KR101571835B1 - Novel endolysin from bacteriophage SPN1S and antibiotic composition thereof - Google Patents
Novel endolysin from bacteriophage SPN1S and antibiotic composition thereof Download PDFInfo
- Publication number
- KR101571835B1 KR101571835B1 KR1020130005668A KR20130005668A KR101571835B1 KR 101571835 B1 KR101571835 B1 KR 101571835B1 KR 1020130005668 A KR1020130005668 A KR 1020130005668A KR 20130005668 A KR20130005668 A KR 20130005668A KR 101571835 B1 KR101571835 B1 KR 101571835B1
- Authority
- KR
- South Korea
- Prior art keywords
- spn1s
- bacteriophage
- endorhysin
- derived
- present
- Prior art date
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K20/00—Accessory food factors for animal feeding-stuffs
- A23K20/10—Organic substances
- A23K20/195—Antibiotics
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L27/00—Spices; Flavouring agents or condiments; Artificial sweetening agents; Table salts; Dietetic salt substitutes; Preparation or treatment thereof
- A23L27/20—Synthetic spices, flavouring agents or condiments
- A23L27/21—Synthetic spices, flavouring agents or condiments containing amino acids
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Polymers & Plastics (AREA)
- Food Science & Technology (AREA)
- Engineering & Computer Science (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Genetics & Genomics (AREA)
- Biophysics (AREA)
- Biochemistry (AREA)
- Nutrition Science (AREA)
- Molecular Biology (AREA)
- Gastroenterology & Hepatology (AREA)
- Mycology (AREA)
- Virology (AREA)
- Animal Husbandry (AREA)
- Zoology (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Food Preservation Except Freezing, Refrigeration, And Drying (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
본 발명은 박테리오파지 SPN1S 유래의 신규 엔도라이신 및 이를 포함하는 세균 사멸용 조성물에 관한 것으로, 본 발명에서 새롭게 확인한 박테리오파지 SPN1S 유래 엔도라이신은 식중독을 일으키는 원인균 중 하나인 살모넬라균 및 넓은 범위의 그람 음성균에 속하는 균주들에 대해 용균작용을 보이고, 적은 양으로 강한 항균 활성을 보이기 때문에, 식품, 의약, 사료 등에 활용될 수 있는 효과가 있다. The present invention relates to a novel endorhynin derived from bacteriophage SPN1S and a composition for killing bacteria containing the same, wherein the bacteriophage SPN1S-derived endorhysin newly identified in the present invention is a bacteriophage derived from salmonella, which is one of causative agents of food poisoning, It exhibits a lytic action against strains and exhibits a strong antimicrobial activity with a small amount, so that it can be used for food, medicines and feed.
Description
본 발명은 박테리오파지 SPN1S 유래의 신규 엔도라이신 및 이를 포함하는 세균 사멸용 조성물에 관한 것이다.
The present invention relates to a novel endorhynin derived from bacteriophage SPN1S and a composition for killing bacteria containing the same.
엔도라이신(endolysin)은 박테리오파지(bacteriophage)가 세균 세포벽의 펩티도글리칸(peptidoglycan)을 분해하기 위해 만드는 효소로서 박테리오파지의 용균 생활사 마지막 단계에 비리온(virion)을 세포 밖으로 방출시킬 때 사용된다. 엔도라이신은 낮은 농도로 세균을 빠르게 사멸시키고, 저항성 균이 잘 형성되지 않는 특징을 가지므로 최근 항생제 대체 물질로 각광받고 있다. Endolysin is an enzyme that bacteriophages make to break down peptidoglycan in bacterial cell walls and is used to release virions out of cells at the last stage of bacterial life cycle of bacteriophage. Endorylsin is rapidly emerging as a substitute for antibiotics because it rapidly kills bacteria at low concentrations and does not form resistant bacteria.
그런데, 대부분의 엔도라이신에 대한 연구는 그람 양성균에 대한 것으로 편중되어 있으며, 그람 음성균에 대한 엔도라이신에 대한 보고는 극히 드문 실정이다. 따라서, 그람 음성균에 효과적인 박테리오파지 혹은 더 많은 엔도라이신이 분리되고 그 특성 분석이 이루어져야 한다.
However, most studies on endorhynes are biased toward Gram-positive bacteria, and reports of endorhysin on Gram-negative bacteria are extremely rare. Therefore, bacteriophages or more endorhysins that are effective against Gram-negative bacteria must be isolated and characterized.
본 발명에서는 그람 음성균에 효과적인 박테리오파지 또는 그 유래의 엔도라이신을 발굴하여 제공하고자 한다.
In the present invention, bacteriophages effective for Gram-negative bacteria or endorhynin derived therefrom are to be discovered and provided.
본 발명은 서열번호 2의 아미노산 서열을 갖는 엔도라이신을 제공한다. 본 발명에서는 식품 산업에서 경제적으로 악영향을 미치는 그람 음성 병원성 미생물을 제어할 수 있고, 항생제 내성 세균을 제어할 수 있는 새로운 항균 물질을 분리하고자 하였는데, 실험 결과 박테리오파지 SPN1S(KCTC 12279BP) 유전체로부터 그람 음성 세균, 특히 대장균에 강한 사멸 효과를 나타내는 엔도라이신을 분리하고, 본 발명을 완성하였다.The present invention provides endorhysin having the amino acid sequence of SEQ ID NO: 2. In the present invention, it was attempted to isolate a new antimicrobial substance capable of controlling gram-negative pathogenic microorganisms which have an economically adverse effect on the food industry and can control the antibiotic-resistant bacteria. As a result, it was found from the experiment that bacteriophage SPN1S (KCTC 12279BP) , In particular, endorhysin exhibiting a killing effect strong against E. coli was isolated and the present invention was completed.
한편, 본 발명에 있어서, 상기 엔도라이신은, 세포외막 투과성을 높이기 위해 바람직하게 상기 엔도라이신의 C-말단에 펩타이드의 헬릭스(helix) 형태의 펩타이드가 부가된 것이 좋다. 그람 음성균의 경우, 세포외막이 엔도라이신의 도입을 방해하므로 EDTA와 같은 세포외막의 투과성을 높여주는 물질이 필요하다. 그런데, 본 발명에서와 같이 C-말단에 헬릭스 구조를 형성시키면, 세포외막 투과성이 높아져, 굳이 EDTA와 같은 물질을 부가적으로 사용할 필요가 없다. On the other hand, in the present invention, in order to enhance the permeability of the endo-lysine, it is preferable that the helix-type peptide of the peptide is added to the C-terminal of the endorhysin. In the case of Gram-negative bacteria, a substance that enhances the permeability of the extracellular membrane such as EDTA is required because the extracellular membrane interferes with the introduction of endorhynes. However, when a helical structure is formed at the C-terminal as in the present invention, the permeability of the extracellular membrane is increased, and it is not necessary to additionally use a substance such as EDTA.
본 발명 엔도라이신의 말단에 특정 아미노산 서열의 중합체(폴리펩타이드)를 결합시킬 수 있는 방법은 당업계의 유전공학적 지식을 이용하여 용이하게 할 수 있기 때문에 이에 대한 구체적인 기재는 생략하기로 한다. 다만, 일 예로는 부가하고자 하는 폴리펩타이드 서열을 암호화하는 핵산 서열을 서열번호 1의 핵산서열에 중합한 후, 발현시킴으로써 달성 가능하다. Since the method of binding a polymer (polypeptide) of a specific amino acid sequence to the end of the present invention can be facilitated by using the genetic engineering knowledge in the art, a detailed description thereof will be omitted. However, one example can be achieved by polymerizing the nucleic acid sequence encoding the polypeptide sequence to be added to the nucleic acid sequence of SEQ ID NO: 1, followed by expression.
한편, 본 발명에 있어서, 서열번호 2의 아미노산 서열은 바람직하게 서열번호 1의 핵산 서열로 암호화되는 것이 좋다.In the present invention, it is preferable that the amino acid sequence of SEQ ID NO: 2 is preferably encoded by the nucleic acid sequence of SEQ ID NO: 1.
한편, 본 발명은 상기 본 발명의 엔도라이신을 함유하는 식품 첨가제 또는 식품 조성물을 제공한다. 상기 식품 첨가제 또는 식품 조성물은 본 발명의 엔도라이신을 함유하기만 하면 그 제형에 특별히 국한되지 않으나, 대상 식품은 그람 음성균에 의해 오염되기 쉬운 식품이면 더욱 바람직하다.On the other hand, the present invention provides a food additive or a food composition containing the endorhysin of the present invention. The food additive or food composition is not particularly limited as long as it contains the endorhysin of the present invention, but it is more preferable that the subject food is a food prone to contamination by Gram-negative bacteria.
또한, 본 발명은 상기 본 발명의 엔도라이신을 함유하는 사료 첨가제 또는 사료 조성물을 제공한다. 상기 사료 첨가제 또는 사료 조성물은 본 발명의 엔도라이신을 함유하기만 하면 그 제형에 국한되지는 않으나, 그람 음성균에 감염되기 쉬운 가축을 대상으로 하는 사료 첨가제 또는 사료이면 더욱 바람직하다.
The present invention also provides a feed additive or feed composition containing the endorhysin of the present invention. The feed additive or feed composition is not limited to the formulation as long as it contains the endorhysin of the present invention, but is more preferably a feed additive or a feed which is intended for livestock that is susceptible to Gram-negative bacteria.
본 발명에서 새롭게 확인한 박테리오파지 SPN1S 유래 엔도라이신은 식중독을 일으키는 원인균 중 하나인 살모넬라균 및 넓은 범위의 그람 음성균에 속하는 균주들에 대해 용균작용을 보이고, 적은 양으로 강한 항균 활성을 보이기 때문에, 식품, 의약, 사료 등에 활용될 수 있는 효과가 있다.
Endoryline derived from bacteriophage SPN1S newly identified in the present invention shows a lytic action against strains belonging to Salmonella and a wide range of Gram-negative bacteria, which are one of the causative bacteria of food poisoning, and exhibits a strong antibacterial activity in a small amount. , Feed and the like.
도 1은 박테리오파지 SPN1S 유래 엔도라이신(서열번호 2)의 농도에 따른 대장균 사멸 효과(a) 및 라이소자임(lysozyme)과의 비교 결과(b)를 보여준다.
도 2는 박테리오파지 SPN1S 유래 엔도라이신(서열번호 2)과 EDTA의 동시 처리에 따른 대장균의 사멸 효과를 보여준다.
도 3은 여러 가지 pH(a, b)와 온도(c, d)에서, 박테리오파지 SPN1S 유래 엔도라이신(서열번호 2)의 안정성 및 항균 효과를 보이기 위한 최적 조건을 보여준다.
도 4는 박테리오파지 SPN1S 유래 엔도라이신(서열번호 2)의 처리에 따른 대장균 펩티도글리칸의 분해 정도(a) 및 환원당 생성량(b)을 보여준다.
도 5는 박테리오파지 SPN1S 유래 엔도라이신(서열번호 2)의 작용 지점을 보여준다.
도 6은 C-말단에 2차 헬릭스(helix) 구조를 가지도록 변형시킨 박테리오파지 SPN1S 유래 엔도라이신의 용균 작용을 보여준다.Brief Description of the Drawings Fig. 1 shows the effect (a) of killing an Escherichia coli according to the concentration of endor lysine (SEQ ID NO: 2) derived from bacteriophage SPN1S and the comparison result (b) with lysozyme.
Figure 2 shows the killing effect of E. coli following the simultaneous treatment of bacteriophage SPN1S-endoredysin (SEQ ID NO: 2) and EDTA.
FIG. 3 shows the optimum conditions for showing the stability and antibacterial effect of endor lysine (SEQ ID NO: 2) derived from bacteriophage SPN1S at various pH (a, b) and temperature (c, d).
Fig. 4 shows the degree of decomposition (a) and the amount of reducing sugar (b) of E. coli peptidoglycan according to the treatment of endor lysine (SEQ ID NO: 2) derived from bacteriophage SPN1S.
Figure 5 shows the point of action of endoryline (SEQ ID NO: 2) from bacteriophage SPN1S.
Fig. 6 shows the lytic action of endorhysin from the bacteriophage SPN1S modified to have a secondary helix structure at the C-terminus.
이하, 본 발명의 내용을 하기 실시예를 통해 더욱 상세히 설명하고자 한다. 다만, 본 발명의 권리범위가 하기 실시예에만 한정되는 것은 아니고, 그와 등가의 기술적 사상의 변형까지를 포함한다.
Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the scope of the present invention is not limited to the following embodiments, and includes modifications of equivalent technical ideas.
[[ 실시예Example 1: One: SPN1SSPN1S 엔도라이신의Endocrine 분리] detach]
(1) SPN1S 엔도라이신 발현벡터의 구축 및 형질전환 (1) Construction and Transformation of SPN1S Endorylase Expression Vector
자연계에서 분리한 박테리오파지 SPN1S(KCTC 12279BP)의 전체 유전체를 해독하고 이를 알려진 유전 정보와 비교하여 엔도라이신 후보 유전자(서열번호 1)를 찾았다. 이 유전자의 용균 활성을 확인하기 위해 pET15b 플라스미드와의 재조합 플라스미드를 구축하고자 하였다. 이를 위해 같은 제한 효소를 처리한 엔도라이신 유전자와 pET15b 플라스미드를 T4 리가아제를 사용하여 라이게이션(ligation) 시키고 이를 재조합 플라스미드 pET15b-lys라 명명하고 엔도라이신 발현벡터로 사용하였다. pETb-lys 발현벡터를 대장균 BL21 Star (DE3) 에 형질전환(transformation) 하고, 발현 벡터를 갖는 클론(clone)을 항생제 선택 배지에서 선별하였다.
The entire genome of the bacteriophage SPN1S (KCTC 12279BP) isolated from the natural world was decoded and compared with known genetic information to find an endorhysin candidate gene (SEQ ID NO: 1). To confirm the lytic activity of this gene, a recombinant plasmid with pET15b plasmid was constructed. For this, the endor lysine gene and the pET15b plasmid, which had been treated with the same restriction enzyme, were ligated using T4 ligase and named as a recombinant plasmid pET15b-lys and used as an endorylase expression vector. pETb-lys expression vector was transformed into E. coli BL21 Star (DE3), and a clone having an expression vector was selected on an antibiotic selection medium.
(2) 엔도라이신의 발현 및 분리 (2) Expression and isolation of endorhysin
pET15b-lys 재조합 플라스미드로 형질전환된 대장균을 LB 액체 배지에 접종 한 후 600 nm 파장에서의 흡광도가 1.0이 될 때, 최종 농도가 1 mM 이 되도록 IPTG(Isopropyl β-D-1-thiogalactopyranoside)를 첨가하여 재조합 엔도라이신의 발현을 유도하였다. 4시간 동안 더 배양한 후 원심분리하고 세포 침전물을 파쇄하여 원심분리를 한 뒤, 상등액을 Ni-NTA 레진을 사용하여 박테리오파지 SPN1S 유래 엔도라이신(서열번호 2)을 순수분리하였다.
When Escherichia coli transformed with pET15b-lys recombinant plasmid was inoculated into LB liquid medium, IPTG (Isopropyl beta-D-1-thiogalactopyranoside) was added so that the final concentration became 1 mM when the absorbance at 600 nm wavelength became 1.0 To induce the expression of recombinant endorhysin. After further incubation for 4 hours, the cells were centrifuged, and the cell precipitate was disrupted. The supernatant was centrifuged and the endor lysine (SEQ ID NO: 2) derived from bacteriophage SPN1S was purified using Ni-NTA resin.
[[ 실시예Example 2: 2: SPN1SSPN1S 유래 origin 엔도라이신의Endocrine 용균 효과 확인] Confirmation of lytic effect]
(1) SPN1S 유래 엔도라이신의 용균 효과 (1) lytic effect of endorhysin from SPN1S
0.1 M의 EDTA를 처리하여 세포 외막의 투과성을 높인 대장균에, 다양한 농도의 SPN1S 유래 엔도라이신을 넣었을 때, 버퍼만 첨가한 대조군에 비해 흡광도가 효과적으로 감소함을 확인할 수 있었다 (도 1의 a). 나노 그람 단위의 미량을 처리하였을 때에도 효과적인 용균 효과를 확인하였으며, SPN1S 유래 엔도라이신의 농도가 높아질수록 용균 효과는 점점 증가하였고, 라이소자임(lysozyme)과 비교했을 때, 약 30배씩이나 높은 용균 활성을 보였다 (도 1의 b).
It was confirmed that the absorbance was effectively reduced when E. coli treated with 0.1 M EDTA was added with various concentrations of SPN1S-derived endorhysin, compared with the control group added with buffer alone (Fig. 1 (a)). The effective lytic effect was confirmed even when the amount of nanograms was treated. The higher the concentration of endocrine lysine from SPN1S, the greater the lytic effect, and the lytic activity was about 30 times higher than that of lysozyme (Fig. 1 (b)).
(2) SPN1S 유래 엔도라이신의 세균 사멸 효과 (2) Bacterial killing effect of endolysin from SPN1S
살아있는 세균에 대한 용균 효과를 확인하기 위해 SPN1S 유래 엔도라이신과 EDTA를 동시에 처리 한 후 생균수를 측정하였다. SPN1S 유래 엔도라이신의 농도가 높을수록(도 2의 a) 그리고 세포 외막의 투과성을 높이기 위한 EDTA의 농도가 높을수록 그리고 처리 시간이 길어질수록(도 2의 b) 높은 세균 사멸 효과를 나타내었다 (도 2). 도 2의 a는 다양한 농도의 엔도라이신을 2시간 동안, 도 2의 b는 50 ng/μl 농도의 엔도라이신을 다양한 시간 동안 처리한 결과이다.
To confirm the lytic effect on living bacteria, the number of viable cells was measured after treatment of endolysin and EDTA from SPN1S at the same time. As shown in FIG. 2 (a), the concentration of EDTA derived from SPN1S was higher (FIG. 2), and the higher the concentration of EDTA and the longer treatment time (FIG. 2 b) 2). Fig. 2 (a) shows the result of treatment of various concentrations of endorhysin for 2 hours, and Fig. 2 (b) shows the results of treating endorhynes at a concentration of 50 ng /
[[ 실시예Example 3: 3: SPN1SSPN1S 유래 origin 엔도라이신의Endocrine 특성 분석] Characteristic Analysis]
(1) SPN1S 유래 엔도라이신의 용균 작용을 위한 최적 조건 (1) Optimal conditions for the lytic action of endorhysin from SPN1S
SPN1S 유래 엔도라이신의 안정성을 확인하기 위해, 여러 가지 pH와 온도에서 SPN1S 유래 엔도라이신을 일정 시간 보관한 후 남아 있는 용균 활성을 측정하였다. 그 결과, pH 2를 제외한 대부분의 pH (pH 4.3~pH 10.5)에서 용균활성이 유지됨을 확인할 수 있었다 (도 3의 a). 또한, 다양한 온도에서 30분 보관하였을 때 비교적 높은 열 안정성을 보임을 확인할 수 있었다. 다만, 보관 온도가 높아질수록 용균 활성이 감소하였다 (도 3의 b). To confirm the stability of endorhysin from SPN1S, endolysin from SPN1S was stored for several hours at various pH and temperature, and the remaining lytic activity was measured. As a result, it was confirmed that the lytic activity was maintained in most of the pH (pH 4.3 to pH 10.5) except pH 2 (Fig. 3 (a)). In addition, it was confirmed that when stored at various temperatures for 30 minutes, the thermal stability was relatively high. However, as the storage temperature increased, the lytic activity decreased (FIG. 3b).
한편, 여러 가지 pH 또는 온도에서 항균 활성을 측정하여 보았는데, 중성 및 알칼리 범위의 pH (pH 7.0~pH 10.5)에서 항균 활성이 지속되었으며, 37℃에서 최적 활성을 보였다 (도 3의 c, d).
On the other hand, the antimicrobial activity was measured at various pHs or temperatures, and the antimicrobial activity persisted at a pH ranging from neutral to alkaline (pH 7.0 to pH 10.5) and showed optimal activity at 37 ° C (c, d in FIG. 3) .
(2) SPN1S 유래 엔도라이신의 항균 활성 범위 (2) Antimicrobial activity range of endorhysin from SPN1S
다양한 세균에 대한 SPN1S 유래 엔도라이신의 감수성을 확인하였다. 그람 음성균은 EDTA를 전처리하여 세포 외막의 투과성을 높여 사용하였다. 그 결과, 표 1에 나타난 것처럼 SPN1S 유래 엔도라이신은 대장균, 살모넬라균을 비롯한 실험에 사용된 모든 그람 음성균에 대하여 항균 효과를 보였으며 특히 대장균에 대하여 강한 활성을 보였다. 다만, 그람 양성균은 사멸시키지 못하였다. The susceptibility of SPN1S-derived endorhysin to various bacteria was confirmed. Gram - negative bacteria were pretreated with EDTA to increase the permeability of the extracellular membrane. As a result, as shown in Table 1, endorhyns derived from SPN1S showed antimicrobial activity against all Gram-negative bacteria used in the experiments including Escherichia coli, Salmonella, and especially Escherichia coli. However, Gram positive bacteria could not be killed.
따라서, 본 발명의 SPN1S 유래 엔도라이신은 그람 음성균에 대한 넒은 범위의 항균 활성 범위를 가진다고 할 수 있었다. Therefore, it can be said that the endorhysin derived from SPN1S of the present invention has a wide range of antimicrobial activity against Gram-negative bacteria.
[표 1][Table 1]
본 발명 SPN1S 엔도라이신의 항균 범위The antimicrobial range of the present invention SPN1S endorhysin
(3) SPN1S 유래 엔도라이신의 작용 지점 확인(3) Confirmation of the action point of endorhysin from SPN1S
세균의 펩티도글리칸에서 SPN1S 유래 엔도라이신이 작용하는 지점을 확인하기 위해 대장균의 펩티도글리칸을 분리하였다. 분리된 펩티도글리칸(도 4에서 PG로 표시)의 흡광도가 SPN1S 유래 엔도라이신(도 4에서 'end'로 표시)의 처리에 따라 감소하였는데 (도 4의 a), 이를 통해 SPN1S 유래 엔도라이신이 세균의 펩티도글리칸을 자르는 효소임을 다시 한 번 확인할 수 있었다. The peptidoglycan of E. coli was isolated in order to confirm the point where endolysin from SPN1S acts in the peptidoglycan of bacteria. The absorbance of the separated peptidoglycan (denoted PG in FIG. 4) decreased with the treatment of endolysin from SPN1S (indicated as 'end' in FIG. 4) (FIG. 4 a) It was once again confirmed that this enzyme is an enzyme that cleaves peptidoglycan of this bacterium.
또한, 펩티도글리칸의 분해물에서 환원당의 양이 증가하였으므로 (도 4의 b), SPN1S 유래 엔도라이신이 대장균 펩티도글리칸의 당 결합을 자르는 효소임을 확인할 수 있었다. In addition, since the amount of reducing sugar in the digest of the peptidoglycan was increased (Fig. 4 (b)), it was confirmed that the endor lysine derived from SPN1S is an enzyme that cleaves the sugar bond of the E. coli peptidoglycan.
또한, 무라미다아제(muramidase)의 기질인 '4-Methylumbelliferyl β-D-N,N′,N″-triacetylchitotrioside'와 글루코사미니다아제(glucosaminidase)의 기질인 '4-Nitrophenyl N-acetyl-β-D-glucosaminide'에, SPN1S 유래의 엔도라이신을 처리하였는데, SPN1S 유래 엔도라이신은 라이소자임과 같이 대장균 펩티도글리칸의 'N-acetyl muramic acid'와 'N-acetyl glucosamic acid' 사이의 글리코사이드(glycoside) 결합을 자르는 무라미다아제 활성을 가짐을 확인할 수 있었다 (도 5의 a). 또한, SPN1S 유래 엔도라이신(도 5에서 'end'로 표시)의 활성이 라이소자임(도 5에서 'lys'로 표시)보다 약 30배 높음을 재확인할 수 있었다. In addition, '4-Methylumbelliferyl β-DN, N', N "-triacetylchitotrioside 'which is a substrate of muramidase and' 4-Nitrophenyl N-acetyl-β-D-glucosaminidase ', which is a substrate of glucosaminidase, endosylate from SPN1S was treated with glucosaminide, and endorhysin from SPN1S was treated with glycoside bond between 'N-acetyl muramic acid' and 'N-acetyl glucosamic acid' of Escherichia coli peptidoglycan such as lysozyme (Fig. 5 (a)). ≪ tb > < TABLE > In addition, it was confirmed that the activity of endolysin (indicated as 'end' in FIG. 5) derived from SPN1S was about 30 times higher than that of lysozyme (labeled 'lys' in FIG. 5).
한편, SPN1S 유래 엔도라이신의 작용 지점은 도 5의 b에 도식화하였다.
On the other hand, the point of action of the endocrine derived from SPN1S is illustrated in Fig.
[[ 실시예Example 4: 4: SPN1SSPN1S 엔도라이신의Endocrine 개량] improvement]
박테리오파지의 엔도라이신을 그람 음성균에 처리할 때에는 일반적으로 그람 음성균의 세포 외막이 엔도라이신의 도입을 방해하므로, EDTA와 같이 세포 외막의 투과성을 높여주는 물질이 필요하다. When treating endorphins of bacteriophage with Gram-negative bacteria, the extracellular membranes of Gram-negative bacteria generally interfere with the introduction of endorhynes, and substances such as EDTA that enhance the permeability of the extracellular membrane are needed.
본 실시예에서는 SPN1S 유래 엔도라이신의 세포 외막 투과성을 높이기 위해 SPN1S 엔도라이신 C-말단이 헬릭스(helix) 구조를 갖도록 C-말단에 몇 개의 염기서열(CTTACTAAAA TCGCTAAAAA G)을 붙였으며, 실시예 1과 같은 방법으로 개량된 SPN1S 유래 엔도라이신을 순수분리하였다. In this Example, several base sequences (CTTACTAAAA TCGCTAAAAA G) were attached to the C-terminus such that the C-terminus of SPN1S endorillac had a helix structure in order to enhance the extracellular permeability of endorhysin from SPN1S. In the same manner, the purified endocrine lysine from SPN1S was purified.
개량된 엔도라이신(도 6에서 'end-2'로 표시)은 세포 외막의 투과성을 높이지 않은 조건(EDTA가 없는 조건)에서도 세균을 용균시키는 활성을 보였으며, EDTA로 세포외막의 투과성을 높였을 때에는 개량 전의 SPN1S 엔도라이신(도 6에서 'end'로 표시)보다 훨씬 큰 항균 활성을 보였다 (도 6).
The improved endolysin (labeled "end-2" in FIG. 6) showed the activity of lysing bacteria even under the condition of not increasing the permeability of the extracellular membrane (condition without EDTA), and increasing the permeability of the extracellular membrane with EDTA , The antimicrobial activity was much greater than that of SPN1S endoryline (indicated by 'end' in FIG. 6) before the improvement (FIG. 6).
<110> SNU R&DB FOUNDATION <120> Novel endolysin from bacteriophage SPN1S and antibiotic composition thereof <130> AP-2013-0001 <160> 2 <170> KopatentIn 2.0 <210> 1 <211> 630 <212> DNA <213> Bacteriophage SPN 1S (KCTC 12279BP) <400> 1 atggacatta accagttccg gcgcgcatcc ggtatcaatg agcaactggc cgcgcgctgg 60 tttccgcata ttactactgc catgaatgag tttggcatta ccaaaccaga tgaccaggca 120 atgtttatcg cacaggtcgg gcatgagtcc ggaggattta cccggttaca ggaaaacttt 180 aactacagcg ttaacggact gtccgggttt atccgtgccg ggcgcatcac tccagaccag 240 gccaacgcac ttggccggaa aacatatgag aagtctcttc ctctggaacg ccagcgcgcg 300 atcgccaatc ttgtttacag caagcgcatg ggaaataacg gcccgggcga cgggtggaat 360 tatcgcggtc gtggacttat ccagatcact ggtctgaaca actaccggga ttgcggtaat 420 ggtctgaagg ttgatctagt cgctcagcct gaactgctgg cgcaggatga atacgcggcc 480 agaagcgcgg catggttctt ctccagtaaa ggttgcatga agtacaccgg tgacctggtg 540 cgcgtcacgc agattatcaa tggtggccag aacggtatcg acgaccggcg tacgcgttac 600 gccgctgccc gtaaggtgct ggcgttatga 630 <210> 2 <211> 209 <212> PRT <213> Bacteriophage SPN 1S (KCTC 12279BP) <400> 2 Met Asp Ile Asn Gln Phe Arg Arg Ala Ser Gly Ile Asn Glu Gln Leu 1 5 10 15 Ala Ala Arg Trp Phe Pro His Ile Thr Thr Ala Met Asn Glu Phe Gly 20 25 30 Ile Thr Lys Pro Asp Asp Gln Ala Met Phe Ile Ala Gln Val Gly His 35 40 45 Glu Ser Gly Gly Phe Thr Arg Leu Gln Glu Asn Phe Asn Tyr Ser Val 50 55 60 Asn Gly Leu Ser Gly Phe Ile Arg Ala Gly Arg Ile Thr Pro Asp Gln 65 70 75 80 Ala Asn Ala Leu Gly Arg Lys Thr Tyr Glu Lys Ser Leu Pro Leu Glu 85 90 95 Arg Gln Arg Ala Ile Ala Asn Leu Val Tyr Ser Lys Arg Met Gly Asn 100 105 110 Asn Gly Pro Gly Asp Gly Trp Asn Tyr Arg Gly Arg Gly Leu Ile Gln 115 120 125 Ile Thr Gly Leu Asn Asn Tyr Arg Asp Cys Gly Asn Gly Leu Lys Val 130 135 140 Asp Leu Val Ala Gln Pro Glu Leu Leu Ala Gln Asp Glu Tyr Ala Ala 145 150 155 160 Arg Ser Ala Ala Trp Phe Phe Ser Ser Lys Gly Cys Met Lys Tyr Thr 165 170 175 Gly Asp Leu Val Arg Val Thr Gln Ile Ile Asn Gly Gly Gln Asn Gly 180 185 190 Ile Asp Asp Arg Arg Thr Arg Tyr Ala Ala Ala Arg Lys Val Leu Ala 195 200 205 Leu <110> SNU R & DB FOUNDATION <120> Novel endolysin from bacteriophage SPN1S and antibiotic composition thereof <130> AP-2013-0001 <160> 2 <170> Kopatentin 2.0 <210> 1 <211> 630 <212> DNA <213> Bacteriophage SPN 1S (KCTC 12279BP) <400> 1 atggacatta accagttccg gcgcgcatcc ggtatcaatg agcaactggc cgcgcgctgg 60 tttccgcata ttactactgc catgaatgag tttggcatta ccaaaccaga tgaccaggca 120 atgtttatcg cacaggtcgg gcatgagtcc ggaggattta cccggttaca ggaaaacttt 180 aactacagcg ttaacggact gtccgggttt atccgtgccg ggcgcatcac tccagaccag 240 gccaacgcac ttggccggaa aacatatgag aagtctcttc ctctggaacg ccagcgcgcg 300 atcgccaatc ttgtttacag caagcgcatg ggaaataacg gcccgggcga cgggtggaat 360 tatcgcggtc gtggacttat ccagatcact ggtctgaaca actaccggga ttgcggtaat 420 ggtctgaagg ttgatctagt cgctcagcct gaactgctgg cgcaggatga atacgcggcc 480 agaagcgcgg catggttctt ctccagtaaa ggttgcatga agtacaccgg tgacctggtg 540 cgcgtcacgc agattatcaa tggtggccag aacggtatcg acgaccggcg tacgcgttac 600 gccgctgccc gtaaggtgct ggcgttatga 630 <210> 2 <211> 209 <212> PRT <213> Bacteriophage SPN 1S (KCTC 12279BP) <400> 2 Met Asp Ile Asn Gln Phe Arg Arg Ala Ser Gly Ile Asn Glu Gln Leu 1 5 10 15 Ala Ala Arg Trp Phe Pro His Ile Thr Thr Ala Met Asn Glu Phe Gly 20 25 30 Ile Thr Lys Pro Asp Asp Gln Ala Met Phe Ile Ala Gln Val Gly His 35 40 45 Glu Ser Gly Gly Phe Thr Arg Leu Gln Glu Asn Phe Asn Tyr Ser Val 50 55 60 Asn Gly Leu Ser Gly Phe Ile Arg Ala Gly Arg Ile Thr Pro Asp Gln 65 70 75 80 Ala Asn Ala Leu Gly Arg Lys Thr Tyr Glu Lys Ser Leu Pro Leu Glu 85 90 95 Arg Gln Arg Ala Ile Ala Asn Leu Val Tyr Ser Lys Arg Met Gly Asn 100 105 110 Asn Gly Pro Gly Asp Gly Trp Asn Tyr Arg Gly Arg Gly Leu Ile Gln 115 120 125 Ile Thr Gly Leu Asn Asn Tyr Arg Asp Cys Gly Asn Gly Leu Lys Val 130 135 140 Asp Leu Val Ala Gln Pro Glu Leu Leu Ala Gln Asp Glu Tyr Ala Ala 145 150 155 160 Arg Ser Ala Ala Trp Phe Phe Ser Ser Lys Gly Cys Met Lys Tyr Thr 165 170 175 Gly Asp Leu Val Arg Val Thr Gln Ile Ile Asn Gly Gly Gln Asn Gly 180 185 190 Ile Asp Asp Arg Arg Thr Arg Tyr Ala Ala Ala Arg Lys Val Leu Ala 195 200 205 Leu
Claims (7)
Endoryline having a peptide of the form of helix added to enhance the extracellular permeability at the C-terminal of the amino acid sequence of SEQ ID NO: 2.
상기 서열번호 2의 아미노산 서열은,
서열번호 1의 핵산 서열로 암호화되는 것을 특징으로 하는 엔도라이신.
3. The method of claim 2,
The amino acid sequence of SEQ ID NO:
Characterized in that it is encoded by the nucleic acid sequence of SEQ ID NO: 1.
A food additive containing endorhysin of claim 2.
A food composition comprising the endorhysin of claim 2.
A feed additive comprising endorhysin of claim 2.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| KR1020130005668A KR101571835B1 (en) | 2013-01-18 | 2013-01-18 | Novel endolysin from bacteriophage SPN1S and antibiotic composition thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| KR1020130005668A KR101571835B1 (en) | 2013-01-18 | 2013-01-18 | Novel endolysin from bacteriophage SPN1S and antibiotic composition thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| KR20140093403A KR20140093403A (en) | 2014-07-28 |
| KR101571835B1 true KR101571835B1 (en) | 2015-11-25 |
Family
ID=51739600
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| KR1020130005668A KR101571835B1 (en) | 2013-01-18 | 2013-01-18 | Novel endolysin from bacteriophage SPN1S and antibiotic composition thereof |
Country Status (1)
| Country | Link |
|---|---|
| KR (1) | KR101571835B1 (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2021235876A1 (en) | 2020-05-22 | 2021-11-25 | 주식회사 라이센텍 | Novel polypeptide, fusion polypeptide, and antibiotic against gram-negative bacteria comprising same |
| US20220387563A1 (en) * | 2021-04-19 | 2022-12-08 | Lysentech Co., Ltd. | Novel polypeptides and antibiotics against Gram-negative bacterium comprising the same |
| KR20230027467A (en) | 2021-08-19 | 2023-02-28 | 박선규 | Pharmaceutical composition for antibiotics comprising bacteriophage endolysin and plant extract |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR101200333B1 (en) * | 2011-12-05 | 2012-11-12 | 서울대학교산학협력단 | Thermal stable endolysin LysBPS13, food composition and feed composition thereof |
-
2013
- 2013-01-18 KR KR1020130005668A patent/KR101571835B1/en active IP Right Grant
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR101200333B1 (en) * | 2011-12-05 | 2012-11-12 | 서울대학교산학협력단 | Thermal stable endolysin LysBPS13, food composition and feed composition thereof |
Non-Patent Citations (3)
| Title |
|---|
| Appl. Microbiol. Biotechnol. (2011) 90, 529-539. |
| LIM, J. "Characterization of Endolysin from Salmonella Typhimurium-infecting Bacteriophage for Bio-control agents" 서울대학교 박사학위 논문(공개일: 2012. 8.).* |
| NCBI GenBank Locus No. AEX26904(공개일: 2012. 1. 10.).* |
Also Published As
| Publication number | Publication date |
|---|---|
| KR20140093403A (en) | 2014-07-28 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Lim et al. | Characterization of endolysin from a Salmonella Typhimurium-infecting bacteriophage SPN1S | |
| CN102482655B (en) | Antimicrobial | |
| US20120244595A1 (en) | Artificial peptidoglycan lysing enzymes and peptidoglycan binding proteins | |
| US10174300B2 (en) | Polypeptide | |
| WO2010011960A2 (en) | Triple acting antimicrobials that are refractory to resistance development | |
| Wang et al. | The Vibrio parahaemolyticus-infecting bacteriophage qdvp001: genome sequence and endolysin with a modular structure | |
| KR101796279B1 (en) | Endolysin with novel host binding domain from Staphylococcus aureus bacteriophage SA11 and antibiotic composition thereof | |
| CN110036105B (en) | Modified peptides | |
| US8790639B2 (en) | Enhanced antimicrobial lytic activity of a chimeric Ply187 endolysin | |
| CN108531469B (en) | Bacillus cereus bacteriophage lyase and preparation method and application thereof | |
| KR101571835B1 (en) | Novel endolysin from bacteriophage SPN1S and antibiotic composition thereof | |
| US9789167B2 (en) | Polypeptide mixes with antibacterial activity | |
| US20120171188A1 (en) | Artificial Peptidoglycan Lysing Enzymes and Peptidoglycan Binding Proteins | |
| EP2582813B1 (en) | Methods of generating and screening for lytic chimeric polypeptides | |
| US20150247138A1 (en) | Enhanced Staphylolytic Activity of the Staphylococcus aureus Bacteriophage vB_SauS-philPLA88 Virion-Associated Peptidoglycan Hydrolase HydH5: Fusions, Deletions and Synergy with LysH5 | |
| Sanz et al. | Structural studies of the lysozyme coded by the pneumococcal phage Cp‐1: Conformational changes induced by choline | |
| WO2010003943A1 (en) | New enzymatic active enzyme against clostridium | |
| KR101177358B1 (en) | Endolysin lysb4 with peptidase activity, food composition and feed composition thereof | |
| EP3186370B1 (en) | Enzyme with high lytic activity against enterococcus cell and a method of modification of the gene thereof, enabling overproduction of active enzyme in bacterial cells | |
| CN114908077A (en) | Phage lyase, chimeric substance and application thereof | |
| Ram et al. | Cost effective purification of intein based syntetic cationic antimicrobial peptide expressed in cold shock expression system using salt inducible E. coli GJ1158 | |
| Malinicova et al. | Peptidoglycan hydrolase enterolysin a recognizes lipoteichoic acid chains in the cell walls of sensitive bacteria | |
| Sunthornthummas et al. | Genomic characterisation of Lacticaseibacillus paracasei phage ΦT25 and preliminary analysis of its derived endolysin | |
| Jarábková et al. | SH3 binding domains from phage endolysins: How to use them for detection of Gram-positive pathogens | |
| Wang et al. | Identification and characterization of the two-component cell lysis cassette encoded by temperate bacteriophage uPYB5 of Lactobacillus fermentum |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| A201 | Request for examination | ||
| E902 | Notification of reason for refusal | ||
| E90F | Notification of reason for final refusal | ||
| E701 | Decision to grant or registration of patent right | ||
| GRNT | Written decision to grant | ||
| FPAY | Annual fee payment |
Payment date: 20181101 Year of fee payment: 4 |