KR20230060470A - Novel Lactobacillus plantarum D2-3 strain and use thereof - Google Patents
Novel Lactobacillus plantarum D2-3 strain and use thereof Download PDFInfo
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- KR20230060470A KR20230060470A KR1020220138143A KR20220138143A KR20230060470A KR 20230060470 A KR20230060470 A KR 20230060470A KR 1020220138143 A KR1020220138143 A KR 1020220138143A KR 20220138143 A KR20220138143 A KR 20220138143A KR 20230060470 A KR20230060470 A KR 20230060470A
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- tannin
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- lactobacillus plantarum
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Abstract
Description
본 발명은 신규한 락토바실러스 플란타럼(Lactobacillus plantarum) D2-3 균주 및 이의 용도에 관한 것이다.The present invention relates to a novel Lactobacillus plantarum D2-3 strain and its use.
탄닌(tannin)은 천연에 존재하는 단백질을 수용액으로부터 침전시킬 수 있는 수용성 페놀계 생성물이다. 탄닌은 크게 묽은 산과 가열하면 가수분해되는 가수분해성 탄닌 및 가수분해형 탄닌과 중합하여 물에 불용성인 phlobaphene을 형성하는 축합형 탄닌으로 구분할 수 있다. 탄닌은 많은 미생물의 증식을 억제하고, 미생물의 공격에 내성을 가진다. 일반적으로 곰팡이, 효모 및 일부 호기성 박테리아가 탄닌 분해에 가장 적합하나, 일부 혐기성 박테리아에 의한 탄닌 분해도 보고된 바 있다. Tannins are water-soluble phenolic products that can precipitate naturally occurring proteins from aqueous solutions. Tannins can be classified into hydrolysable tannins that are hydrolyzed when heated with dilute acids and condensed tannins that polymerize with hydrolyzed tannins to form water-insoluble phlobaphene. Tannins inhibit the growth of many microorganisms and have resistance to microbial attack. In general, molds, yeasts, and some aerobic bacteria are most suitable for tannin degradation, but tannin degradation by some anaerobic bacteria has also been reported.
탄닌은 영양성분 및 미네랄 흡수를 방해하는 항영양소로도 알려져 있으나, 건강에 이로운 효과도 다수 보고되었다. 대표적으로, 항산화 활성이 우수하여 염증을 억제하여 혈관, 심장 및 암 질환 개선 효과가 있고, 균류, 효모, 박테리아 및 바이러스의 성장을 억제하며, 이외에도 요로 감염, 피부 질환, 당뇨 등에 대한 개선 효과를 나타낸다. 따라서, 탄닌의 적절한 섭취는 인간 건강에 유익할 수 있다.Tannins are also known as anti-nutrients that hinder absorption of nutrients and minerals, but many beneficial effects on health have also been reported. Representatively, it has excellent antioxidant activity, suppresses inflammation, improves blood vessel, heart and cancer diseases, inhibits the growth of fungi, yeast, bacteria and viruses, and shows improvement effects on urinary tract infections, skin diseases, diabetes, etc. . Therefore, adequate intake of tannins can be beneficial to human health.
본 발명의 목적은 기탁번호 KCTC 15134BP로 기탁된 락토바실러스 플란타럼(Lactobacillus plantarum) D2-3 균주를 제공하는 것이다.An object of the present invention is to provide a Lactobacillus plantarum D2-3 strain deposited under accession number KCTC 15134BP.
본 발명의 다른 목적은 탄닌 화합물에 기탁번호 KCTC 15134BP로 기탁된 락토바실러스 플란타럼(Lactobacillus plantarum) D2-3 균주 추출물 또는 이의 배양액을 처리하여 탄닌 가수분해물을 수득하는 단계를 포함하는, 탄닌 가수분해방법을 제공하는 것이다.Another object of the present invention is tannin hydrolysis comprising the step of obtaining a tannin hydrolyzate by treating a Lactobacillus plantarum D2-3 strain extract or a culture thereof deposited with accession number KCTC 15134BP in a tannin compound. is to provide a way
본 발명의 또 다른 목적은 상기 탄닌 가수분해방법을 통해 수득한 탄닌 가수분해물을 유효성분으로 포함하는, SOD1(superoxide dismutase 1) G93A 돌연변이 단백질 필라멘트 형성 억제용 조성물을 제공하는 것이다.Another object of the present invention is to provide a composition for inhibiting filament formation of SOD1 (superoxide dismutase 1) G93A mutant protein, containing tannin hydrolyzate obtained through the tannin hydrolysis method as an active ingredient.
본 발명의 또 다른 목적은 EGCG(epigallocatechin gallte), 갈릭산(gallic acid), 탄닌산(tannic acid) 및 엘라그산(ellagic acid)으로 이루어진 군에서 선택된 하나 이상의 탄닌 화합물을 유효성분으로 포함하는, SOD1(superoxide dismutase 1) G93A 돌연변이 단백질 필라멘트 형성 억제용 조성물을 제공하는 것이다.Another object of the present invention is SOD1, comprising at least one tannin compound selected from the group consisting of epigallocatechin gallte (EGCG), gallic acid, tannic acid and ellagic acid as an active ingredient ( superoxide dismutase 1) To provide a composition for suppressing filament formation of G93A mutant protein.
본 발명의 또 다른 목적은 EGCG(epigallocatechin gallte), 갈릭산(gallic acid), 탄닌산(tannic acid) 및 엘라그산(ellagic acid)으로 이루어진 군에서 선택된 하나 이상의 탄닌 화합물에 락토바실러스 플란타럼(Lactobacillus plantarum) 균주를 첨가하고 공동배양한 배양액을 유효성분으로 포함하는, SOD1(superoxide dismutase 1) G93A 돌연변이 단백질 필라멘트 형성 억제용 조성물을 제공하는 것이다.Another object of the present invention is to add one or more tannin compounds selected from the group consisting of epigallocatechin gallte (EGCG), gallic acid, tannic acid and ellagic acid to Lactobacillus plantarum ( Lactobacillus plantarum ). ) to provide a composition for inhibiting SOD1 (superoxide dismutase 1) G93A mutant protein filament formation, comprising a culture solution co-cultured with the addition of the strain as an active ingredient.
상기 목적을 달성하기 위해, 본 발명은 기탁번호 KCTC 15134BP로 기탁된 락토바실러스 플란타럼(Lactobacillus plantarum) D2-3 균주를 제공한다.In order to achieve the above object, the present invention provides a Lactobacillus plantarum D2-3 strain deposited with accession number KCTC 15134BP.
또한, 본 발명은 탄닌 화합물에 기탁번호 KCTC 15134BP로 기탁된 락토바실러스 플란타럼(Lactobacillus plantarum) D2-3 균주 추출물 또는 이의 배양액을 처리하여 탄닌 가수분해물을 수득하는 단계를 포함하는, 탄닌 가수분해방법을 제공한다.In addition, the present invention is a tannin hydrolysis method comprising the step of obtaining a tannin hydrolyzate by treating a Lactobacillus plantarum D2-3 strain extract or a culture thereof deposited with accession number KCTC 15134BP in a tannin compound. provides
또한, 본 발명은 상기 탄닌 가수분해방법을 통해 수득한 탄닌 가수분해물을 유효성분으로 포함하는, SOD1(superoxide dismutase 1) G93A 돌연변이 단백질 필라멘트 형성 억제용 조성물을 제공한다.In addition, the present invention provides a composition for inhibiting filament formation of SOD1 (superoxide dismutase 1) G93A mutant protein, comprising tannin hydrolyzate obtained through the tannin hydrolysis method as an active ingredient.
또한, 본 발명은 EGCG(epigallocatechin gallte), 갈릭산(gallic acid), 탄닌산(tannic acid) 및 엘라그산(ellagic acid)으로 이루어진 군에서 선택된 하나 이상의 탄닌 화합물을 유효성분으로 포함하는, SOD1(superoxide dismutase 1) G93A 돌연변이 단백질 필라멘트 형성 억제용 조성물을 제공한다.In addition, the present invention is a superoxide dismutase (SOD1) containing at least one tannin compound selected from the group consisting of epigallocatechin gallte (EGCG), gallic acid, tannic acid and ellagic acid as an active ingredient. 1) Provided is a composition for inhibiting G93A mutant protein filament formation.
또한, 본 발명은 EGCG(epigallocatechin gallte), 갈릭산(gallic acid), 탄닌산(tannic acid) 및 엘라그산(ellagic acid)으로 이루어진 군에서 선택된 하나 이상의 탄닌 화합물에 락토바실러스 플란타럼(Lactobacillus plantarum) 균주를 첨가하고 공동배양한 배양액을 유효성분으로 포함하는, SOD1(superoxide dismutase 1) G93A 돌연변이 단백질 필라멘트 형성 억제용 조성물을 제공한다.In addition, the present invention relates to one or more tannin compounds selected from the group consisting of epigallocatechin gallte (EGCG), gallic acid, tannic acid and ellagic acid, Lactobacillus plantarum strain It provides a composition for inhibiting SOD1 (superoxide dismutase 1) G93A mutant protein filament formation, containing the co-cultured culture medium as an active ingredient.
본 발명에 따르면, 탄닌 화합물에 신규한 락토바실러스 플란타럼(Lactobacillus plantarum) D2-3 균주를 처리하여 수득한 탄닌 가수분해물이 SOD1(superoxide dismutase 1) G93A 돌연변이 단백질 필라멘트 형성을 효과적으로 억제하는 것을 확인함으로써, SOD1 G93A 돌연변이 단백질 필라멘트 형성 억제용 조성물로서 유용하게 활용될 수 있다.According to the present invention, by confirming that the tannin hydrolyzate obtained by treating the novel Lactobacillus plantarum D2-3 strain with a tannin compound effectively inhibits SOD1 (superoxide dismutase 1) G93A mutant protein filament formation , SOD1 G93A mutant protein can be usefully utilized as a composition for inhibiting filament formation.
도 1은 락토바실러스 플란타럼 D2-3 균주의 16S rRNA 시퀀싱 결과이다.
도 2는 락토바실러스 플란타럼 D2-3 균주를 포함한 9개의 락토바실러스 플란타럼 균주의 탄네이즈 발현량을 비교 및 분석한 결과이다.
도 3은 락토바실러스 플란타럼 D2-3 균주의 탄네이즈 효소의 PCR 산물을 전기영동으로 분석한 결과이다.
도 4는 pET15b 벡터로의 락토바실러스 플란타럼 D2-3 탄네이즈 유전자 클로닝 결과를 전기영동으로 분석한 결과이다.
도 5는 락토바실러스 플란타럼 D2-3 균주의 탄네이즈 유전자 클로닝 염기서열을 분석한 결과이다.
도 6은 락토바실러스 플란타럼 D2-3 균주의 탄네이즈 효소를 대량발현시켜 SDS-PAGE(Sodium dodecyl-sulfate polyacrylamide gel electrophoresis)로 분석한 결과이다.
도 7은 정제된 탄네이즈 효소를 SDS-PAGE(Sodium dodecyl-sulfate polyacrylamide gel electrophoresis)로 분석한 결과이다.
도 8은 정제된 탄네이즈 효소를 MALDI-TOF MS(Matrix-assisted laser desorption ionization time-of-flight mass spectrometry)로 분석한 결과이다.
도 9는 정제된 락토바실러스 플란타럼 D2-3 균주의 탄네이즈 효소의 탄닌산 분해능을 클리어 존(clear zone)을 통해 분석한 결과이다.
도 10은 정제된 락토바실러스 플란타럼 D2-3 균주의 탄네이즈 효소에 의한 EGCG 분해물을 HPLC를 통해 분석한 결과이다.
도 11은 탄닌계 천연물에 의한 SOD1(superoxide dismutase 1) 야생형 단백질의 필라멘트 형성 저해 활성을 Thioflavin T assay(ThT)로 분석한 결과이다.
도 12는 탄닌계 천연물에 의한 SOD1 G93A 돌연변이 단백질의 필라멘트 형성 저해 활성을 Thioflavin T assay로 분석한 결과이다.
도 13은 탄닌계 천연물에 의한 SOD1 단백질의 필라멘트 형성 저해 활성을 Negative-staining 전자현미경(electron microscopy; EM)으로 분석한 결과이다.
도 14는 탄닌계 천연물에 의한 SOD1 G93A 돌연변이 단백질의 필라멘트 형성 저해 활성을 SK-N-SH 신경세포에서 분석한 결과이다.
도 15는 락토바실러스 균주들과 탄닌계 천연물의 배양 산물에 의한 SOD1 G93A 돌연변이 단백질의 필라멘트 형성 저해 활성을 Thioflavin T assay로 분석한 결과이다.1 is a 16S rRNA sequencing result of Lactobacillus plantarum D2-3 strain.
Figure 2 is a result of comparing and analyzing tannaise expression levels of nine Lactobacillus plantarum strains, including the Lactobacillus plantarum D2-3 strain.
Figure 3 is a result of analyzing the PCR product of the tannaise enzyme of the Lactobacillus plantarum D2-3 strain by electrophoresis.
FIG. 4 shows the result of cloning the Lactobacillus plantarum D2-3 tannaise gene into the pET15b vector by electrophoresis.
5 is a result of analyzing the nucleotide sequence of the tannaise gene cloning of the Lactobacillus plantarum D2-3 strain.
6 is a result of mass expression of tannase enzyme of Lactobacillus plantarum D2-3 strain and analysis by SDS-PAGE (sodium dodecyl-sulfate polyacrylamide gel electrophoresis).
7 is a result of analyzing the purified tannaise enzyme by SDS-PAGE (sodium dodecyl-sulfate polyacrylamide gel electrophoresis).
8 is a result of analyzing purified tannaise enzyme by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS).
9 is a result of analyzing the tannic acid degradation ability of the tannase enzyme of the purified Lactobacillus plantarum D2-3 strain through a clear zone.
10 is a result of HPLC analysis of EGCG decomposition products by the tannaise enzyme of the purified Lactobacillus plantarum D2-3 strain.
11 is a result of analyzing the filament formation inhibitory activity of SOD1 (superoxide dismutase 1) wild-type protein by a tannin-based natural product by Thioflavin T assay (ThT).
12 is a result of analyzing the filament formation inhibitory activity of the SOD1 G93A mutant protein by a tannin-based natural product by Thioflavin T assay.
13 is a result of analyzing the filament formation inhibitory activity of SOD1 protein by a tannin-based natural product with a negative-staining electron microscope (electron microscopy; EM).
14 is a result of analyzing the filament formation inhibitory activity of the SOD1 G93A mutant protein by tannin-based natural products in SK-N-SH neurons.
15 is a result of analyzing the filament formation inhibitory activity of the SOD1 G93A mutant protein by the culture product of Lactobacillus strains and tannin-based natural products by Thioflavin T assay.
이하, 본 발명을 보다 상세하게 설명한다.Hereinafter, the present invention will be described in more detail.
본 발명은 기탁번호 KCTC 15134BP로 기탁된 락토바실러스 플란타럼(Lactobacillus plantarum) D2-3 균주를 제공한다.The present invention provides a Lactobacillus plantarum D2-3 strain deposited under accession number KCTC 15134BP.
상기 균주는 탄닌산 분해효소(tannase) 활성을 나타낼 수 있다.The strain may exhibit tannase activity.
또한, 본 발명은 탄닌 화합물에 기탁번호 KCTC 15134BP로 기탁된 락토바실러스 플란타럼(Lactobacillus plantarum) D2-3 균주 추출물 또는 이의 배양액을 처리하여 탄닌 가수분해물을 수득하는 단계를 포함하는, 탄닌 가수분해방법을 제공한다.In addition, the present invention is a tannin hydrolysis method comprising the step of obtaining a tannin hydrolyzate by treating a Lactobacillus plantarum D2-3 strain extract or a culture thereof deposited with accession number KCTC 15134BP in a tannin compound. provides
상기 탄닌 화합물은 EGCG(epigallocatechin gallte), 갈릭산(gallic acid), 탄닌산(tannic acid) 및 엘라그산(ellagic acid)으로 이루어진 군에서 선택된 하나 이상일 수 있으나, 이에 한정되는 것은 아니다.The tannin compound may be at least one selected from the group consisting of epigallocatechin gallte (EGCG), gallic acid, tannic acid, and ellagic acid, but is not limited thereto.
또한, 본 발명은 상기 탄닌 가수분해방법을 통해 수득한 탄닌 가수분해물을 유효성분으로 포함하는, SOD1(superoxide dismutase 1) G93A 돌연변이 단백질 필라멘트 형성 억제용 조성물을 제공한다.In addition, the present invention provides a composition for inhibiting filament formation of SOD1 (superoxide dismutase 1) G93A mutant protein, comprising tannin hydrolyzate obtained through the tannin hydrolysis method as an active ingredient.
상기 탄닌 가수분해물은 EGCG(epigallocatechin gallte), 갈릭산(gallic acid), 탄닌산(tannic acid) 및 엘라그산(ellagic acid)으로 이루어진 군에서 선택된 하나 이상일 수 있으나, 이에 한정되는 것은 아니다.The tannin hydrolyzate may be one or more selected from the group consisting of epigallocatechin gallte (EGCG), gallic acid, tannic acid, and ellagic acid, but is not limited thereto.
또한, 본 발명은 EGCG(epigallocatechin gallte), 갈릭산(gallic acid), 탄닌산(tannic acid) 및 엘라그산(ellagic acid)으로 이루어진 군에서 선택된 하나 이상의 탄닌 화합물을 유효성분으로 포함하는, SOD1(superoxide dismutase 1) G93A 돌연변이 단백질 필라멘트 형성 억제용 조성물을 제공한다.In addition, the present invention is a superoxide dismutase (SOD1) containing at least one tannin compound selected from the group consisting of epigallocatechin gallte (EGCG), gallic acid, tannic acid and ellagic acid as an active ingredient. 1) Provided is a composition for inhibiting G93A mutant protein filament formation.
또한, 본 발명은 EGCG(epigallocatechin gallte), 갈릭산(gallic acid), 탄닌산(tannic acid) 및 엘라그산(ellagic acid)으로 이루어진 군에서 선택된 하나 이상의 탄닌 화합물에 락토바실러스 플란타럼(Lactobacillus plantarum) 균주를 첨가하고 공동배양한 배양액을 유효성분으로 포함하는, SOD1(superoxide dismutase 1) G93A 돌연변이 단백질 필라멘트 형성 억제용 조성물을 제공한다.In addition, the present invention relates to one or more tannin compounds selected from the group consisting of epigallocatechin gallte (EGCG), gallic acid, tannic acid and ellagic acid, Lactobacillus plantarum strain It provides a composition for inhibiting SOD1 (superoxide dismutase 1) G93A mutant protein filament formation, containing the co-cultured culture medium as an active ingredient.
이하, 본 발명의 이해를 돕기 위하여 실시예를 들어 상세하게 설명하기로 한다. 다만 하기의 실시예는 본 발명의 내용을 예시하는 것일 뿐 본 발명의 범위가 하기 실시예에 한정되는 것은 아니다. 본 발명의 실시예는 당업계에서 평균적인 지식을 가진 자에게 본 발명을 보다 완전하게 설명하기 위해 제공되는 것이다.Hereinafter, examples will be described in detail to aid understanding of the present invention. However, the following examples are merely illustrative of the contents of the present invention, but the scope of the present invention is not limited to the following examples. The embodiments of the present invention are provided to more completely explain the present invention to those skilled in the art.
[실시예 1] 균주 분리 및 동정[Example 1] Strain Isolation and Identification
전통발효식품을 확보하여 확보된 샘플로부터 LBS(Lactobacillus Selection) 선택배지를 활용하여 락토바실러스 플란타럼 균주 분리배양을 수행하였다. 선택배지를 통해 분리한 균주의 16S rRNA에 대한 PCR 및 시퀀싱을 통해, 도 1에 나타난 바와 같이, 상기 분리한 균주가 락토바실러스 플란타럼(Lactobacillus plantarum D2-3; 이하 D2-3이라 함) 균주임을 확인하였다. 상기 D2-3 균주의 16S rRNA 서열은 하기 표 1과 같다.Isolated culture of Lactobacillus plantarum strains was performed using LBS (Lactobacillus Selection) selective medium from samples obtained by securing traditional fermented foods. Through PCR and sequencing of the 16S rRNA of the strain isolated through the selective medium, as shown in Figure 1, the isolated strain is Lactobacillus plantarum ( Lactobacillus plantarum D2-3; hereinafter referred to as D2-3) strain It was confirmed that The 16S rRNA sequence of the D2-3 strain is shown in Table 1 below.
[[ 실시예Example 2] D2-3 균주의 2] D2-3 strain 탄네이즈tannaise 유전자 발현 측정 Gene expression measurement
상기 분리한 D2-3 균주의 탄네이즈 효소 발현량을 확인하기 위해, 8개의 서로 다른 전통발효식품 유래 락토바실러스 플란타럼 균주 8종 및 ATCC(American Type Culture Collection)로부터 분양받은 표준균주의 탄네이즈 효소 발현량을 비교 및 분석하였다. 탄네이즈 효소 발현량은 실시간으로 정량이 가능한 qRT-PCR을 활용하여 균주별 상대적인 mRNA 발현량을 비교하였다. qRT-PCR에 사용한 프라이머 세트는 선행연구(Natalia et al., (2014) Applied and Environmental Microbiology, 80(10), 2991-2997)를 참고하여 제작하였다. 상기 제작한 프라이머 세트의 프라이머 서열은 하기 표 2와 같다. 그 결과, 도 2에 나타난 바와 같이, D2-3 균주를 포함한 총 9개의 균주 중에서, D2-3 균주의 탄네이즈 발현량이 가장 우수한 것을 확인하였다.In order to confirm the expression level of the tannaise enzyme of the isolated D2-3 strain, 8 strains of Lactobacillus plantarum derived from 8 different traditional fermented foods and the tannaise of the standard strain distributed from ATCC (American Type Culture Collection) Enzyme expression levels were compared and analyzed. The tannaise enzyme expression level was compared with the relative mRNA expression level for each strain using qRT-PCR, which can be quantified in real time. The primer set used for qRT-PCR was prepared by referring to a previous study (Natalia et al., (2014) Applied and Environmental Microbiology, 80(10), 2991-2997). Primer sequences of the prepared primer set are shown in Table 2 below. As a result, as shown in FIG. 2 , it was confirmed that the tannaise expression level of the D2-3 strain was the highest among a total of 9 strains including the D2-3 strain.
[실시예 3] D2-3 균주의 탄네이즈 유전자 증폭[Example 3] Tannaise gene amplification of strain D2-3
D2-3 균주의 탄네이즈 효소 염기서열을 NCBI GenBank database (http://www.ncbi.nlm.nih.gov/genbank/)를 통해 확보하였다. 확보한 염기서열을 바탕으로 해당 유전자의 양 끝에 특정 제한효소(XhoI 및 BamHI)를 붙여 탄네이즈 유전자 증폭을 위한 프라이머 세트를 제작하였다. 상기 D2-3 균주의 탄네이즈 효소 염기서열은 하기 표 3과 같다. 그 후, 도 3에 나타난 바와 같이, PCR을 이용하여 D2-3 균주의 탄네이즈 효소 증폭산물을 얻어 전기영동으로 확인하였다. PCR 조건은 하기 표 4와 같다.The tannaise enzyme nucleotide sequence of strain D2-3 was obtained through the NCBI GenBank database (http://www.ncbi.nlm.nih.gov/genbank/). Based on the obtained nucleotide sequence, a primer set for tannaise gene amplification was prepared by attaching specific restriction enzymes (XhoI and BamHI) to both ends of the gene. The tannaise enzyme nucleotide sequence of the D2-3 strain is shown in Table 3 below. Thereafter, as shown in FIG. 3, PCR was used to obtain tannaise enzyme amplification products of the D2-3 strain and confirmed by electrophoresis. PCR conditions are shown in Table 4 below.
[실시예 4] pET15b 벡터로의 탄네이즈 유전자 클로닝 [Example 4] Cloning of tannaise gene into pET15b vector
PCR로 증폭한 탄네이즈 유전자를 두 가지 제한효소(XhoI 및 BamHI)로 double digestion하여 insert DNA를 확보하였다. 확보된 insert DNA를 Quick Ligation Kit (BioLabs, USA)를 이용하여 pET15b 벡터 내 XhoI 및 BamHI 부분으로 클로닝 후, 대장균[E. coli BL21(DE3)]에 형질전환(transformation)을 수행하였고, 암피실린(ampicillin)이 포함된 고체 배지에서 콜로니를 선발하여 플라스미드를 분리 및 정제하였다. 상기 플라스미드를 확인하기 위해, XhoI 및 BamHI로 double digestion을 한 후, 전기영동으로 확인한 결과, 도 4에 나타난 바와 같이, 형질전환이 잘 되었음을 확인하였다.The tannaise gene amplified by PCR was double digested with two restriction enzymes (XhoI and BamHI) to obtain insert DNA. After cloning the obtained insert DNA into the XhoI and BamHI parts in the pET15b vector using the Quick Ligation Kit (BioLabs, USA), transformation was performed in E. coli [ E. coli BL21(DE3)], and ampicillin ), the plasmid was isolated and purified by selecting colonies on a solid medium containing. In order to confirm the plasmid, after double digestion with XhoI and BamHI, as a result of confirming by electrophoresis, as shown in FIG. 4, it was confirmed that the transformation was successful.
[실시예 5] 클로닝된 탄네이즈 유전자 시퀀스 확인 [Example 5] Confirmation of cloned tannaise gene sequence
클로닝이 완료되어 전기영동으로 확인한 형질전환물(transformants)을 도 5에 나타난 바와 같이, pET15b 벡터의 universal primer를 이용하여 최종 클로닝 산물의 시퀀스를 확인하였다. 상기 universal primer의 서열은 하기 표 5와 같다.As shown in FIG. 5, the transformants confirmed by electrophoresis after cloning was completed were confirmed by using the universal primer of the pET15b vector to confirm the sequence of the final cloning product. The sequence of the universal primer is shown in Table 5 below.
[실시예 6] 탄네이즈 대량발현[Example 6] Mass expression of tannaise
클로닝된 탄네이즈 유전자의 대량발현을 위해, 5ml LB(50μg/ml 암피실린 함유) 배지에 D2-3 균주의 탄네이즈 유전자가 도입된 대장균[E. coli BL21(DE3)]을 접종하여 37℃에서 12시간 배양하여 종균(seed culture)을 제작하였다. 신선한 LB 배지 100ml(50μg/ml 암피실린 함유)에 종균을 1% 접종하여 sub-culture를 진행하였다. Sub-culture의 OD600이 0.6~0.8이 될 때까지 37℃에서 배양 후 IPTG를 최종 농도 1mM이 되도록 넣어 준 후, 30℃에서 24시간 대량발현 하였다. 도 6에 나타난 바와 같이, SDS-PAGE를 통해 탄네이즈 효소가 대량발현되었음을 확인하였다.For mass expression of the cloned tannaise gene, E. coli [ E. coli BL21(DE3)] into which the tannaise gene of the D2-3 strain was introduced was inoculated into 5ml LB (containing 50 μg/ml ampicillin) medium and incubated at 37°C for 12 days. A seed culture was prepared by culturing for a period of time. Sub-culture was performed by inoculating 1% of the spawn in 100 ml of fresh LB medium (containing 50 μg/ml ampicillin). After incubation at 37℃ until the OD 600 of the sub-culture is 0.6~0.8, IPTG was added to a final concentration of 1 mM, followed by mass expression at 30℃ for 24 hours. As shown in FIG. 6, it was confirmed through SDS-PAGE that the tannase enzyme was expressed in large quantities.
[실시예 7] 발현된 탄네이즈 효소의 분리 및 정제 [Example 7] Isolation and purification of expressed tannase enzyme
탄네이즈 효소의 대량발현을 위해 사용된 pET15b 벡터에는 His-Tag이 코딩되어 있기 때문에, 외래 유전자 발현시 His-Tag를 이용하여 정제할 수 있다. 따라서, 대량발현된 탄네이즈 효소를 얻기 위해, 먼저 대량발현한 배양액을 4℃ 및 10,000 × g 조건에서 30분동안 원심 분리하여 상등액을 제거하였다. 상등액을 제거하고 남은 균 펠렛(pellet)을 lysis buffer[50mM 모노나트륨 인산염(NaH2PO4), 300mM 염화나트륨(NaCl) 및 10mM 이미다졸(imidazole)] 5ml로 재현탁(resuspension)한 후, 200W에서 10sec/10sec pulse로 총 10분간 sonication하여 균주를 파쇄하였다. 파쇄한 균주의 상등액을 얻기 위해 4℃ 및 10,000 × g 조건에서 30분 동안 원심 분리하여 상등액만을 분리하였다. 탄네이즈 효소만를 정제하기 위해, lysis buffer 조성에서 이미다졸 농도를 각각 50mM 및 500mM로 바꾸어 wash buffer 및 elution buffer를 제작하였다. Ni-NTA 컬럼에 용량의 5배에 해당하는 lysis buffer를 흘려보낸 후, 분리한 상등액 시료를 천천히 흘려보냈다. 컬럼 용량의 10배의 wash buffer를 천천히 흘려보내 컬럼과 결합하지 않은 다른 단백질들을 제거하였다. 컬럼 용량의 5배의 elution buffer를 천천히 흘려보내 His-Tag이 붙어있는 탄네이즈 효소를 정제하여 도 7에 나타난 바와 같이, SDS-PAGE로 정제물을 확인하였다. 탄네이즈 효소가 불순물 없이 정제되었는지 확인하기 위해, MALDI-TOF MS로 분석한 결과, 도 8에 나타난 바와 같이, 단일 피크가 검출되는 것을 확인함으로써, 불순물 없이 잘 정제되었음을 확인하였다.Since the His-Tag is encoded in the pET15b vector used for the large-scale expression of the tanase enzyme, His-Tag can be used for purification when exogenous genes are expressed. Therefore, in order to obtain a large amount of tannase enzyme, first, the supernatant was removed by centrifuging the mass-expressed culture medium at 4° C. and 10,000 × g for 30 minutes. After removing the supernatant, the remaining bacterial pellet was resuspended in 5ml of lysis buffer [50mM monosodium phosphate (NaH 2 PO 4 ), 300mM sodium chloride (NaCl) and 10mM imidazole], and then at 200W. The strain was disrupted by sonication for a total of 10 minutes with a 10sec/10sec pulse. In order to obtain the supernatant of the disrupted strain, only the supernatant was separated by centrifugation for 30 minutes at 4 ° C and 10,000 × g conditions. In order to purify only the tannaise enzyme, a wash buffer and an elution buffer were prepared by changing the imidazole concentration to 50 mM and 500 mM, respectively, in the composition of the lysis buffer. After flowing lysis buffer corresponding to 5 times the capacity to the Ni-NTA column, the separated supernatant sample was slowly flowed. 10 times the column volume of wash buffer was slowly flowed to remove other proteins not bound to the column. Elution buffer 5 times the column capacity was slowly flowed to purify the His-Tag-attached tannase enzyme, and as shown in FIG. 7, the purified product was confirmed by SDS-PAGE. In order to confirm that the tannaise enzyme was purified without impurities, as a result of MALDI-TOF MS analysis, as shown in FIG. 8, it was confirmed that a single peak was detected, thereby confirming that the enzyme was well purified without impurities.
[실시예 8] 정제 탄네이즈 효소의 탄닌산 분해능 분석 [Example 8] Analysis of tannic acid degradation ability of purified tannase enzyme
정제한 탄네이즈 효소의 탄닌산 분해능을 확인하기 위해, 고체배지 상에서 탄닌산을 가수분해하는지 분석하였다. 0.5% 효모 추출물을 함유한 BHI 고체배지 위에 정제수에 녹인 2% 탄닌산 용액을 고르게 분주한 후, 20분간 반응시켜 불투명한 배지를 만들었다. 그 위에 정제한 D2-3 탄네이즈 효소를 dotting하여 클리어 존(clear zone)을 통해 탄닌산 분해 여부를 확인한 결과, 도 9에 나타난 바와 같이, 정제 탄네이즈 효소에 의해 탄닌산 분해 활성이 나타나는 것을 확인하였다. 또한, 정제 탄네이즈 효소에 의한 EGCG(Epigallocatechin gallte) 분해물을 HPLC로 분석한 결과, 도 10에 나타난 바와 같이, EGCG가 갈릭산(gallic acid)으로 분해되는 것을 확인하였다.In order to confirm the tannic acid decomposition ability of the purified tannase enzyme, it was analyzed whether the tannic acid was hydrolyzed on a solid medium. A 2% tannic acid solution dissolved in purified water was evenly dispensed on the BHI solid medium containing 0.5% yeast extract, and then reacted for 20 minutes to make an opaque medium. As a result of confirming tannic acid decomposition through a clear zone by dotting the purified D2-3 tannaise enzyme thereon, it was confirmed that tannic acid decomposition activity was exhibited by the purified tannaise enzyme, as shown in FIG. 9 . In addition, as a result of HPLC analysis of EGCG (Epigallocatechin gallte) decomposition by purified tannaise enzyme, as shown in FIG. 10, it was confirmed that EGCG was decomposed into gallic acid.
[[ 실시예Example 9] 9] 탄닌에on tannins 의한 by superoxidesuperoxide dismutasedismutase 1( One( SOD1SOD1 ) 야생형 단백질의 필라멘트 형성 저해 분석) Analysis of inhibition of filament formation by wild-type protein
탄닌계 천연물의 superoxide dismutase 1(이하 SOD1이라 함) 야생형 단백질 및 SOD1 G93A 돌연변이 단백질의 필라멘트 형성 저해 활성을 확인하기 위해, Thioflavin T assay(ThT) 및 Negative-staining 전자현미경(EM)을 통해 확인하였다. 그 결과, 도 11, 12 및 13에 나타난 바와 같이, 탄닌계 천연물이 SOD1 G93A 돌연변이 단백질의 필라멘트 형성 저해 활성을 나타내는 것을 확인하였다. 또한, 도 14에 나타난 바와 같이, SK-N-SH 신경세포에서도 탄닌계 천연물이 SOD1 G93A 돌연변이 단백질의 필라멘트 형성 저해 활성을 나타내는 것을 확인하였다.In order to confirm the filament formation inhibitory activity of superoxide dismutase 1 (hereinafter referred to as SOD1) wild-type protein and SOD1 G93A mutant protein of tannin-based natural products, Thioflavin T assay (ThT) and negative-staining electron microscope (EM) were confirmed. As a result, as shown in FIGS. 11, 12 and 13, it was confirmed that the tannin-based natural product showed the filament formation inhibitory activity of the SOD1 G93A mutant protein. In addition, as shown in FIG. 14, it was confirmed that the tannin-based natural product exhibited the filament formation inhibitory activity of the SOD1 G93A mutant protein in SK-N-SH nerve cells.
[[ 실시예Example 10] 10] 락토바실러스Lactobacillus 균주 및 strains and 탄닌계Tannin system 천연물의 배양 산물에 의한 by culture products of natural products SOD1SOD1 G93A 돌연변이 단백질 필라멘트 형성 저해 분석 G93A mutant protein filament formation inhibition assay
락토바실러스 람노서스(Lactobacillus rhamnosus) 및 락토바실러스 플란타럼(Lactobacillus plantarum) 균주의 탄닌계 천연물 공동배양 산물에 의한 SOD1 G93A 돌연변이 단백질의 필라멘트 형성 저해 활성을 확인하기 위해, Thioflavin T assay(ThT)을 수행하였다. 그 결과, 도 15에 나타난 바와 같이, EGCG, 탄닌산 (tannic acid) 및 갈릭산(gallic acid)에 락토바실러스 플란타럼 균주를 첨가하고 배양한 배양액이 상기 균주와 공동배양을 하지 않은 탄닌계 천연물보다 우수한 SOD1 G93A 돌연변이 단백질 필라멘트 형성 저해 활성을 나타내는 것을 확인하였다.Lactobacillus rhamnosus ( Lactobacillus rhamnosus ) and Lactobacillus plantarum ( Lactobacillus plantarum ) In order to confirm the filament formation inhibitory activity of the SOD1 G93A mutant protein by co-culture products of tannin-based natural products, Thioflavin T assay (ThT) was performed did As a result, as shown in FIG. 15, the culture solution obtained by adding and culturing the Lactobacillus plantarum strain to EGCG, tannic acid and gallic acid was superior to the tannin-based natural product not co-cultured with the strain. It was confirmed that the SOD1 G93A mutant protein showed excellent filament formation inhibitory activity.
이상으로 본 발명 내용의 특정한 부분을 상세히 기술하였는 바, 당업계의 통상의 지식을 가진 자에게 있어서, 이러한 구체적 기술은 단지 바람직한 실시양태일 뿐이며, 이에 의해 본 발명의 범위가 제한되는 것이 아닌 점은 명백하다. 즉, 본 발명의 실질적인 범위는 첨부된 청구항들과 그것들의 등가물에 의하여 정의된다.Having described specific parts of the present invention in detail above, it is clear to those skilled in the art that these specific descriptions are only preferred embodiments, and the scope of the present invention is not limited thereby. do. That is, the substantial scope of the present invention is defined by the appended claims and their equivalents.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20190005157A (en) | 2016-03-28 | 2019-01-15 | 신유알엑스 인터내셔널 (타이완) 코포레이션 | Compositions Containing Tannic Acid and Uses Thereof |
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