TLC에 제공했다.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.

TLC 플레이트는 Merck사 제조의 TLC 셀룰로오스 플레이트 No.1.05716.0001을 이용했다. 구성 아미노산의 검출에서는 1차원째를 이소프로판올 : 아세트산 : 초순수 = 75 : 10 : 15의 전개 용매로 전개하고, 2차원째를 메탄올 : 피리딘 : 10 N 염산 : 초순수 = 64 : 8 : 2 : 14로 전개했다. 전개 후 TLC 플레이트에 닌히드린 시약을 스프레이하고, 세포벽 펩티도글리칸에 포함되는 아미노산을 검출했다. 또한, 디아미노피멜산의 이성형(異性型)의 검출은 동일한 TLC 플레이트를 이용하여, 메탄올 : 피리딘 : 10 N 염산 : 초순수 = 64 : 8 : 2 : 14로 전개하고, 표준품과의 이동도의 비교에 의해 특정했다. 그 결과, 세포벽 펩티도글리칸의 아미노산 조성으로서 알라닌, 글루타민산 및 메소형 디아미노피멜산(meso-DAP)을 포함하는 것이 확인되었다.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. 메나퀴논E. menaquinone

1) 시료 조정1) Sample adjustment

약 500 mg의 건조 균체에 20 mL의 클로로포름 : 메탄올 = 2 : 1 액을 첨가하고, 냉암소에서 하룻밤, 서서히 교반했다. 이 현탁액을 여과하고, 여과액을 탕욕 온도 30℃의 로터리 이배퍼레이터로 농축 건고했다. 건고물을 3 mL의 아세톤에 용해시키고, 질소 가스로 약 200 μL로 농축했다. Merck사 제조의 HPTLC 실리카겔 60 F254 플레이트 No.1.05628.0001에 이 농축액을 제공하고, 톨루엔을 전개 용매로 하여 전개했다. 전개 후에 TLC 플레이트를 건조하고, 메나퀴논의 밴드를 UV 램프(254 nm)하에서 확인하고, 그 부분의 실리카겔을 스패출러로 긁어내어, 시험관에 넣었다. 긁어낸 실리카겔에 4 mL의 아세톤을 첨가하여 서서히 교반하고, 필터 여과하여 실리카겔 입자를 제거했다. 얻어진 상청을 질소 가스에 의해 약 200 μL까지 농축하여, HPLC의 시료로 했다.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) HPLC에 의한 분석2) Analysis by HPLC

시료 25 μL를 HPLC에 주입했다. 컬럼은 ODS 컬럼(SHISEIDO사 제조 CAPCELL PAK C18 UG120 S-5)을 이용하고, 전개 용매에는 메탄올 : 이소프로판올 = 2 : 1 액을 이용했다. 이동 속도 1.5 mL/분, 컬럼 온도 40℃에서 전개하고, UV(270 nm)로 검출했다. HPLC의 기계는 HEWLETT PACKART사 제조의 SERIES 1100을 이용했다. 이 분석을 복수회 행한 바, MK7의 함유량이 1회째에는 83.9%이고, 2회째에는 85.2%였기 때문에, 주요한 메나퀴논이 MK7인 것이 확인되었다.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 함량F. GC content

1) DNA 조정 1) DNA regulation

게놈 DNA의 추출은 참고 문헌 1의 방법에 따라 행했다.Genomic DNA was extracted according to the method of Reference 1.

즉, 0.9 g∼1.6 g의 습균체를 Saline-0.1 M EDTA(pH 8.0)로 세정 후, 5 mL의 동일 용액에 현탁했다. 난백 리소자임을 종농도 1 mg/mL로 첨가하여 35℃에서 용균시킨 후, 용균액을 동결시켰다. 동결품에 등량의 Tris-SDS(pH 9.0)를 첨가하고, 60℃의 탕욕에서 융해하고, 10분간 유지했다. 그 후, 빙랭하고, 등량의 물 포화 페놀을 첨가하여 10분간 진탕했다. 원심하여 상청을 회수하고, 2배량의 에탄올을 첨가하고 섬유형의 DNA를 유리 막대로 권취했다. 권취한 DNA를 70%, 90%, 99.5%의 에탄올로 단계적으로 세정하고, 페놀을 제거했다. DNA를 건조 후, 적량의 0.1XSSC 용액으로 용해하고, RNase를 첨가하여 35℃에서 30분 이상 반응시키고, RNA를 제거했다. 빙랭 후, 1/5량의 물 포화 페놀을 첨가하고, 동일하게 진탕, 원심, 에탄올 침전, 페놀 제거, 0.1XSSC 용액으로 용해, RNase 처리를 행했다.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.

RNase 처리 후의 DNA를 페놀 처리하고, 원심하여 DNA 용해 획분을 분리 채취하고, DNA 용해 획분에 1/10량의 아세테이트-EDTA와 0.6배량의 이소프로판올을 첨가하고 DNA를 재차 권취했다. 권취한 DNA를 70%, 90%, 99.5%의 에탄올로 세정하고, 건조 후, DNA 농도가 500 ㎍/mL 이상이 되도록 0.1XSSC 용액으로 용해했다. DNA의 순도가 낮은 경우에는, 페놀에 의한 단백질의 제거와 RNase 처리를 반복했다.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.

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

2) 시약2) Reagent

(1) Saline-0.1 M EDTA(NaCl : 8.77 g, EDTA·2Na·2H2O : 37.22 g, pH : 8.0, 초순수 : 총액량이 1000 mL가 되도록 첨가했다)(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(Tris : 12.1 g, SDS : 20.0 g, pH 9.0, 초순수 : 총액량이 1000 mL가 되도록 첨가했다)(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) 20XSSC(NaCl : 175 g, 시트르산삼나트륨 : 77.4 g, 초순수 : 총액량이 1000 mL가 되도록 첨가했다. 0.1XSSC와 1XSSC는 20XSSC를 초순수로 희석하여 작성했다)(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) 아세테이트-EDTA(A액 : CH3COONa·3H2O : 81.65 g/초순수 200 mL, B액 : CH3COOH : 36.03 g/초순수 200 mL. A액과 B액을 혼합하여 pH 7.0으로 조정했다. EDTA·2Na·2H2O : 74.45 mg/조정액 200 mL로서 아세테이트-EDTA로 했다)(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) GC 함량의 측정 3) Measurement of GC content

GC 함량의 측정은 DNA GC kit(야마사 간장 주식회사 제조)를 이용했다. 7 ㎍ 상당량의 DNA를 20 μL의 0.1XSSC에 용해하고, 100℃에서 10분간 열변성시키고, 즉시 빙랭했다. 빙랭 후, 20 μL의 4 U/mL 뉴클레아제 P1 용액을 첨가하고, 50℃에서 1시간 반응시켰다. 뉴클레아제 P1 처리 후, 20 μL의 2.4 U/mL 알칼리포스파타아제 용액을 첨가하고, 37℃에서 1시간 반응시킨 것을 HPLC에 제공했다.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.

HPLC의 컬럼은 ODS 컬럼(SHISEIDO사 제조 CAPCELL PAK C18 UG120 S-5)을 이용하고, 전개 용매에는 0.02 M NH4H2PO4 : 아세토니트릴 = 20 : 1 액을 이용했다. 이동 속도 1.0 mL/분, 컬럼 온도 40℃에서 전개하고, UV(270 nm)로 검출했다. DNA GC kit에 부속의 스탠다드를 기준으로 하여 YM17주의 GC 함량을 계산한 바, GC 함량은 51.4%였다.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%.

[표 2-1][Table 2-1]

Figure pat00002
Figure pat00002

[표 2-2][Table 2-2]

Figure pat00003
Figure pat00003

[표 2-3][Table 2-3]

Figure pat00004
Figure pat00004

[표 2-4][Table 2-4]

Figure pat00005
Figure pat00005

[표 2-5][Table 2-5]

Figure pat00006
Figure pat00006

G. 16S rRNA 유전자의 염기 서열의 해석G. Analysis of base sequence of 16S rRNA gene

1) PCR 반응 1) PCR reaction

YM17주의 16S rRNA 유전자를 콜로니 다이렉트 PCR 법에 의해 증폭했다. PCR 반응에는 Tks Gflex(등록 상표) DNA Polymerase(다카라바이오사)와 EU10F 프라이머(서열표 서열번호 2), EU1500R 프라이머(서열표 서열번호 3)를 이용했다. PCR 반응액의 조성은 Tks Gflex(등록 상표) DNA Polymerase의 설명서에 기재되어 있는 혼합 비율에 따랐다. PCR 반응은 94℃에서 1분의 열처리를 1사이클 행한 후, 98℃에서 10초, 55℃에서 15초, 68℃에서 1분 30초의 반응을 30사이클 행했다.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) 시퀀스 반응2) Sequence response

얻어진 PCR 반응물은 Wizard(등록 상표) SV Gel 및 PCR Clean-Up System(Promega사)에 의해 정제했다. 정제 산물과 EU10F 프라이머, EU520F 프라이머(서열표 서열번호 4), EU907R 프라이머(서열표 서열번호 5), 또는 EU1500R 프라이머를 이용하여 시퀀스 반응을 행하여, 단리 균주의 16S rRNA 유전자의 염기 서열을 결정했다.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.

YM17주의 16S rRNA 유전자의 염기 서열을 서열표 서열번호 1에 나타냈다.The nucleotide sequence of the 16S rRNA gene of YM17 is shown in SEQ ID NO: 1.

YM17주의 16S rRNA 유전자 염기 서열에 관해, 유전자 데이터베이스인 The National Center for Biotechnology Information의 BLAST 호몰로지 검색(http://blast.ncbi.nlm.nih.gov/Blast.cgi)을 행한 결과, YM17주의 16S rRNA 유전자와 가장 높은 상동성을 나타내는 종은 메커카리마이세스 아스포로포리게넨스(Mechercharimyces asporophorigenens)이고, 상동성은 91%였다.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%.

계속해서 YM17주에 근연종의 16S rRNA 유전자를 The National Center for Biotechnology Information의 유전자 데이터베이스로부터 수집하여, 계통 해석을 행했다.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.

수집한 16S rRNA 유전자 염기 서열을 Clustal X 2.0.12 프로그램에 의해 얼라이먼트하고, 근린 결합법에 의해 계통수를 작성했다. 얻어진 계통수를 도 2에 도시했다. 괄호 안의 숫자는 GenBank의 액세션 번호를 나타내고, YM16은 본 발명과 동일한 방법으로 본 발명자들이 단리한 균주를 나타낸다.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. YM17주의 동정과 분류H. Identification and Classification of YM17 State

1) YM17주1) YM17 week

도 2의 계통 해석에 나타낸 바와 같이 YM17주의 16S rRNA 유전자는 바실라지과(Bacillaceae 과)나 알리시클로바실라지과(Alicyclobacillaceae 과), 서모액티노마이세타지과(Thermoactinomycetaceae 과)와는 크게 분리되는 분기를 형성한다.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).

YM17주가 나타내는 간균의 형태나 세포 내에서 아포 형성하는 형태는 바실라지과(Bacillaceae 과)나 알리시클로바실라지과(Alicyclobacillaceae 과)에서 보이는 성질이고, 사상성의 균사를 형성하는 서모액티노마이세타지과(Thermoactinomycetaceae 과)와는 분명히 상이하다.The form of bacillus and the form of apolipoprotein expressed in the YM17 strain are the ones seen in the Bacillaceae and Alicyclobacillaceae, and the Thermoactinomycetaceae , which form mycelia of mating , ).

YM17주와 바실라지과(Bacillaceae 과), 및 알리시클로바실라지과(Alicyclobacillaceae 과)에 속하는 종의 16S rRNA 유전자 서열의 상동성을 비교했다. 결과를 표 3에 나타낸다. 모든 속에서 상동성은 89% 이하가 되고, YM17주는 바실라지과(Bacillaceae 과), 및 알리시클로바실라지과(Alicyclobacillaceae 과)에 속하는 종과는 분류학적으로 크게 상이하다. 도 2의 계통수에 있어서 YM17주와 분기가 가까운 알리시클로바실러스 아시도칼다리우스(Alicyclobacillus acidocaldarius), 칼디테리콜라 사츠멘시스(Calditerricola satsumensis), 칼달칼리바실러스 서마룸(Caldalkalibacillus thermarum), 칼디바실러스 데빌리스(Caldibacillus debilis)와 YM17주의 분류학적 성질의 비교를 행했다. 결과를 표 4에 나타낸다.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.

표 4 중 YM17주 이외의 데이터는 International Journal of Systematic and Evolutionary Microbiology(2002), 52, 1681-1685, Journal of General Microbiology(1971), 67, 9-15, International Journal of Systematic and Evolutionary Microbiology(2011), 61, 631-636, International Journal of Systematic and Evolutionary Microbiology(2006), 56, 1217-1221, 및 International Journal of Systematic and Evolutionary Microbiology(2004), 54, 2197-2201로부터 얻었다.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.

알리시클로바실러스속(Alicyclobacillus 속)은 주요한 지방산에 ω-시클로헥산(Cyclohexane)-C17:0, ω-시클로헥산-C19:0을 갖는 것이 속의 특징적인 성질로, YM17주와 상이하다. 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.

칼디테리콜라속(Calditerricola 속)은 75℃ 이상에서 생육할 수 있는 고도 호열성 세균으로 YM17주와 상이하다. Calditoricola (genus Calditerricola ) is a highly thermophilic bacterium that can grow above 75 ℃ and is different from YM17 strain.

칼달칼리바실러스속(Caldalkalibacillus 속)은 그램 염색이 양성인 것, DNA의 염기 조성이 45%인 것이 YM17주와 상이하다.The genus Caldarkalibacillus (genus Caldalkalibacillus ) is gram-positive, and the base composition of DNA is 45% different from that of YM17.

칼디바실러스속(Caldibacillus 속)은 65℃ 이상에서도 생육하고, 글루코오스로부터의 산 생성이 보이지 않기 때문에, YM17주와 상이하다.The genus Caldibacillus (the genus Caldibacillus ) grows above 65 ° C and differs from the YM17 strain because no acid production from glucose is seen.

이상으로부터 YM17주는 신과(프라테리바실라지과), 신속(프라테리바실러스속)의 신종으로 판정하고, YM17주를 프라테리바실러스 플라보알버스(Frateribacillus flavoalbus)(이하, 간단히 프라테리바실러스 플라보알버스로 기재하는 경우가 있음)로 명명했다.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 ) ).

Figure pat00007
Figure pat00007

Figure pat00008
Figure pat00008

[실시예 2][Example 2]

프라테리바실러스 플라보알버스(Frateribacillus flavoalbus)종의 분리 Isolation of Frateribacillus flavoalbus species

상기한 실시예 1에 의해, 프라테리바실러스 플라보알버스 종은 이미 알려진 과·속·종과 상이한 것으로 나타났다. 그래서, 프라테리바실러스 플라보알버스 종의 특성을 명확히 하기 위해, YM17주 이외의 프라테리바실러스 플라보알버스 종에 속하는 균주의 분리를 행했다.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. 프라테리바실러스 플라보알버스 종의 분리1. Isolation of Prateri Bacillus flavobus species

가고시마현 소오시의 퇴비화장 및 가고시마시의 퇴비화장의 퇴비를 분리원으로 하여 프라테리바실러스 플라보알버스 종의 균주의 분리를 시도했다.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.

상기한 실시예 1에서의 YM17주의 배양 성상을 참고로 하여, CYC 한천 배지에 7% 또는 10%의 염화나트륨과 50 ㎍/mL 암피실린을 첨가 또는 무첨가한 것을 분리 배지로 하고, 55℃의 배양 온도에서 6일간 배양하고 균의 분리를 행한 바, 사상성 세균의 다수의 콜로니와 간균형의 세균의 콜로니가 분리 배지 상에 나타났다.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.

이 중에서 간균형의 세균의 콜로니를 분리하고, 다음의 2.에 나타낸 프라테리바실러스 플라보알버스 종을 검출하기 위한 특이적 PCR에 의해 판정을 행한 바, 표 5에 나타낸 바와 같이, 합계 12 균주가 프라테리바실러스 플라보알버스 종으로 판정되었다.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.

특히 CYC 한천 배지에 7% 염화나트륨과 50 ㎍/mL 암피실린을 첨가한 배지에서의 분리 빈도가 높고, 이 배지를 이용하여 55℃에서 배양함으로써 프라테리바실러스 플라보알버스 종을 효율적으로 분리하는 것이 가능했다. 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 .

분리한 12 균주에 관해, 각각 NaCl_20131016_04주, YM17-2주, YM17-3주, YM17-4주, NaCl_20131016_19주, YM17-5주, NaCl_20131016_24주, NaCl_20131016_28주, NaCl_20131016_30주, NaCl_20131016_34주, NaCl_20131016_37주 또는 NaCl_20131016_38주로 했다.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.

이 중, YM17-2주, YM17-3주, YM17-4주 및 YM17-5주에 관해서는, 본 출원인의 신청에 의해, 독립행정법인제품평가기술기반기구(NITE) 특허 미생물 기탁 센터에 부다페스트 조약에 기초하여 국제 기탁되어 있다. 이하에, 기탁을 특정하는 내용을 기재한다.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.

YM17-2주YM17-2 week

(1) 기탁기관명 : 독립행정법인제품평가기술기반기구 특허 미생물 기탁 센터(1) Depositary Institution: Independent administrative corporation Product evaluation Technology-based organization Patented microorganism deposit center

(2) 연락처 : 우편 292-0818 치바현 키사라츠시 카즈사 카마타리 2-5-8(2) Contact: Postal 292-0818 2-5-8 Matsuri, Kazusaka, Kisaratsu, Chiba Prefecture

전화 번호 0438-20-5580Phone number 0438-20-5580

(3) 수탁 번호 : NITE BP-01948(3) Accession number: NITE BP-01948

(4) 식별을 위한 표시 : YM17-2(4) Indication for identification: YM17-2

(5) 원기탁일 : 2014년 10월 6일(5) Date of donation: October 6, 2014

YM17-3주YM17-3 week

(1) 기탁기관명 : 독립행정법인제품평가기술기반기구 특허 미생물 기탁 센터(1) Depositary Institution: Independent administrative corporation Product evaluation Technology-based organization Patented microorganism deposit center

(2) 연락처 : 우편 292-0818 치바현 키사라츠시 카즈사 카마타리 2-5-8(2) Contact: Postal 292-0818 2-5-8 Matsuri, Kazusaka, Kisaratsu, Chiba Prefecture

전화 번호 0438-20-5580Phone number 0438-20-5580

(3) 수탁 번호 : NITE BP-01949(3) Accession number: NITE BP-01949

(4) 식별을 위한 표시 : YM17-3(4) Indication for identification: YM17-3

(5) 원기탁일 : 2014년 10월 6일(5) Date of donation: October 6, 2014

YM17-4주YM17-4 week

(1) 기탁기관명 : 독립행정법인제품평가기술기반기구 특허 미생물 기탁 센터(1) Depositary Institution: Independent administrative corporation Product evaluation Technology-based organization Patented microorganism deposit center

(2) 연락처 : 우편 292-0818 치바현 키사라츠시 카즈사 카마타리 2-5-8(2) Contact: Postal 292-0818 2-5-8 Matsuri, Kazusaka, Kisaratsu, Chiba Prefecture

전화 번호 0438-20-5580Phone number 0438-20-5580

(3) 수탁 번호 : NITE BP-01950(3) Accession number: NITE BP-01950

(4) 식별을 위한 표시 : YM17-4(4) Indication for identification: YM17-4

(5) 원기탁일 : 2014년 10월 6일(5) Date of donation: October 6, 2014

YM17-5주YM17-5 week

(1) 기탁기관명 : 독립행정법인제품평가기술기반기구 특허 미생물 기탁 센터(1) Depositary Institution: Independent administrative corporation Product evaluation Technology-based organization Patented microorganism deposit center

(2) 연락처 : 우편 292-0818 치바현 키사라츠시 카즈사 카마타리 2-5-8(2) Contact: Postal 292-0818 2-5-8 Matsuri, Kazusaka, Kisaratsu, Chiba Prefecture

전화 번호 0438-20-5580Phone number 0438-20-5580

(3) 수탁 번호 : NITE BP-01925(3) Accession number: NITE BP-01925

(4) 식별을 위한 표시 : YM17-5(4) Indication for identification: YM17-5

(5) 원기탁일 : 2014년 8월 28일(5) Date of donation: August 28, 2014

2. 프라테리바실러스 플라보알버스 종을 검출하기 위한 특이적 PCR 2. Specific PCR for detection of Prateri Bacillus flavobus species

프라테리바실러스 플라보알버스 종을 검출하기 위한 특이적 PCR을 다음과 같이 행했다. 우선, YM17주의 16S rRNA 유전자에 특이적인 염기 서열을 이용하여, TIP155F1 프라이머(5'-ATACCGGATAAGAGGCTTTT-3')(서열표 서열번호 6)와 TIP155R1 프라이머(5'-TGGTACCGTCACGCTAAGA-3')(서열표 서열번호 7)를 작성했다.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).

PCR 반응에는 Tks Gflex(등록 상표) DNA Polymerase와 TIP155F1 프라이머, TIP155R1 프라이머 및 분리 균주의 DNA를 이용했다. PCR 반응액의 조성은 Tks Gflex(등록 상표) DNA Polymerase의 설명서에 기재되어 있는 혼합 비율에 따랐다. PCR 반응은 94℃에서 1분의 열처리를 1사이클 행한 후, 98℃에서 10초, 55℃에서 15초, 68℃에서 1분의 반응을 25사이클 행하고, 전기 영동으로 328 bp 부근에 증폭 산물이 확인된 균주를 프라테리바실러스 플라보알버스 종으로 판정했다.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.

Figure pat00009
Figure pat00009

3. 분리 균주의 분류학적 해석3. Taxonomic analysis of isolated strains

상기 1. 및 2.에 의해 분리한 12 균주의 분류학적 성질을 YM17주에 관한 실시예 1과 동일한 방법으로 조사하고, 결과를 표 2-1∼표 2-5에 나타냈다.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. 분리 균주의 16S rRNA 유전자의 염기 서열의 해석4. Analysis of the base sequence of the 16S rRNA gene of the isolate

상기 1. 및 2.에 의해 분리한 12 균주의 16S rRNA 유전자의 염기 서열을 YM17주에 관한 실시예 1과 동일한 방법으로 조사했다. 각 주의 16S rRNA 유전자의 염기 서열은 서열표 서열번호 8∼19에 각각 나타냈다. 서열번호 8은 NaCl_20131016_04주, 서열번호 9는 YM17-2주, 서열번호 10은 YM17-3주, 서열번호 11은 YM17-4주, 서열번호 12는 NaCl_20131016_19주, 서열번호 13은 YM17-5주, 서열번호 14는 NaCl_20131016_24주, 서열번호 15는 NaCl_20131016_28주, 서열번호 16은 NaCl_20131016_30주, 서열번호 17은 NaCl_20131016_34주, 서열번호 18은 NaCl_20131016_37주, 서열번호 19는 NaCl_20131016_38주에 대응한다.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.

YM17주의 16S rRNA 유전자와 이들의 12 균주의 16S rRNA 유전자의 염기 서열의 상동성을 표 6에 나타냈다. 표 6에 나타낸 바와 같이, 분리한 12 균주의 16S rRNA 유전자의 염기 서열은 YM17주의 16S rRNA 유전자의 염기 서열과 98.7% 이상의 상동성이 있었다.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.

Figure pat00010
Figure pat00010

5. DNA-DNA 하이브리다이제이션 5. DNA-DNA hybridization

상기 1. 및 2.에 의해 분리한 12 균주와 YM17주의 게놈 DNA를 이용하여 DNA-DNA 하이브리다이제이션 시험을 행했다. 또한, 네거티브 컨트롤로서 근연속인 칼디테리콜라 사츠멘시스(Calditerricola satsumensis) YMO81주의 DNA를 이용했다. DNA의 조정은 [실시예 1]의 GC 함량 측정에서 조정한 방법과 동일한 방법에 의해 행했다.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].

얻어진 게놈 DNA는 어느 것이나 농도가 500 ㎍/mL 이상, OD260/OD280 값이 1.8 이상, OD260/OD230 값이 2.0 이상으로 RNA의 혼입은 없고, DNA-DNA 하이브리다이제이션 시험에 이용할 수 있는 높은 품질이었다.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 .

DNA-DNA 하이브리다이제이션 시험은 참고 문헌 2와 미생물의 분류·동정 실험법(슈프링가·재팬 발행)에 기재된 방법에 따랐다.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).

우선, DNA 플레이트의 작성을 행했다. 게놈 DNA 용액을 0.1XSSC로 희석하여, 100 ㎍/mL로 했다. 희석한 DNA 용액을 100℃에서 5분간 가열하여 단일쇄로 한 후, 즉시 빙랭했다. 열변성한 DNA 용액을 PBS-Mg 용액으로 10배 희석하여, 10 ㎍/mL로 했다. 10 ㎍/mL로 한 DNA 용액을 100 μL씩 마이크로플레이트의 웰에 나누어 주입하고, 마이크로플레이트에 시일을 하여 30℃에서 3시간 이상 정치했다. 그 후, 웰 내의 DNA 용액을 버리고, 시험용 DNA 플레이트로 했다. 백그라운드 컨트롤에는 연어 정자 DNA를 이용했다.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.

DNA 플레이트의 준비와 병행하여, DNA의 포토비오틴 표지를 행했다. 500 ㎍/mL의 DNA 용액을 30 μL 조정하고, 초음파 세정기로 10분간 처리하고 DNA를 절단했다. 절단한 DNA 용액에 25 μL의 1 mg/mL 포토비오틴액을 첨가하고, DNA를 완전히 용해한 후, 빙랭하면서 250 W의 저압 수은 램프로 30분간 조사하고, DNA를 포토비오틴 표지했다. 여분의 포토비오틴을 제거하기 위해, 조사 후의 DNA 용액에 540 μL의 0.1 M Tris-HCl 버퍼(buffer)(pH 9.0)를 첨가한 후, 600 μL의 2-부탄올을 첨가하여 혼합했다. 15,000 rpm으로 5초간 원심하고, 과잉의 포토비오틴이 포함되는 상청을 버렸다. 2-부탄올을 첨가하고 과잉의 포토비오틴을 제거하는 작업은 합계 2번 행했다. 포토비오틴 표지한 DNA 용액을 100℃에서 5분간 가열한 후, 즉시 빙랭하여, 하이브리다이제이션에 이용했다.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.

다음으로 하이브리다이제이션 조작을 행했다. DNA 플레이트의 각 웰에 200 μL의 예비하이브리다이제이션 혼합물(Prehybridization mixture)을 나누어 주입하고, 37℃에서 15분간 정치했다. 열변성시킨 300 μL의 포토비오틴 표지 DNA 용액을 6 mL의 하이브리다이제이션 혼합물(Hybridization mixture)과 혼합했다. DNA 플레이트의 각 웰로부터 예비하이브리다이제이션 혼합물을 제거하고, 포토비오틴 표지 DNA를 포함하는 하이브리다이제이션 혼합물을 100 μL씩 나누어 주입하고, DNA 플레이트를 시일했다. 이 DNA 플레이트를 46℃에서 2시간 이상 정치하고, 하이브리다이제이션 처리를 행했다. 하이브리다이제이션 처리 후, 포토비오틴 표지 DNA를 포함하는 하이브리다이제이션 혼합물을 버리고, 300 μL의 1XSSC 용액으로 각 웰을 세정하는 작업을 합계 3번 행했다.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.

세정 후의 각 웰에 100 μL의 스트렙타비딘-효소(Streptavidin-enzyme) 용액을 나누어 주입했다. 37℃에서 30분간 정치한 후, 스트렙타비딘-효소 용액을 버렸다. 100 μL의 1XSSC 용액으로 각 웰 세정한 후, 또한 300 μL의 1XSSC 용액으로 각 웰을 합계 3회 세정했다. 세정 후의 각 웰에 100 μL의 형광 기질 용액을 첨가하고, 마이크로플레이트 리더로 형광 강도를 측정했다(여기 파장 360 nm, 측정 파장 450 nm). 37℃에서 보온하고, 1분마다 형광 강도를 측정했다. DNA의 각 조합의 형광 강도는 6웰의 반복 중에서 중앙치 4웰의 평균치를 산출하고, 연어 정자 DNA를 부착한 웰의 평균 형광 강도치를 백그라운드치로서 감산하여 산출했다. 상동치는 동일한 균주의 DNA를 웰 및 표지에 이용한 호모의 수치에 대한 백분율로 표시했다.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.

참고 문헌 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 : p224-229References 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

시약reagent

(1) PBS-Mg 용액(MgCl2 : 0.95 g/PBS 100 mL)(1) PBS-Mg solution (MgCl2: 0.95 g / 100 mL of PBS)

(2) 0.1 M Tris-HCl 버퍼(Tris : 12.1 g, EDTA·2Na : 0.4 g, pH 9.0, 초순수 : 총액량이 1000 mL가 되도록 첨가했다)(2) 0.1 M Tris-HCl buffer (Tris: 12.1 g, EDTA 占 Na: 0.4 g, pH 9.0, ultrapure water:

(3) 예비하이브리다이제이션 혼합물(20XSSC : 1 mL, 50XDenhardt액 : 1 mL, 10 mg/mL 변성 연어 DNA : 0.1 mL, 포름아미드 : 5 mL, 초순수 : 2.9 mL)(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) 하이브리다이제이션 혼합물(20XSSC : 1 mL, 50XDenhardt액 : 1 mL, 10 mg/mL 변성 연어 DNA : 0.1 mL, 포름아미드 : 5 mL, 황산덱스트란 : 0.25 g, 초순수 : 2.8 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) 스트렙타비딘-효소 용액(스트렙토아비딘-β-D-갈락토시다아제 컨쥬게이트 : 10 μL, 0.5% 소혈청 알부민(fraction V)/PBS 용액 : 10 mL)(5) Streptavidin-enzyme solution (10 μL of streptavidin-β-D-galactosidase conjugate, 0.5% bovine serum albumin / PBS solution: 10 mL)

(6) 형광 기질 용액(10 mg/mL 4-메틸움벨리페릴-β-D-갈락토피라노시드 용액 : 100 μL, PBS+1 mmol MgCl2 용액 : 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)

DNA-DNA 하이브리다이제이션 시험의 결과를 표 7에 나타냈다.The results of the DNA-DNA hybridization test are shown in Table 7.

표 7에 나타낸 바와 같이, YM-17주, YM17-2주, YM17-3주, YM17-4주, YM17-5주, NaCl_20131016_37주 및 NaCl_20131016_38주의 상호의 DNA-DNA 상동치는 모두 70% 이상이었다.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%.

참고 문헌 3에 기재된 바와 같이, 미생물 분류학에 있어서 DNA-DNA 상동치가 70% 이상이면 동종으로 여겨지고 있는 점에서, 이들 8주가 동종인 것이 확인되었다.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.

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

Figure pat00011
Figure pat00011

6. 프라테리바실러스 플라보알버스의 성질6. Properties of Pratery Bacillus Flavo Albus

상기한 각 해석, 시험에 의해, 프라테리바실러스 플라보알버스(Frateribacillus flavoalbus)는 다음 1)∼10)의 특징을 성질로서 갖는 종인 것이 확인되었다.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) 그램 염색이 음성이고, 포자를 형성함1) Gram stain is negative, forming spores

2) 옥시다아제 테스트는 음성, 카탈라아제 테스트는 양성이고 호기성임2) oxydase test is negative, catalase test is positive and aerobic

3) 1%의 당 및 2.0%의 NaCl을 포함하는 LB 배지(pH 8.0)에, 상기 당으로서 D-갈락토오스 또는 수크로오스를 첨가한 경우에는 산 생성을 하지 않지만, D-글루코오스, 락토오스, D-만니톨, 글리세린 또는 N-아세틸글루코사민을 첨가한 경우에는 산 생성을 함3) 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) 지적 생육 온도는 45℃∼55℃이고, 35℃ 이하 또는 65℃ 이상에서는 증식이 확인되지 않음4) Intellectual growth temperature is 45 ℃ ~ 55 ℃. No growth is observed at 35 ℃ or above or above 65 ℃

5) 지적 pH는 7.0∼8.0이고, pH 5.7 이하 또는 pH 10.0 이상에서는 증식이 확인되지 않음5) 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) CYC 배지에 NaCl을 종농도 7%까지 첨가해도 증식이 확인됨6) Proliferation was confirmed by addition of NaCl to the CYC medium up to a concentration of 7%.

7) 주요한 메나퀴논은 MK7임7) The main menaquinone is MK7

8) 세포벽 펩티도글리칸의 아미노산 조성으로서 알라닌, 글루타민산, 및 메소형 디아미노피멜산(meso-DAP)을 포함함8) The amino acid composition of the cell wall peptidoglycan includes alanine, glutamic acid, and a small diamino pimelic acid (meso-DAP).

9) 게놈 DNA의 GC 함량은 51.2%∼52.4%임9) GC content of genomic DNA is 51.2% ~ 52.4%

10) 가장 주요한 균체 지방산은 iso C16:0임10) The most important fungal fatty acid is iso C16: 0

7. 프라테리바실러스 플라보알버스 종 내의 아종 분류 7. Classification of subspecies in Prateri Bacillus flavobus species

YM17주와 분리한 12주(NaCl_20131016_ 04주, YM17-2주, YM17-3주, YM17-4주, NaCl_20131016_19주, YM17-5주, NaCl_20131016_24주, NaCl_20131016_28주, NaCl_20131016_30주, NaCl_20131016_34주, NaCl-20131016_37주 및 NaCl_20131016_38주)의 16S rRNA 유전자에 의한 계통수를 도 3에 도시했다. 도 3에 도시된 바와 같이 YM17주와 YM17-5주의 16S rRNA 유전자는 다른 균주와 분기되었다.(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 week 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.

또한, 표 8에 나타낸 바와 같이 YM17주와 YM17-5주의 주요한 균체 지방산 상위 4종이 iso-C16:0, anteiso-C17:0, anteiso-C15:0, iso-C17:0인 데 대하여, YM17-2주, YM17-3주, YM17-4주, NaCl_20131016_37주 및 NaCl_20131016_38주는 iso-C16:0, C16:0, anteiso-C17:0, anteiso-C15:0으로 상이했다. 이 때문에, YM17주·YM17-5주와 그 밖의 균주는 프라테리바실러스 플라보알버스로 동종이지만, 상이한 아종이었다.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.

Figure pat00012
Figure pat00012

이상의 결과로부터, YM17주와 YM17-5주의 2주를 프라테리바실러스 플라보알버스 아종 플라보알버스(Frateribacillus flavoalbus subsp. flavoalbus)로 명명하고, NaCl_20131016_04주, YM17-2주, YM17-3주, YM17-4주, NaCl_20131016_19주, NaCl_20131016_24주, NaCl_20131016_28주, NaCl_20131016_30주, NaCl_20131016_34주, NaCl_20131016_37주 및 NaCl_20131016_38주의 11주를 프라테리바실러스 플라보알버스 아종 피메타리움(Frateribacillus flavoalbus subsp. fimetarium)으로 명명했다.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 .

즉, 프라테리바실러스 플라보알버스(Frateribacillus flavoalbus)는 프라테리바실러스 플라보알버스 아종 플라보알버스(Frateribacillus flavoalbus subsp. flavoalbus)와, 프라테리바실러스 플라보알버스 아종 피메타리움(Frateribacillus flavoalbus subsp. fimetarium)의 2 아종으로 이루어지는 신속·신종이었다.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. 프라테리바실러스 플라보알버스의 단백질 분해 활성8. Proteolytic activity of Pratery Bacillus flavobus

다음으로 프라테리바실러스 플라보알버스(Frateribacillus flavoalbus)의 단백질 분해 활성을 조사했다. 기본 배지(염화나트륨 : 2.0%, 효모 엑기스 : 0.1%)에 2.0%의 젤라틴 또는 0.4%의 카제인을 첨가하고 pH를 8.0으로 조정한 후, 1.5%의 한천을 첨가하고, 120℃에서 20분간 오토클레이브 멸균을 하여, 시험 배지로 했다.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.

시험 배지에 YM17주, NaCl_20131016_04주, YM17-2주, YM17-3주, YM17-4주, NaCl_20131016_19주, YM17-5주, NaCl_20131016_24주, NaCl_20131016_28주, NaCl_20131016_30주, NaCl_20131016_34주, NaCl_20131016_37주 또는 NaCl_20131016_38주를 도포하고, 55℃에서 7일간 배양했다. 배양 후, 시험 배지에 염산-염화제2수은 용액(염화제2수은 15 g, 농염산 20 mL, 증류수 100 mL)을 적하하고, 할로 형성에 의해, 단백질의 분해 활성을 조사했다. 시험 결과, 13 균주 전부에서, 젤라틴 및 카제인의 분해 활성이 관찰되었다.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. 프라테리바실러스 플라보알버스의 퇴비 환경에서의 생육과 유기물 분해 활성9. Growth and decomposition of organic matter in the compost environment of Pratery Bacillus flavobus

프라테리바실러스 플라보알버스(Frateribacillus flavoalbus)의 퇴비 중에서의 생육과 유기물 분해 활성을 조사했다.The growth and organic degradation activity of Frateribacillus flavoalbus in compost was investigated.

120℃, 30분간의 오토클레이브 멸균을 2번 한 10 g 퇴비에 대하여, 필터 여과 멸균한 10% 효모 엑기스 용액 1 mL, 1% 카제인 용액 또는 멸균수 3 mL, 2% NaCl을 포함하는 LB 배지(pH 8.0)를 이용하여 55℃에서 1일간 배양한 균의 배양액 1 mL를 첨가하고, 수분이 퇴비 전체에 고루 퍼지도록 멸균한 약수저로 혼합했다. 혼합물은 기상이 충분히 있는 상태에서 용기를 밀폐하고, 55℃에서 3일간 배양(이하 퇴비 배양이라고 함)했다. 균의 배양액 대신에 멸균한 2% NaCl을 포함하는 LB 배지(pH 8.0)를 첨가한 것을 네거티브 컨트롤로 했다. 시험균으로서, YM17주, YM17-2주, YM17-3주, YM17-4주, YM17-5주, NaCl_20131016_37주 및 NaCl_20131016_38주를 이용했다.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.

퇴비 배양 3일 후의 배양물을 현미경으로 관찰한 바, YM17주, YM17-2주, YM17-3주, YM17-4주, YM17-5주, NaCl_20131016_37주 또는 NaCl_20131016_38주의 배양액을 첨가한 시험군에서는 전체에서 양호한 균의 생육이 확인되었다. 한편, 균의 배양물을 첨가하지 않은 네거티브 컨트롤에서는 균체가 보이지 않았다. 도 4에 YM17주 또는 네거티브 컨트롤의 카제인 첨가 퇴비 배양 3일 후의 현미경 사진을 나타낸다.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.

도 4의 A의 동그라미로 표시된 바와 같이, YM17주에 관해서는 균체의 양호한 생육을 확인할 수 있었다. 한편, 도 4의 B에 도시된 바와 같이, 네거티브 컨트롤에서는 퇴비의 고형물만이 관찰되고, 균체는 보이지 않았다.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.

퇴비 배양 3일 후의 배양물로부터 단백질을 추출했다. 우선, 배양물에 30 mL의 Tris-HCl(pH 8.0)을 첨가하여 현탁액으로 하고, 실온에서 3시간 정치했다. 정치 후의 현탁액을 여과(여과지 : 아도반테크 도요, 형식 NO.5B, 110 mm)하고, 여과액을 회수했다. 남은 잔류물을 Tris-HCl(pH 8.0)로 세정하도록 하고, 여과액을 총량 30 mL로 조정했다. 30 mL의 여과액에 14.2 g의 황산암모늄을 첨가하고, 서서히 교반하여, 황산암모늄을 용해시켰다. 황산암모늄 용해 후, 빙상에서 하룻밤 정치했다. 정치 후의 액을 4℃, 10,000 g으로 30분간 원심하여, 상청을 제거하고 침전물을 회수했다. 침전물을 1 mL의 Tris-HCl(pH 8.0)로 용해시키고, 용해물을 투석막(Spectrum Laboratories, Inc., 스펙트라/포어(등록 상표) 1, MW 6,000-8,000)에 넣고, Tris-HCl(pH 8.0) 중에서 투석하여 탈염 처리를 했다. 탈염 후, Tris-HCl(pH 8.0)로 조정하여, 액량을 2 mL로 하고, 자이모그램의 제공 시료로 했다.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.

자이모그래피용의 전기 영동 겔에는 기질로서 종농도 0.1%의 젤라틴을 첨가했다. 탈염 후의 단백질 용해액 15 μL와 로딩 버퍼(Loading Buffer) 5 μL를 혼합하여, 20 μL의 혼합액을 자이모그래피에 제공했다.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.

전기 영동 후의 겔을 0.2% TritonX를 포함하는 Tris-HCl(pH 8.0) 중에 10분간 둔 후, 본 버퍼를 버리고, TritonX를 포함하지 않는 Tris-HCl(pH 8.0)로 2번 세정했다. 세정 후의 겔을 2 mM의 CaCl2 및 2 mM의 MgSO4를 포함하는 20 mL의 Tris-HCl(pH 8.0)에 침지하여 55℃에서 16시간 정치하고, 활성 배양을 행했다. 활성 배양 후, 2 mM의 CaCl2 및 2 mM의 MgSO4를 포함하는 20 mL의 Tris-HCl(pH 8.0)을 버리고 겔을 30분간 쿠마시 염색하고, 쿠마시 염색 후에 탈색을 하고, 단백질의 분해 활성을 조사했다.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.

자이모그래피의 결과, 카제인 무첨가의 퇴비 배양으로부터 추출한 단백질도 젤라틴 분해 활성을 나타냈지만, 카제인 첨가의 퇴비 배양으로부터 추출한 단백질은 보다 강한 활성을 나타냈다. 또한, 균의 배양액을 첨가하지 않은 네거티브 컨트롤에서는, 카제인 첨가의 유무에 관계없이 퇴비 배양으로부터 추출한 단백질은 젤라틴 분해 활성을 나타내지 않았다.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.

균의 배양액을 첨가한 경우, 카제인 무첨가의 퇴비 배양의 경우에도 단백질의 분해 활성이 보인 것은, 기재로 한 퇴비 중에 단백질이 잔존하고 있고, 활성이 유도되고 있기 때문이었다. 또한, 카제인을 첨가하여 퇴비 배양을 함으로써, 활성 유도가 보다 강하게 나타나고 있었다.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.

이상의 결과로부터, YM17주, YM17-2주, YM17-3주, YM17-4주, YM17-5주, NaCl_20131016_37주 또는 NaCl_20131016_38주 등의 프라테리바실러스 플라보알버스(Frateribacillus flavoalbus)종은 퇴비 중에서도 생육하여, 단백질 분해 활성을 갖는 것이 밝혀졌다. 도 5에 젤라틴 자이모그래피의 결과를 도시했다. 도 5의 A는, 카제인 무첨가 퇴비 배양으로부터 추출한 단백질을 이용한 자이모그래피의 결과이고, 도 5의 B는 카제인 첨가 퇴비 배양으로부터 추출한 단백질을 이용한 자이모그래피의 결과이다.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.

시약reagent

(1) Tris-HCl(pH 8.0)(Tris : 6.1 g, 농염산으로 pH 8.0으로 조정, 초순수 : 총액량이 1000 mL가 되도록 첨가했다)(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) 자이모그램용 전기 영동 겔(2) Electrophoresis gel for zymogram

세퍼레이트층(30% 아크릴아미드 : 2000 μL, 1.5 M Tris-HCl(pH 8.5) : 1500 μL, 초순수 : 1800 μL, 10% SDS : 60 μL, 10% APS : 50 μL, TEMED : 5 μL, 1% 젤라틴 : 600 μL)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)

스태킹층(30% 아크릴아미드 : 450 μL, 0.5 M Tris-HCl(pH 6.5) : 750 μL, 초순수 : 1700 μL, 10% SDS : 30 μL, 10% APS : 50 μL, TEMED : 5 μL)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)

세퍼레이트층이 고화된 후, 스태킹층을 중층하여 고화시킨다.After the separate layer is solidified, the stacking layer is layered and solidified.

(3) 10X 자이모그램용 전기 영동 버퍼(Tris : 30.3 g, 글리신 : 144 g, 초순수 : 총액량이 1000 mL가 되도록 첨가했다). 자이모그램의 전기 영동에서는 10X 자이모그램용 전기 영동 버퍼를 초순수로 희석하여 1X 버퍼로 하고, 종농도 0.1%가 되도록 SDS를 첨가했다.(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%.

본 발명에 의해 얻어진 신규 미생물은, 신속 프라테리바실러스속(Frateribacillus 속)의 신종으로서, 시뇨, 오니 등의 유기 폐기물의 처리 등에 유용하고, 또한, 여러가지 용도에 사용할 수 있다.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.

[수탁번호][Access number]

[기탁 생물 재료에 대한 언급][Reference to Deposited Biomaterials]

(1) 기탁기관명 : 독립행정법인제품평가기술기반기구 특허 미생물 기탁 센터(1) Depositary Institution: Independent administrative corporation Product evaluation Technology-based organization Patented microorganism deposit center

(2) 연락처 : 우편 292-0818 치바현 키사라츠시 카즈사 카마타리 2-5-8(2) Contact: Postal 292-0818 2-5-8 Matsuri, Kazusaka, Kisaratsu, Chiba Prefecture

전화 번호 0438-20-5580Phone number 0438-20-5580

(3) 수탁 번호 : NITE BP-01764(3) Accession number: NITE BP-01764

(4) 식별을 위한 표시 : YM17(4) Indication for identification: YM17

(5) 원기탁일 : 2013년 11월 29일(5) Date of original deposit: November 29, 2013

[기탁 생물 재료에 대한 언급][Reference to Deposited Biomaterials]

(1) 기탁기관명 : 독립행정법인제품평가기술기반기구 특허 미생물 기탁 센터(1) Depositary Institution: Independent administrative corporation Product evaluation Technology-based organization Patented microorganism deposit center

(2) 연락처 : 우편 292-0818 치바현 키사라츠시 카즈사 카마타리 2-5-8(2) Contact: Postal 292-0818 2-5-8 Matsuri, Kazusaka, Kisaratsu, Chiba Prefecture

전화 번호 0438-20-5580Phone number 0438-20-5580

(3) 수탁 번호 : NITE BP-01948(3) Accession number: NITE BP-01948

(4) 식별을 위한 표시 : YM17-2(4) Indication for identification: YM17-2

(5) 원기탁일 : 2014년 10월 6일 (5) Date of donation: October 6, 2014

[기탁 생물 재료에 대한 언급][Reference to Deposited Biomaterials]

(1) 기탁기관명 : 독립행정법인제품평가기술기반기구 특허 미생물 기탁 센터(1) Depositary Institution: Independent administrative corporation Product evaluation Technology-based organization Patented microorganism deposit center

(2) 연락처 : 우편 292-0818 치바현 키사라츠시 카즈사 카마타리 2-5-8(2) Contact: Postal 292-0818 2-5-8 Matsuri, Kazusaka, Kisaratsu, Chiba Prefecture

전화 번호 0438-20-5580Phone number 0438-20-5580

(3) 수탁 번호 : NITE BP-01949(3) Accession number: NITE BP-01949

(4) 식별을 위한 표시 : YM17-3(4) Indication for identification: YM17-3

(5) 원기탁일 : 2014년 10월 6일(5) Date of donation: October 6, 2014

[기탁 생물 재료에 대한 언급][Reference to Deposited Biomaterials]

(1) 기탁기관명 : 독립행정법인제품평가기술기반기구 특허 미생물 기탁 센터(1) Depositary Institution: Independent administrative corporation Product evaluation Technology-based organization Patented microorganism deposit center

(2) 연락처 : 우편 292-0818 치바현 키사라츠시 카즈사 카마타리 2-5-8(2) Contact: Postal 292-0818 2-5-8 Matsuri, Kazusaka, Kisaratsu, Chiba Prefecture

전화 번호 0438-20-5580Phone number 0438-20-5580

(3) 수탁 번호 : NITE BP-01950(3) Accession number: NITE BP-01950

(4) 식별을 위한 표시 : YM17-4(4) Indication for identification: YM17-4

(5) 원기탁일 : 2014년 10월 6일(5) Date of donation: October 6, 2014

[기탁 생물 재료에 대한 언급][Reference to Deposited Biomaterials]

(1) 기탁기관명 : 독립행정법인제품평가기술기반기구 특허 미생물 기탁 센터(1) Depositary Institution: Independent administrative corporation Product evaluation Technology-based organization Patented microorganism deposit center

(2) 연락처 : 우편 292-0818 치바현 키사라츠시 카즈사 카마타리 2-5-8(2) Contact: Postal 292-0818 2-5-8 Matsuri, Kazusaka, Kisaratsu, Chiba Prefecture

전화 번호 0438-20-5580Phone number 0438-20-5580

(3) 수탁 번호 : NITE BP-01925(3) Accession number: NITE BP-01925

(4) 식별을 위한 표시 : YM17-5(4) Indication for identification: YM17-5

(5) 원기탁일 : 2014년 8월 28일(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 aatacatgca agtcgcgcgg gtgaagctga ccgccccttc gggggaggat ggcggaacca 60 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                          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)

프라테리바실러스속(Frateribacillus 속)의 신종이고, 다음 1)∼10)의 특성을 갖는, 프라테리바실러스 플라보알버스(Frateribacillus flavoalbus).
1) 그램 염색이 음성인 간균이고, 포자를 형성함
2) 옥시다아제 테스트는 음성, 카탈라아제 테스트는 양성이고 호기성임
3) 1%의 당 및 2.0%의 NaCl을 포함하는 LB 배지(pH 8.0)에, 상기 당으로서 D-갈락토오스 또는 수크로오스를 첨가한 경우에는 산 생성을 하지 않지만, D-글루코오스, 락토오스, D-만니톨, 글리세린 또는 N-아세틸글루코사민을 첨가한 경우에는 산 생성을 함
4) 지적 생육 온도는 45℃∼55℃이고, 35℃ 이하 또는 65℃ 이상에서는 증식이 확인되지 않음
5) 지적 pH는 7.0∼8.0이고, pH 5.7 이하 또는 pH 10.0 이상에서는 증식이 확인되지 않음
6) CYC 배지에 NaCl을 종농도 7%까지 첨가해도 증식이 확인됨
7) 주요한 메나퀴논은 MK7임
8) 세포벽 펩티도글리칸의 아미노산 조성으로서 알라닌, 글루타민산, 및 메소형 디아미노피멜산(meso-DAP)을 포함함
9) 게놈 DNA의 GC 함량은 51.2%∼52.4%임
10) 가장 주요한 균체 지방산은 iso-C16:0임
Plastic Terry Bacillus swine, and the following: 1) having the characteristics of to 10), Bacillus Plastic Plastic Terry beam albus (Frateribacillus flavoalbu s) 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 2.0% 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
제1항에 있어서, 서열번호 1, 또는 서열번호 8∼서열번호 19 중 어느 것에 나타낸 염기 서열에 대하여 98.7% 이상의 상동성을 갖는 염기 서열로 이루어지는 16S rRNA 유전자를 갖는, 프라테리바실러스 플라보알버스(Frateribacillus flavoalbus).The vector according to any one of claims 1 to 6, 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 any one of SEQ ID NO: 1 to SEQ ID NO: 19, Frateribacillus flavoalbus ). 제1항 또는 제2항에 있어서, YM17주(NITE BP-01764), YM17-2주(NITE BP-01948), YM17-3주(NITE BP-01949), YM17-4주(NITE BP-01950) 또는 YM17-5주(NITE BP-01925)와의 DNA-DNA 하이브리다이제이션 상동치가 70% 이상인, 프라테리바실러스 플라보알버스(Frateribacillus flavoalbus).The method according to any one of claims 1 to 3, further comprising the steps of: YM17 strain (NITE BP-01764), YM17-2 strain (NITE BP-01948), YM17-3 strain (NITE BP- ) or YM17-5 state (NITE BP-01925) with DNA-DNA hybridization homology values, plastic Terry Bacillus Plastic beam albus (Frateribacillus flavoalbus 70% or more). 제1항 내지 제3항 중 어느 한 항에 있어서, 또한, 균체 지방산으로서, anteiso-C15:0, C16:0, anteiso-C17:0, iso-C17:0 중 어느 1종 이상을 포함하는, 프라테리바실러스 플라보알버스(Frateribacillus flavoalbus).The microcapsule according to any one of claims 1 to 3, further comprising at least one of anteiso-C15: 0, C16: 0, anteiso-C17: 0 and iso-C17: Frateribacillus flavoalbus ( Frateribacillus flavoalbus ). 제1항 내지 제4항 중 어느 한 항에 있어서, 균체 지방산으로서 iso-C16:0에 더하여, anteiso-C15:0, anteiso-C17:0, iso-C17:0을 포함하는 프라테리바실러스 플라보알버스 아종 플라보알버스(Frateribacillus flavoalbus subsp. flavoalbus)인, 프라테리바실러스 플라보알버스(Frateribacillus flavoalbus).The method according to any one of claims 1 to 4, wherein in addition to iso-C16: 0 as a fungus fatty acid, a Prater Bacillus flavor comprising anteiso-C15: 0, anteiso-C17: 0, iso-C17: Albers subspecies Plastic beam albus (Frateribacillus flavoalbus subsp. flavoalbus) which, plastic Terry Bacillus Plastic beam albus (Frateribacillus flavoalbus). 제1항 내지 제4항 중 어느 한 항에 있어서, 균체 지방산으로서 iso-C16:0에 더하여, anteiso-C15:0, C16:0, anteiso-C17:0을 포함하는, 프라테리바실러스 플라보알버스 아종 피메타리움(Frateribacillus flavoalbus subsp. fimetarium)인, 프라테리바실러스 플라보알버스(Frateribacillus flavoalbus).The method according to any one of claims 1 to 4, wherein in addition to iso-C16: 0 as a fungus fatty acid, a mixture of antelope-C15: 0, C16: 0, anteiso-C17: 0, subspecies blood meth Solarium (Frateribacillus flavoalbus subsp. fimetarium) which, plastic Terry Bacillus Plastic beam albus (Frateribacillus flavoalbus). 제1항 내지 제6항 중 어느 한 항에 있어서, 유기 폐기물의 처리에 사용되는, 프라테리바실러스 플라보알버스(Frateribacillus flavoalbus).7. Frateribacillus flavoalbus according to any one of claims 1 to 6, used in the treatment of organic wastes. 제7항에 있어서, 유기 폐기물이 시뇨, 유기성 오니, 식품 잔사물, 또는 동물 분뇨인, 프라테리바실러스 플라보알버스(Frateribacillus flavoalbus).The method of claim 7, wherein the organic waste is sinyo, organic sludge, food glass object, or animal manure, plastic Terry Bacillus Plastic beam albus (Frateribacillus flavoalbus). 제1항 내지 제8항 중 어느 한 항에 있어서, YM17주(NITE BP-01764), NaCl_20131016_04주, YM17-2주(NITE BP-01948), YM17-3주(NITE BP-01949), YM17-4주(NITE BP-01950), NaCl_20131016_19주, YM17-5주(NITE BP-01925), NaCl_20131016_24주, NaCl_20131016_28주, NaCl_20131016_30주, NaCl_20131016_34주, NaCl_20131016_37주 또는 NaCl_20131016_38주 중 어느 것인, 프라테리바실러스 플라보알버스(Frateribacillus flavoalbus).9. The method according to any one of claims 1 to 8, further comprising the steps of: YM17 strain (NITE BP-01764), NaCl_20131016_04 strain, YM17-2 strain (NITE BP-01948), YM17-3 strain (NITE BP- 4 weeks (NITE BP-01950), NaCl_20131016_19 weeks, YM17-5 weeks (NITE BP-01925), NaCl_20131016_24 weeks, NaCl_20131016_28 weeks, NaCl_20131016_30 weeks, NaCl_20131016_34 weeks, NaCl_20131016_37 weeks or NaCl_20131016_38 weeks, Fraser ( Frateribacillus flavoalbus ). 제1항 내지 제9항 중 어느 한 항에 기재된 프라테리바실러스 플라보알버스(Frateribacillus flavoalbus)를 사용하는 유기 폐기물의 처리 방법.A method for treating organic waste using the Frateribacillus flavoalbus according to any one of claims 1 to 9. 제1항 내지 제9항 중 어느 한 항에 기재된 프라테리바실러스 플라보알버스(Frateribacillus flavoalbus)를 사용하는 단백질의 분해 방법.10. A method for degrading a protein using the Frateribacillus flavoalbus according to any one of claims 1 to 9.
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