KR20230127005A - Novel Bacillus subtilis strain IJK1-4 and uses thereof - Google Patents
Novel Bacillus subtilis strain IJK1-4 and uses thereof Download PDFInfo
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- 244000063299 Bacillus subtilis Species 0.000 title claims abstract description 33
- 235000014469 Bacillus subtilis Nutrition 0.000 title claims abstract description 33
- 108020004465 16S ribosomal RNA Proteins 0.000 claims abstract description 11
- 230000001461 cytolytic effect Effects 0.000 claims abstract description 11
- 239000002773 nucleotide Substances 0.000 claims abstract description 9
- 125000003729 nucleotide group Chemical group 0.000 claims abstract description 9
- 239000006227 byproduct Substances 0.000 claims description 11
- 230000000813 microbial effect Effects 0.000 claims description 7
- 239000002689 soil Substances 0.000 claims description 4
- 238000000354 decomposition reaction Methods 0.000 claims description 2
- 239000003795 chemical substances by application Substances 0.000 claims 1
- 244000005700 microbiome Species 0.000 abstract description 15
- 241000207199 Citrus Species 0.000 description 11
- 235000020971 citrus fruits Nutrition 0.000 description 11
- 230000000694 effects Effects 0.000 description 8
- 241000894006 Bacteria Species 0.000 description 5
- 238000002360 preparation method Methods 0.000 description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 5
- 239000012153 distilled water Substances 0.000 description 4
- 239000002609 medium Substances 0.000 description 4
- 238000012545 processing Methods 0.000 description 4
- LWFUFLREGJMOIZ-UHFFFAOYSA-N 3,5-dinitrosalicylic acid Chemical compound OC(=O)C1=CC([N+]([O-])=O)=CC([N+]([O-])=O)=C1O LWFUFLREGJMOIZ-UHFFFAOYSA-N 0.000 description 3
- 239000000872 buffer Substances 0.000 description 3
- 238000006243 chemical reaction Methods 0.000 description 3
- 239000000446 fuel Substances 0.000 description 3
- 238000000034 method Methods 0.000 description 3
- 238000012163 sequencing technique Methods 0.000 description 3
- 241000675108 Citrus tangerina Species 0.000 description 2
- GLNADSQYFUSGOU-GPTZEZBUSA-J Trypan blue Chemical compound [Na+].[Na+].[Na+].[Na+].C1=C(S([O-])(=O)=O)C=C2C=C(S([O-])(=O)=O)C(/N=N/C3=CC=C(C=C3C)C=3C=C(C(=CC=3)\N=N\C=3C(=CC4=CC(=CC(N)=C4C=3O)S([O-])(=O)=O)S([O-])(=O)=O)C)=C(O)C2=C1N GLNADSQYFUSGOU-GPTZEZBUSA-J 0.000 description 2
- 238000002835 absorbance Methods 0.000 description 2
- 238000011481 absorbance measurement Methods 0.000 description 2
- 239000001913 cellulose Substances 0.000 description 2
- 229920002678 cellulose Polymers 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 239000007375 cmc medium Substances 0.000 description 2
- 239000005515 coenzyme Substances 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 235000011389 fruit/vegetable juice Nutrition 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 238000003260 vortexing Methods 0.000 description 2
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 1
- 229920001817 Agar Polymers 0.000 description 1
- 241000193830 Bacillus <bacterium> Species 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 239000001888 Peptone Substances 0.000 description 1
- 108010080698 Peptones Proteins 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 229940041514 candida albicans extract Drugs 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 238000003912 environmental pollution Methods 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 235000012041 food component Nutrition 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 239000011259 mixed solution Substances 0.000 description 1
- 244000052769 pathogen Species 0.000 description 1
- 235000019319 peptone Nutrition 0.000 description 1
- 238000003672 processing method Methods 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 239000001509 sodium citrate Substances 0.000 description 1
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 description 1
- 239000012064 sodium phosphate buffer Substances 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 238000003911 water pollution Methods 0.000 description 1
- 239000012138 yeast extract Substances 0.000 description 1
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N1/00—Microorganisms, 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
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B09—DISPOSAL OF SOLID WASTE; RECLAMATION OF CONTAMINATED SOIL
- B09B—DISPOSAL OF SOLID WASTE NOT OTHERWISE PROVIDED FOR
- B09B3/00—Destroying solid waste or transforming solid waste into something useful or harmless
- B09B3/60—Biochemical treatment, e.g. by using enzymes
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B09—DISPOSAL OF SOLID WASTE; RECLAMATION OF CONTAMINATED SOIL
- B09B—DISPOSAL OF SOLID WASTE NOT OTHERWISE PROVIDED FOR
- B09B2101/00—Type of solid waste
- B09B2101/70—Kitchen refuse; Food waste
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/07—Bacillus
- C12R2001/125—Bacillus subtilis ; Hay bacillus; Grass bacillus
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Abstract
본 발명이 해결하고자 하는 과제는 셀룰로오스 분해 활성이 있는, 서열번호 1로 기재되는 염기서열을 갖는 16S rDNA를 포함하는 신규 미생물 바실러스 서브틸리스 (Bacillus subtilis) IJK1-4 균주를 제공하는 것이다.An object to be solved by the present invention is to provide a novel microorganism Bacillus subtilis IJK1-4 strain containing 16S rDNA having a nucleotide sequence represented by SEQ ID NO: 1 having cellulolytic activity.
Description
본 발명은 신규 미생물 바실러스 서브틸리스 (Bacillus subtilis) IJK1-4 균주 및 이의 용도에 관한 것이다.The present invention relates to a novel microorganism Bacillus subtilis ( Bacillus subtilis ) IJK1-4 strain and its use.
감귤 가공 부산물인 감귤박은 연간 6~7만여톤 생상되고 있다. 현재 감귤 가공 부산물을 처리하기 위한 주된 방법으로는 매립이 이용되고 있으나, 이는 환경 및 수질 오염의 원인으로 지목되고 있다. 이러한 문제를 해결하기 위해 감귤 가공 부산물의 처리 방법으로 연료화, 사료화 등의 방법이 이용되고 있으나, 연료화는 시설 초기 투자 비용이 막대하고 다른 연료와 비교해 효율성이 떨어진다는 단점이 있으며, 사료화의 경우에는 부패성 병원균의 발생, 영양성분의 불균형 등의 문제점이 있다. 또한 비상품과 농산물이나 시장 등에서 농산 부산물이 발생되어 이에 대한 처리 문제도 발생하고 있다.About 60,000 to 70,000 tons of tangerine peel, a by-product of tangerine processing, are produced annually. Currently, landfill is used as the main method for disposing of citrus processing by-products, but this is pointed out as a cause of environmental and water pollution. In order to solve this problem, methods such as fuel conversion and feed conversion are used as processing methods for citrus processing by-products. However, conversion into fuel has the disadvantage that the initial investment cost for the facility is enormous and the efficiency is low compared to other fuels. There are problems such as occurrence of pathogens and imbalance of nutritional components. In addition, agricultural by-products are generated from non-commodities, agricultural products, and markets, so there is a problem with their disposal.
이에, 감귤박을 포함하는 농산 부산물을 처리하기 위한 친환경적인 방법이 필요한 실정이다.Accordingly, there is a need for an environmentally friendly method for processing agricultural by-products including citrus peels.
본 발명이 해결하고자 하는 과제는 셀룰로오스 분해 활성이 있는, 서열번호 1로 기재되는 염기서열을 갖는 16S rDNA를 포함하는 신규 미생물 바실러스 서브틸리스 (Bacillus subtilis) IJK1-4 균주를 제공하는 것이다.An object to be solved by the present invention is to provide a novel microorganism Bacillus subtilis IJK1-4 strain containing a 16S rDNA having a nucleotide sequence represented by SEQ ID NO: 1 having cellulolytic activity.
또한, 상기 신규 미생물 바실러스 서브틸리스 (Bacillus subtilis) IJK1-4 균주의 균체 또는 배양물을 포함하는 농산부산물 분해용 미생물 제제를 제공하는 것이다.In addition, it is to provide a microbial preparation for decomposing agricultural by-products comprising a cell or culture of the novel microorganism Bacillus subtilis IJK1-4 strain.
본 발명의 일 실시형태는 서열번호 1로 기재되는 염기서열을 갖는 16S rDNA를 포함하는 신규 미생물 바실러스 서브틸리스 (Bacillus subtilis) IJK1-4 균주이다.One embodiment of the present invention is a novel microorganism Bacillus subtilis IJK1-4 strain containing 16S rDNA having the nucleotide sequence shown in SEQ ID NO: 1.
상기 균주는 수탁번호 KFCC11914P로 기탁된 것일 수 있다.The strain may be deposited under accession number KFCC11914P.
상기 균주는 셀룰로오스 분해 활성을 가질 수 있다.The strain may have cellulolytic activity.
상기 균주는 pH 4~9에서 셀룰로오스 분해 활성을 나타낼 수 있다.The strain may exhibit cellulolytic activity at pH 4-9.
상기 균주는 제주 토양에서 분리된 것일 수 있다.The strain may be isolated from Jeju soil.
본 발명의 다른 실시형태는 신규 미생물 바실러스 서브틸리스 (Bacillus subtilis) IJK1-4 균주의 균체 또는 배양물을 포함하는 농산부산물 분해용 미생물 제제이다.Another embodiment of the present invention is a microbial preparation for decomposing agricultural by-products comprising a cell or culture of a novel microorganism Bacillus subtilis IJK1-4 strain.
본 발명에 따른 서열번호 1로 기재되는 염기서열을 갖는 16S rRNA를 포함하는 신규 미생물 바실러스 서브틸리스 (Bacillus subtilis) IJK1-4 균주는, 수탁번호 KFCC11914P로 기탁되며, 셀룰로오스 분해 활성을 가지고 있다. 이는 셀룰로오스를 성분으로 갖는 감귤박과 같은 농산 부산물을 분해할 수 있어, 감귤에서 나오는 폐기물과 같은 농산 부산물을 친환경적으로 처리할 수 있다.The novel microorganism Bacillus subtilis IJK1-4 strain containing 16S rRNA having the nucleotide sequence represented by SEQ ID NO: 1 according to the present invention is deposited under accession number KFCC11914P and has cellulolytic activity. This can decompose agricultural by-products, such as citrus peels having cellulose as a component, so that agricultural by-products, such as wastes from citrus, can be treated in an environmentally friendly manner.
도 1은 본 발명에 따른 IJK1-4 균주의 16S rDNA 유전자의 염기서열이다.
도 2는 CMC 배지에서 IJK1-4 균주의 투명환을 나타낸 사진이다.
도 3은 감귤박에서 IJK1-4 균주의 시간에 따른 환원당 변화를 나타낸 도면이다.
도 4는 IJK1-4 균주의 pH에 따른 효소 활성을 나타낸 도면이다.1 is a nucleotide sequence of the 16S rDNA gene of the IJK1-4 strain according to the present invention.
Figure 2 is a photograph showing the transparent ring of IJK1-4 strain in CMC medium.
Figure 3 is a diagram showing the change of reducing sugar over time in IJK1-4 strains in citrus peels.
Figure 4 is a diagram showing the enzyme activity according to the pH of the IJK1-4 strain.
이하, 본 발명이 속하는 기술 분야에서 통상의 지식을 가진 자가 용이하게 실시할 수 있도록 본 발명의 실시예에 대하여 첨부한 도면을 참고로 하여 상세히 설명한다. 그러나 본 발명은 여러 가지 상이한 형태로 구현될 수 있으며 여기에서 설명하는 실시예에 한정되지 않는다. Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings so that those skilled in the art can easily carry out the present invention. However, the present invention may be embodied in many different forms and is not limited to the embodiments described herein.
도 1은 본 발명에 따른 IJK1-4 균주의 16S rDNA 유전자의 염기서열이다.1 is a nucleotide sequence of the 16S rDNA gene of the IJK1-4 strain according to the present invention.
도 1을 참조하면, 본 발명은 서열번호 1로 기재되는 염기서열을 갖는 16S rDNA를 포함하는 신규 미생물 바실러스 서브틸리스 (Bacillus subtilis) IJK1-4 균주로서, 상기 균주는 수탁번호 KFCC11914P로 기탁된 것일 수 있다.Referring to Figure 1, the present invention is a novel microorganism Bacillus subtilis IJK1-4 strain containing 16S rDNA having a nucleotide sequence represented by SEQ ID NO: 1, the strain deposited under accession number KFCC11914P can
TAGAGTTTGGAATCATGGCTCAGGACGAACGCTGGCGGCGTGCCTAATACATGCAAGTCGAGCGGACAGATGGGAGCTTGCTCCCTGATGTTAGCGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCTGTAAGACTGGGATAACTCCGGGAAACCGGGGCTAATACCGGATGGTTGTCTGAACCGCATGGTTCAGACATAAAAGGTGGCTTCGGCTACCACTTACAGATGGACCCGCGGCGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCGACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGATGAAGGTTTTCGGATCGTAAAGCTCTGTTGTTAGGGAAGAACAAGTGCCGTTCAAATAGGGCGGCACCTTGACGGTACCTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGGGCTCGCAGGCGGTTTCTTAAGTCTGATGTGAAAGCCCCCGGCTCAACCGGGGAGGGTCATTGGAAACTGGGGAACTTGAGTGCAGAAGAGGAGAGTGGAATTCCACGTGTAGCGGTGAAATGCGTAGAGATGTGGAGGAACACCAGTGGCGAAGGCGACTCTCTGGTCTGTAACTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGGGGGTTTCCGCCCCTTAGTGCTGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGGTCGCAAGACTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTCTGACAATCCTAGAGATAGGACGTCCCCTTCGGGGGCAGAGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGATCTTAGTTGCCAGCATTCAGTTGGGCACTCTAAGGTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGACAGAACAAAGGGCAGCGAAACCGCGAGGTTAAGCCAATCCCACAAATCTGTTCTCAGTTCGGATCGCAGTCTGCAACTCGACTGCGTGAAGCTGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAGAGTTTGTAACACCCGAAGTCGGTGAGGTAACCTTTATGGAGCCAGCCGCCGAAGGTGGGACAGATGATTGGGGTGAAGTCGTAACAAGGTAACCGTAASEQ ID NO: 1
구체적으로, 본 발명자들은 제주도 토양에서 24종의 균주를 분리하였으며, 분리한 균주의 단일 콜로니를 채취하여 배양하고, Trpan blue가 포함된 CMC 배지에 페이퍼 디스크(paper disk)를 사용하여 스크리닝한 후, 탈색된 투명환이 크고 선명한 균을 선별하여 IJK1-4로 명명하였다(도 2 참조).Specifically, the present inventors isolated 24 strains from Jeju Island soil, collected and cultured a single colony of the isolated strain, screened using a paper disk in CMC medium containing Trpan blue, Bacteria with large and clear discolored rings were selected and named as IJK1-4 (see FIG. 2).
이후, Wizard Genomic DNA Purification Kit(promega)를 이용하여 DNA를 추출한 후 Bioneer에 24F, 1492R universal primer를 사용하여 시퀀싱을 의뢰한 후, NCBI BLAST를 이용하여 미생물을 동정하였고, 그 결과 IJK1-4는 바실러스 서브틸리스 (Bacillus subtilis) 속에 속하는 신규한 균주인 것을 확인하였고, 이를 한국 미생물 보존 센터에 2021년 11월 12일자로 기탁하여 수탁번호 KFCC11914를 부여받았다. Afterwards, DNA was extracted using the Wizard Genomic DNA Purification Kit (promega), and sequencing was requested using 24F, 1492R universal primers in Bioneer, and microorganisms were identified using NCBI BLAST. As a result, IJK1-4 was bacillus It was confirmed that it was a new strain belonging to the subtilis ( Bacillus subtilis ), and it was deposited with the Korea Microorganism Conservation Center on November 12, 2021, and was given accession number KFCC11914.
본 발명에 따른 균주는 감귤박을 포함하는 배양 환경에서 시간에 따른 환원당 변화를 나타내어 셀룰로오스 분해 활성을 가지고 있음을 확인하였다(도 3 참조). 본 발명의 균주는 pH 4~9 범위에서 셀룰로오스 분해 활성을 나타낼 수 있으며, 약 pH 5에서 최적 분해 활성을 나타낼 수 있다 (도 4 참조).It was confirmed that the strain according to the present invention has cellulolytic activity by showing a change in reducing sugar over time in a culture environment including citrus peel (see FIG. 3). The strain of the present invention may exhibit cellulolytic activity in the pH range of 4 to 9, and may exhibit optimal decomposition activity at about pH 5 (see FIG. 4).
본 발명의 다른 실시형태는 신규 미생물 바실러스 서브틸리스 (Bacillus subtilis) IJK1-4 균주의 균체 또는 배양액을 포함하는 미생물 제제로서, 상기 미생물 제제를 이용하여 감귤박과 같은 농산 부산물을 분해 및 처리할 수 있다.Another embodiment of the present invention is a microbial preparation comprising a cell or culture medium of a novel microorganism Bacillus subtilis IJK1-4 strain, wherein agricultural by-products such as citrus peels can be decomposed and treated using the microbial preparation there is.
상기 미생물 제제는 상기 신규 균주의 균체 또는 배양액 중 어느 하나 이상을 포함하는 것으로, 바람직하게는 상기 균주 6×104~5×106cfu/ml을 포함하며, 상기 균주의 생장온도는 25~45℃, 생장 pH는 4~9이고, 상기 조건에서 셀룰로오스 분해활성을 나타낼 수 있다.The microbial preparation includes at least one of the cells or culture of the new strain, preferably the strain 6 × 10 4 ~ 5 × 10 6 cfu / ml, the growth temperature of the strain is 25 ~ 45 ℃, the growth pH is 4 to 9, and can exhibit cellulosic activity under the above conditions.
이하, 본 발명의 이해를 돕기 위하여 바람직한 실시예를 제시하나, 이러한 실시예로 본 발명을 제한하려는 의도는 아님에 유의하여야 한다.Hereinafter, preferred embodiments are presented to aid understanding of the present invention, but it should be noted that the present invention is not intended to be limited to these embodiments.
<실시예 1> 균주의 분리 및 선발<Example 1> Isolation and selection of strains
제주 한림에 위치한 감귤밭 토양을 채취한 후, 증류수와 1:2 중량비로 혼합한 후, 혼합용액을 거름종이로 2회 여과하여 여과한 용액을 LB Agar 배지에 도말하고, 37℃에서 배양하였다. 배양된 균들의 콜로니 모양을 분리하여 표지하고, 생육이 빠른 미생물 24종을 분리하였다. 분리한 균주의 단일 콜로니를 채취하여 LB 액체 배지에 37℃에서 24시간 배양하고, 트리판 블루(Trypan blue)가 포함된 CMC(Carboxymethylcelluose) 배지 (CMC Na 1g, Peptone 5g, Yeast extract 1g, K2HPO4 4g, MgSO4·7H2O 1g, KCl 0.2g, F2SO4·7H2O 0.02g, Agar 15g, 증류수(DW) 1L, 트리판 블루 0.02%)에 페이퍼 디스크(Paper disk)를 사용하여 스크리닝한 후, 탈색된 투명환이 크고 선명한 균을 선별하여 IJK1-4로 명명하였다(도 2 참조)After collecting soil from a citrus field located in Hallim, Jeju, it was mixed with distilled water at a weight ratio of 1:2, and the mixed solution was filtered twice with filter paper, and the filtered solution was spread on LB Agar medium and cultured at 37 ° C. The colony shapes of the cultured bacteria were separated and labeled, and 24 types of fast-growing microorganisms were isolated. A single colony of the isolated strain was collected and cultured in LB liquid medium at 37 ° C for 24 hours, and CMC (Carboxymethylcelluose) medium containing Trypan blue (CMC Na 1g, Peptone 5g, Yeast extract 1g, K 2 HPO 4 4 g, MgSO 4 7H 2 O 1 g, KCl 0.2 g, F 2 SO 4 7H 2 O 0.02 g, Agar 15 g, distilled water (DW) 1 L, trypan blue 0.02%). After screening, bacteria with large and clear discolored rings were selected and named as IJK1-4 (see Fig. 2).
<실시예 2> 바실러스 서브틸리스 (<Example 2> Bacillus subtilis ( Bacillus subtilisBacillus subtilis ) IJK1-4 균주의 동정) Identification of IJK1-4 strains
상기 실시예 1에서 선발한 균주의 동정은 Wizard Genomic DNA Purification Kit(promega)를 이용하여 DNA를 추출한 후, Bioneer에 24F, 1491R universal Primer를 사용하여 시퀀싱을 의뢰한 후, NCBI BLAST를 이용하여 미생물을 동정하였고, 동정된 균주의 16S rDNA는 서열번호 1과 같다. 동정된 균주 IJK1-4는 바실러스 서브틸리스 (Bacillus subtilis) 속의 균으로 확인되었고, 한국 미생물 보존 센터에 2021년 11월 12일자로 기탁하여 수탁번호 KFCC11914를 부여받았다. Identification of the strain selected in Example 1 was performed by extracting DNA using the Wizard Genomic DNA Purification Kit (promega), requesting sequencing using 24F, 1491R universal Primer in Bioneer, and then sequencing the microorganisms using NCBI BLAST. It was identified, and the 16S rDNA of the identified strain is shown in SEQ ID NO: 1. The identified strains IJK1-4 were identified as bacteria of the genus Bacillus subtilis , and were deposited with the Korea Microorganism Conservation Center on November 12, 2021 and given accession number KFCC11914.
<실시예 3> 바실러스 서브틸리스 (<Example 3> Bacillus subtilis ( Bacillus subtilisBacillus subtilis ) IJK1-4 균주의 셀룰로오스 분해 활성 평가 ) Evaluation of cellulose degradation activity of IJK1-4 strains
IJK1-4 균의 배양액 5mL를 감귤 착즙박 20g, 증류수 10mL 가 포함된 50mL 튜브에 접종 한 후, 37℃, 180rpm에서 2, 4, 6, 8, 10, 12, 16일마다 샘플을 채취하여 환원당 변화를 측정하였다. 환원당 변화는 채취한 샘플을 15,000rpm에서 10분간 원심분리한 후, 상층액을 100배 희석하여 시료로 사용하였다. 시료 300㎕, DNS(3,5-dinitrosalicylic acid) 시약 600㎕를 100℃에서 15분 반응한 후, 540nm로 흡광도를 측정하였고, 이에 대한 결과를 도 3에 나타내었다. After inoculating 5mL of the culture medium of the IJK1-4 bacteria into a 50mL tube containing 20g of citrus juice peel and 10mL of distilled water, samples were taken every 2, 4, 6, 8, 10, 12, and 16 days at 37℃ and 180 rpm to obtain reducing sugar Changes were measured. For the change of reducing sugar, the collected sample was centrifuged at 15,000 rpm for 10 minutes, and the supernatant was diluted 100 times and used as a sample. After reacting 300 μl of the sample and 600 μl of DNS (3,5-dinitrosalicylic acid) reagent at 100° C. for 15 minutes, the absorbance was measured at 540 nm, and the results are shown in FIG. 3 .
도 3을 참조하면, 감귤 착즙박에서 12~16일을 배양했을 때의 흡광도 측정값이 높았고, 16일을 배양했을 때, 흡광도 측정값이 가장 높았다.Referring to FIG. 3, the absorbance measurement value was high when cultured for 12 to 16 days in the citrus juice cake, and the absorbance measurement value was the highest when cultured for 16 days.
이로부터 IJK1-4 균주는 셀룰로오스 분해 활성이 있을 뿐 아니라, 장기간 활성이 유지됨음을 알 수 있다.From this, it can be seen that the IJK1-4 strain not only has cellulolytic activity, but also maintains the activity for a long period of time.
<실시예 4> 바실러스 서브틸리스 (<Example 4> Bacillus subtilis ( Bacillus subtilisBacillus subtilis ) IJK1-4 균주의 pH에 따른 활성) Activity according to pH of strain IJK1-4
CMC(Carboxymethylcelluose)가 1중량% 포함된 LB 액체 배지에 IJK1-4 균주를 하루 배양한 후, 15,000rpm에서 10분간 원심분리한 후, 상층액을 조효소로 사용하였다. CMC 1중량% 수용액 50㎕, 조효소 50㎕, 각 pH 별 버퍼(buffer)(200mM sodium citrate buffer pH 4,5, 200mM sodium phosphate buffer pH 6,7, 200mM Tris-HCl buffer pH 8,9) 200㎕를 20초간 볼텍싱(vortexing) 후, 워터배스(waterbath)에서 1시간 동안 반응시켰다. 이후, DNS 시약 600㎕를 넣은 후, 20초간 볼텍싱한 후, 100℃에서 15분간 반응시키고, 흡광도 540nm로 측정하였고, 이에 대한 결과를 도 4에 나타내었다. After culturing the IJK1-4 strain for one day in LB liquid medium containing 1% by weight of CMC (Carboxymethylcelluose), after centrifugation at 15,000 rpm for 10 minutes, the supernatant was used as a coenzyme. 50 μl of CMC 1% by weight aqueous solution, 50 μl of coenzyme, 200 μl of buffers for each pH (200 mM sodium citrate buffer pH 4,5, 200 mM sodium phosphate buffer pH 6,7, 200 mM Tris-HCl buffer pH 8,9) After vortexing for 20 seconds, it was reacted for 1 hour in a water bath. Then, after adding 600 μl of DNS reagent, vortexing for 20 seconds, reacting at 100 ° C. for 15 minutes, and measuring absorbance at 540 nm, the results are shown in FIG. 4 .
도 4를 참조하면, IJK1-4 균주의 활성은 산성에서 염기성 범위(pH 4내지 9)에 걸쳐 폭넓게 유지되며, 약 pH 5에서 1.24U/mL로 가장 높은 것을 확인할 수 있다.Referring to Figure 4, it can be seen that the activity of the IJK1-4 strain is maintained over a wide range from acidic to basic (pH 4 to 9), and is highest at about pH 5 at 1.24 U/mL.
이와 같이, 본 발명에 따른 서열번호 1로 기재되는 염기서열을 갖는 16S rDNA를 포함하는 신규 미생물 바실러스 서브틸리스 (Bacillus subtilis) IJK1-4 균주는 셀룰로오스 분해 활성이 있으며, 약 pH 5에서 최대 활성을 나타내는 특징이 있음을 알 수 있다.As such, the novel microorganism Bacillus subtilis IJK1-4 strain containing 16S rDNA having the nucleotide sequence represented by SEQ ID NO: 1 according to the present invention has cellulolytic activity, and exhibits maximum activity at about pH 5. It can be seen that there are characteristic features.
상기 진술한 본 발명의 설명은 예시를 위한 것이며, 본 발명이 속하는 기술분야의 통상의 지식을 가진 자는 본 발명의 기술적 사상이나 필수적인 특징을 변경하지 않고서 다른 구체적인 형태로 쉽게 변형이 가능하다는 것을 이해할 수 있을 것이다. 그러므로 이상에서 기술한 실시예들은 모든 면에서 예시적인 것이며 한정적이 아닌 것으로 이해해야만 한다.The description of the present invention described above is for illustrative purposes, and those skilled in the art can understand that it can be easily modified into other specific forms without changing the technical spirit or essential features of the present invention. There will be. Therefore, the embodiments described above should be understood as illustrative in all respects and not limiting.
<110> Jeju National University Industry-Academic Cooperation Foundation <120> Novel Bacillus subtilis strain IJK1-4 and uses thereof <130> DP20220038 <160> 1 <170> KoPatentIn 3.0 <210> 1 <211> 1518 <212> DNA <213> Artificial Sequence <220> <223> Bacillus subtilis IJK1-4 16S rDNA <400> 1 tagagtttgg aatcatggct caggacgaac gctggcggcg tgcctaatac atgcaagtcg 60 agcggacaga tgggagcttg ctccctgatg ttagcggcgg acgggtgagt aacacgtggg 120 taacctgcct gtaagactgg gataactccg ggaaaccggg gctaataccg gatggttgtc 180 tgaaccgcat ggttcagaca taaaaggtgg cttcggctac cacttacaga tggacccgcg 240 gcgcattagc tagttggtga ggtaacggct caccaaggcg acgatgcgta gccgacctga 300 gagggtgatc ggccacactg ggactgagac acggcccaga ctcctacggg aggcagcagt 360 agggaatctt ccgcaatgga cgaaagtctg acggagcaac gccgcgtgag tgatgaaggt 420 tttcggatcg taaagctctg ttgttaggga agaacaagtg ccgttcaaat agggcggcac 480 cttgacggta cctaaccaga aagccacggc taactacgtg ccagcagccg cggtaatacg 540 taggtggcaa gcgttgtccg gaattattgg gcgtaaaggg ctcgcaggcg gtttcttaag 600 tctgatgtga aagcccccgg ctcaaccggg gagggtcatt ggaaactggg gaacttgagt 660 gcagaagagg agagtggaat tccacgtgta gcggtgaaat gcgtagagat gtggaggaac 720 accagtggcg aaggcgactc tctggtctgt aactgacgct gaggagcgaa agcgtgggga 780 gcgaacagga ttagataccc tggtagtcca cgccgtaaac gatgagtgct aagtgttagg 840 gggtttccgc cccttagtgc tgcagctaac gcattaagca ctccgcctgg ggagtacggt 900 cgcaagactg aaactcaaag gaattgacgg gggcccgcac aagcggtgga gcatgtggtt 960 taattcgaag caacgcgaag aaccttacca ggtcttgaca tcctctgaca atcctagaga 1020 taggacgtcc ccttcggggg cagagtgaca ggtggtgcat ggttgtcgtc agctcgtgtc 1080 gtgagatgtt gggttaagtc ccgcaacgag cgcaaccctt gatcttagtt gccagcattc 1140 agttgggcac tctaaggtga ctgccggtga caaaccggag gaaggtgggg atgacgtcaa 1200 atcatcatgc cccttatgac ctgggctaca cacgtgctac aatggacaga acaaagggca 1260 gcgaaaccgc gaggttaagc caatcccaca aatctgttct cagttcggat cgcagtctgc 1320 aactcgactg cgtgaagctg gaatcgctag taatcgcgga tcagcatgcc gcggtgaata 1380 cgttcccggg ccttgtacac accgcccgtc acaccacgag agtttgtaac acccgaagtc 1440 ggtgaggtaa cctttatgga gccagccgcc gaaggtggga cagatgattg gggtgaagtc 1500 gtaacaaggt aaccgtaa 1518 <110> Jeju National University Industry-Academic Cooperation Foundation <120> Novel Bacillus subtilis strain IJK1-4 and uses its <130> DP20220038 <160> 1 <170> KoPatentIn 3.0 <210> 1 <211> 1518 <212> DNA <213> artificial sequence <220> <223> Bacillus subtilis IJK1-4 16S rDNA <400> 1 tagagtttgg aatcatggct caggacgaac gctggcggcg tgcctaatac atgcaagtcg 60 agcggacaga tgggagcttg ctccctgatg ttagcggcgg acgggtgagt aacacgtggg 120 taacctgcct gtaagactgg gataactccg ggaaaccggg gctaataccg gatggttgtc 180 tgaaccgcat ggttcagaca taaaaggtgg cttcggctac cacttacaga tggacccgcg 240 gcgcattagc tagttggtga ggtaacggct caccaaggcg acgatgcgta gccgacctga 300 gagggtgatc ggccacactg ggactgagac acggcccaga ctcctacggg aggcagcagt 360 agggaatctt ccgcaatgga cgaaagtctg acggagcaac gccgcgtgag tgatgaaggt 420 tttcggatcg taaagctctg ttgttaggga agaacaagtg ccgttcaaat agggcggcac 480 cttgacggta cctaaccaga aagccacggc taactacgtg ccagcagccg cggtaatacg 540 taggtggcaa gcgttgtccg gaattattgg gcgtaaaggg ctcgcaggcg gtttcttaag 600 tctgatgtga aagcccccgg ctcaaccggg gagggtcatt ggaaactggg gaacttgagt 660 gcagaagagg agagtggaat tccacgtgta gcggtgaaat gcgtagagat gtggaggaac 720 accagtggcg aaggcgactc tctggtctgt aactgacgct gaggagcgaa agcgtgggga 780 gcgaacagga ttagataccc tggtagtcca cgccgtaaac gatgagtgct aagtgttagg 840 gggtttccgc cccttagtgc tgcagctaac gcattaagca ctccgcctgg ggagtacggt 900 cgcaagactg aaactcaaag gaattgacgg gggcccgcac aagcggtgga gcatgtggtt 960 taattcgaag caacgcgaag aaccttacca ggtcttgaca tcctctgaca atcctagaga 1020 taggacgtcc ccttcggggg cagagtgaca ggtggtgcat ggttgtcgtc agctcgtgtc 1080 gtgagatgtt gggttaagtc ccgcaacgag cgcaaccctt gatcttagtt gccagcattc 1140 agttgggcac tctaaggtga ctgccggtga caaaccggag gaaggtgggg atgacgtcaa 1200 atcatcatgc cccttatgac ctgggctaca cacgtgctac aatggacaga acaaagggca 1260 gcgaaaccgc gaggttaagc caatcccaca aatctgttct cagttcggat cgcagtctgc 1320 aactcgactg cgtgaagctg gaatcgctag taatcgcgga tcagcatgcc gcggtgaata 1380 cgttcccggg ccttgtacac accgcccgtc acaccacgag agtttgtaac acccgaagtc 1440 ggtgaggtaa cctttatgga gccagccgcc gaaggtggga cagatgattg gggtgaagtc 1500 gtaacaaggt aaccgtaa 1518
Claims (6)
Novel Bacillus subtilis IJK1-4 strain containing 16S rDNA having the nucleotide sequence represented by SEQ ID NO: 1.
상기 균주는 수탁번호 KFCC11914P로 기탁된 것인, 바실러스 서브틸리스 (Bacillus subtilis) IJK1-4 균주.
According to claim 1,
The strain is deposited with accession number KFCC11914P, Bacillus subtilis ( Bacillus subtilis ) IJK1-4 strain.
상기 균주는 셀룰로오스 분해 활성을 갖는, 바실러스 서브틸리스 (Bacillus subtilis) IJK1-4 균주.
According to claim 1,
The strain has cellulolytic activity, Bacillus subtilis ( Bacillus subtilis ) IJK1-4 strain.
상기 균주는 pH 4~9에서 셀룰로오스 분해 활성을 나타내는, 바실러스 서브틸리스 (Bacillus subtilis) IJK1-4 균주.
According to claim 3,
The strain exhibits cellulolytic activity at pH 4-9, Bacillus subtilis ( Bacillus subtilis ) IJK1-4 strain.
상기 균주는 제주 토양에서 분리된 것인, 바실러스 서브틸리스 (Bacillus subtilis) IJK1-4 균주.
According to claim 1,
The strain is isolated from Jeju soil, Bacillus subtilis ( Bacillus subtilis ) IJK1-4 strain.
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