KR20200070845A - Cinnamoyl esterase from Lactobacillus fermentum J2 and preparation method thereof - Google Patents
Cinnamoyl esterase from Lactobacillus fermentum J2 and preparation method thereof Download PDFInfo
- Publication number
- KR20200070845A KR20200070845A KR1020180158427A KR20180158427A KR20200070845A KR 20200070845 A KR20200070845 A KR 20200070845A KR 1020180158427 A KR1020180158427 A KR 1020180158427A KR 20180158427 A KR20180158427 A KR 20180158427A KR 20200070845 A KR20200070845 A KR 20200070845A
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- South Korea
- Prior art keywords
- cinnamoyl esterase
- strain
- esterase
- cinnamoyl
- lactobacillus
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Abstract
Description
본 발명은 락토바실러스 퍼멘텀(Lactobacillus fermentum) J2 균주(KACC 92245P)에서 유래하는 시나모일 에스터라아제(Cinnamoyl esterase), 이의 제조 방법에 관한 것이다. 더욱 상세하게는, 본 발명은 락토바실러스 퍼멘텀(Lactobacillus fermentum) J2 균주(KACC 92245P)에서 유래하는 시나모일 에스터라아제(Cinnamoyl esterase), 이의 제조 방법, 상기 시나모일 에스터라아제를 포함하는 기능성 식품 조성물, 및 사료용 조성물에 관한 것이다.The present invention relates to a cinnamoyl esterase derived from Lactobacillus fermentum J2 strain (KACC 92245P), and a method for manufacturing the same. In more detail, the present invention is a cinnamoyl esterase (Cinnamoyl esterase) derived from Lactobacillus fermentum J2 strain (KACC 92245P), a method for manufacturing the same, a functional food containing the cinnamoyl esterase It relates to a composition, and a composition for feed.
카페산(caffeic acid), 페룰산(ferulic acid), 시나핀산(sinapic acid), p-쿠말린산(p-coumaric acid)와 같은 하이드록시시남산(hydroxycinnamic acid)은 거의 모든 식물에서 발견되고(Hermann, 1976; Kuhnau, 1976), 주요 부류의 페놀 화합물을 대표한다. 가장 흔한 하이드록시남산(hydroxycinnamic acid)은 카페산이다.Hydroxycinnamic acids such as caffeic acid, ferulic acid, sinapic acid, and p-coumaric acid are found in almost all plants ( Hermann, 1976; Kuhnau, 1976), representing a major class of phenolic compounds. The most common hydroxycinnamic acid is caffeic acid.
식물 세포벽의 다당류는 자연적으로 발생되는 가장 풍부한 유기체 화합물로 커피, 차, 사과, 배과류, 베리류, 아티초크, 가지 등 식물체에는 페놀류에 속하는 기능성 성분인 하이드록시남산(hydroxycinnamic acid)이 폭넓게 존재한다. Polysaccharides on the cell wall of plants are the most abundant naturally occurring organic compounds. Plants such as coffee, tea, apple, pear, berry, artichoke, and eggplant have a wide range of hydroxycinnamic acids, which are functional components belonging to phenols.
하이드록시남산의 일종인 페룰산은 인체 내에서 활성 산소종(ROS)와 같은 자유 라디칼 포집(free radical scavenging), 금속 이온 봉쇄(metal ion chelating) 등의 강한 항산화 작용을 하는 것으로 알려져 있다. 상기 활성 산소종과 같은 자유 라디칼은 DNA 손상, 암, 및 세포 노화 촉진에 관여한다. 국소용 제제에 페룰산을 첨가하면 산화 스트레스를 줄일 수 있고, 피부 미백 과정에서 멜라닌 생성을 억제할 수 있다는 연구가 있으나, 아직 페룰산에 관한 연구는 미미한 편이다. Ferulic acid, a type of hydroxynamic acid, is known to have a strong antioxidant effect in the human body, such as free radical scavenging (ROS) and metal ion chelating. Free radicals, such as free radical species, are involved in promoting DNA damage, cancer, and cellular aging. Studies have shown that adding ferulic acid to topical preparations can reduce oxidative stress and inhibit melanin production in the skin whitening process, but research on ferulic acid is still minor.
인체에서의 흡수 및 작용은 효소적인 작용에 크게 의존되어 유용물질의 흡수율에 지대하게 영향을 미친다고 할 수 있는데, 에틸 페룰레이트를 페룰산으로 가수분해하는 효소를 시나모일 에스터라아제(cinnamoyl esterase)라고 한다.Absorption and action in the human body is highly dependent on the enzymatic action, which can be said to greatly affect the absorption rate of useful substances. Cinnamoyl esterase is an enzyme that hydrolyzes ethyl ferulate to ferulic acid. It is said.
한편, 인간의 장내 미생물은 이와 같은 시나모일 에스터라제 활성을 가진 것으로 알려져 있으나, 현재까지 유산균 유래의 이 효소에 대한 연구와 특이적으로 에틸 페룰레이트를 페룰산으로 전환하는 시나모일 에스터라제의 활성을 가진 균주의 구체적인 특성에 대한 연구와 특히 이를 이용한 식품의 개발에 대한 연구는 미미한 실정이다.On the other hand, human gut microorganisms are known to have such cinnamoyl esterase activity, but until now, research on this enzyme derived from lactic acid bacteria and specifically cinnamoyl esterase converting ethyl ferulate to ferulic acid Studies on the specific characteristics of strains with activity and, in particular, on the development of foods using them are insignificant.
반면, 콩과 (Leguminosae)류, 커피, 배, 사과, 토마토, 녹차 등 다양한 식물체 속에는 항산화 물질 성분인 주요 폴리페놀류로 전술의 클로로젠산, 카페산과 쿠말린산(p-coumalic acid), 시링산(syringic acid), 바닐산(vanillic acid), 페룰산(ferulic acid) 등이 천연적으로 존재하며, 이들은 유리 상태보다는 에스테르 결합을 한 형태를 가진다.On the other hand, it is a major polyphenol that is an antioxidant component in various plants such as legumes, coffee, pear, apple, tomato, green tea, etc., and the above-mentioned chlorogenic acid, caffeic acid and cumalic acid (p-coumalic acid), shiring acid (syringic acid), vanillic acid, ferulic acid, etc. are naturally present, and they have an ester bond rather than a free state.
따라서, 이런 에스테르 결합을 한 형태로 주로 존재하는 에틸 페룰레이트는 시나모일 에스터라제에 의해 유리형태인 페룰산으로써 섭취될 때, 흡수가 용이하고 항산화 효과도 높일 수 있다. Therefore, ethyl ferulate, which is mainly present in one form of such an ester bond, can be easily absorbed and have an antioxidant effect when taken as free ferulic acid by cinnamoyl esterase.
현재까지 하이드록시남산에 연결되어 있는 여러 종류의 당-에스터 결합을 가수분해하는 효소인 시나모일 에스터라제에 관한 연구는 인간의 대장 속에서 발견되는 박테리아 유래에 대한 연구 또는 Aspergillus niger 또는 A. japonicus 등과 같은 곰팡이에 대한 연구가 주로 진행되어 있는 상태로, 유산균 유래의 시나모일 에스터라제의 생산을 위한 발현과 정제에 관한 연구는 매우 미미하다. To date, studies on cinnamoyl esterase, an enzyme that hydrolyzes several types of sugar-ester bonds linked to hydroxynamic acid, are studies of bacterial origin found in the human large intestine or Aspergillus niger or A. japonicus Research on fungi, such as the state, has been mainly conducted, and studies on expression and purification for production of lactic acid bacteria-derived cinnamoyl esterase are very small.
본 발명의 배경기술로는 한국 공개 특허 10-2004-0082007에 페룰산(ferulic acid)을 유효성분으로 하는 항산화, 항노화 또는 항염증용 약학 조성물이 공개되어 있다.As a background technology of the present invention, a pharmaceutical composition for antioxidant, anti-aging or anti-inflammatory using ferulic acid as an active ingredient is disclosed in Korean Patent Publication No. 10-2004-0082007.
본 발명에서는 활성이 우수한 시나모일 에스터라제의 고생산 유산균을 발굴하고, 본 유산균 유래 시나모일 에스터라제를 고효율 제조 방법, 및 시나모일 에스터라제를 포함하는 기능성 식품 조성물, 또는 사료용 조성물을 제공하고자 하였다.In the present invention, a high-production lactic acid bacterium having excellent activity cinnamoyl esterase is discovered, a high-efficiency manufacturing method of cinnamoyl esterase derived from the present lactic acid bacteria, and a functional food composition comprising cinnamoyl esterase, or a composition for feed I wanted to.
본 발명의 목적은 활성이 우수한 시나모일 에스터라아제를 고효율로 생산할 수 있는 락토바실러스 퍼멘텀 J2 균주(KACC 92245P)를 제공하는 것이다.An object of the present invention is to provide a Lactobacillus performance J2 strain (KACC 92245P) capable of producing cinnamoyl esterase having excellent activity with high efficiency.
본 발명의 다른 목적은 락토바실러스 퍼멘텀 J2 균주(KACC 92245P)로부터 유래한, 에틸 페룰레이트에 대해 기질특이성 및 분해율이 개선되어 활성이 우수한 시나모일 에스터라아제를 제공하는 것이다.Another object of the present invention is to provide a cinnamoyl esterase having excellent activity by improving substrate specificity and decomposition rate with respect to ethyl ferulate, derived from the Lactobacillus performance J2 strain (KACC 92245P).
본 발명의 또 다른 목적은 에틸 페룰레이트에서 페룰산으로의 전환율이 개선된 시나모일 에스터라아제를 제공하는 것이다.Another object of the present invention is to provide a cinnamoyl esterase with improved conversion from ethyl ferulate to ferulic acid.
본 발명의 또 다른 목적은 활성이 우수한 시나모일 에스터라아제를 고효율로 생산할 수 있는 시나모일 에스터라아제 제조 방법을 제공하는 것이다.Another object of the present invention is to provide a method for producing cinnamoyl esterase capable of producing cinnamoyl esterase having excellent activity with high efficiency.
본 발명의 또 다른 목적은 락토바실러스 퍼멘텀(Lactobacillus fermentum) J2 균주(KACC 92245P)에서 유래하는 시나모일 에스터라아제(Cinnamoyl esterase)를 포함하는 기능성 식품 조성물을 제공하는 것이다.Another object of the present invention is to provide a functional food composition comprising cinnamoyl esterase derived from Lactobacillus fermentum J2 strain (KACC 92245P).
본 발명의 또 다른 목적은 락토바실러스 퍼멘텀(Lactobacillus fermentum) J2 균주(KACC 92245P)에서 유래하는 시나모일 에스터라아제(Cinnamoyl esterase)를 포함하는 사료용 조성물을 제공하는 것이다.Another object of the present invention is to provide a composition for feed comprising cinnamoyl esterase derived from Lactobacillus fermentum J2 strain (KACC 92245P).
본 발명의 다른 목적 및 이점은 하기의 발명의 상세한 설명, 청구범위 및 도면에 의해 더욱 명확하게 된다.Other objects and advantages of the present invention will be further clarified by the following detailed description of the invention, claims and drawings.
본 발명의 일 측면에 의하면, 시나모일 에스터라아제(Cinnamoyl esterase)를 생산하는 락토바실러스 퍼멘텀(Lactobacillus fermentum) J2 균주(KACC 92245P)가 제공된다.According to an aspect of the present invention, a Lactobacillus fermentum J2 strain (KACC 92245P) producing cinnamoyl esterase is provided.
본 발명의 일 실시예에 의하면, 상기 락토바실러스 퍼멘텀(Lactobacillus fermentum) J2 균주(KACC 92245P)는 서열번호 1로 기재되는 16s rRNA로 구성되는 것을 특징으로 한다.According to an embodiment of the present invention, the Lactobacillus fermentum J2 strain (KACC 92245P) is characterized by comprising 16s rRNA of SEQ ID NO: 1.
본 발명의 다른 측면에 의하면, 본원의 락토바실러스 퍼멘텀(Lactobacillus fermentum) J2 균주(KACC 92245P)로부터 생산된, 시나모일 에스터라아제가 제공된다.According to another aspect of the present invention, a cinnamoyl esterase produced from Lactobacillus fermentum J2 strain (KACC 92245P) of the present application is provided.
본 발명의 일 실시예에 의하면, 상기 시나모일 에스터라아제는 서열번호 5로 기재되는 아미노산 서열을 포함하는 것을 특징으로 한다.According to one embodiment of the invention, the cinnamoyl esterase is characterized in that it comprises the amino acid sequence of SEQ ID NO: 5.
본 발명의 일 실시예에 의하면, 상기 시나모일 에스터라아제는 에틸 페룰레이트(ethyl ferulate)에 대한 기질 특이성이 향상된 것을 특징으로 한다.According to an embodiment of the present invention, the cinnamoyl esterase is characterized in that substrate specificity for ethyl ferulate is improved.
본 발명의 일 실시예에 의하면, 상기 시나모일 에스터라아제는 에틸 페룰레이트(ethyl ferulate)에서 페룰산으로 90% 이상의 전환율을 나타내는 것을 특징으로 한다.According to an embodiment of the present invention, the cinnamoyl esterase is characterized in that it exhibits a conversion rate of at least 90% from ethyl ferulate to ferulic acid.
본 발명의 다른 측면에 의하면, 본원의 시나모일 에스터라아제를 암호화하는 유전자가 제공된다.According to another aspect of the present invention, a gene encoding a cinnamoyl esterase of the present application is provided.
본 발명의 일 실시예에 의하면, 상기 유전자는 서열번호 2로 기재되는 폴리뉴클레오티드로 구성되는 것을 특징으로 한다.According to an embodiment of the present invention, the gene is characterized by being composed of the polynucleotide of SEQ ID NO: 2.
본 발명의 다른 측면에 의하면, 본원의 시나모일 에스터라아제를 암호화하는 유전자의 DNA를 증폭하기 위한 서열번호 3 및 4로 이루어진 프라이머 세트가 제공된다.According to another aspect of the present invention, there is provided a primer set consisting of SEQ ID NOs: 3 and 4 for amplifying the DNA of the gene encoding the cinnamoyl esterase of the present application.
본 발명의 다른 측면에 의하면, (1) 제1항 또는 제2항의 시나모일 에스터라아제(Cinnamoyl esterase)를 생산하는 락토바실러스 퍼멘텀(Lactobacillus fermentum) J2 균주를 배양하는 단계; 및 (2) 상기 (1) 단계의 배양액으로부터 시나모일 에스터라아제(Cinnamoyl esterase)를 분리하는 단계를 포함하는, 시나모일 에스터라아제의 제조 방법이 제공된다.According to another aspect of the present invention, (1) culturing the Lactobacillus fermentum ( Lactobacillus fermentum ) J2 strain to produce the cinnamoyl esterase of
본 발명의 일 실시예에 의하면, 상기 (1) 단계에서, 락토바실러스 퍼멘텀 J2 균주를 45시간 내지 96시간 동안 배양할 수 있다.According to an embodiment of the present invention, in step (1), the Lactobacillus performance J2 strain can be cultured for 45 hours to 96 hours.
본 발명의 일 실시예에 의하면, 상기 (1) 단계에서, 락토바실러스 퍼멘텀 J2 균주를 pH 6.5 내지 7.5에서 배양할 수 있다.According to an embodiment of the present invention, in step (1), the Lactobacillus performance J2 strain may be cultured at pH 6.5 to 7.5.
본 발명의 일 실시예에 의하면, 상기 (1) 단계에서, 락토바실러스 퍼멘텀 J2 균주를 30℃ 내지 40℃에서 배양할 수 있다.According to an embodiment of the present invention, in step (1), the Lactobacillus performance J2 strain may be cultured at 30°C to 40°C.
본 발명의 일 실시예에 의하면, 상기 방법에서 상기 시나모일 에스터라아제는 에틸 페룰레이트(ethyl ferulate)에 대한 기질 특이성이 향상된 것을 특징으로 한다.According to an embodiment of the present invention, in the above method, the cinnamoyl esterase is characterized in that substrate specificity for ethyl ferulate is improved.
본 발명의 일 실시예에 의하면, 상기 방법에서 상기 시나모일 에스터라아제는 에틸 페룰레이트(ethyl ferulate)에서 페룰산으로 90% 이상의 전환율을 나타내는 것을 특징으로 한다. According to one embodiment of the present invention, in the above method, the cinnamoyl esterase is characterized in that it exhibits a conversion rate of at least 90% from ethyl ferulate to ferulic acid.
본 발명의 다른 측면에 의하면, 본원의 시나모일 에스터라아제(Cinnamoyl esterase)를 생산하는 신규한 락토바실러스 퍼멘텀(Lactobacillus fermentum) J2 균주, 이의 파쇄물 및 상기 균주 배양물로 이루어진 군에서 선택된 하나 이상을 유효성분으로 포함하는 기능성 식품 조성물이 제공된다.According to another aspect of the present invention, the novel Lactobacillus fermentum ( Lactobacillus fermentum ) J2 strain producing the cinnamoyl esterase (Cinnamoyl esterase) of the present application, at least one selected from the group consisting of the crushed material and the strain culture Provided is a functional food composition comprising as an active ingredient.
본 발명의 다른 측면에 의하면, 본원의 시나모일 에스터라제를 유효성분으로 포함하는 기능성 식품 조성물이 제공된다.According to another aspect of the present invention, there is provided a functional food composition comprising the cinnamoyl esterase of the present application as an active ingredient.
본 발명의 다른 측면에 의하면, 본원의 시나모일 에스터라아제(Cinnamoyl esterase)를 생산하는 신규한 락토바실러스 퍼멘텀(Lactobacillus fermentum) J2 균주, 이의 파쇄물 및 상기 균주 배양물로 이루어진 군에서 선택된 하나 이상을 유효성분으로 포함하는 사료용 조성물이 제공된다.According to another aspect of the present invention, the novel Lactobacillus fermentum ( Lactobacillus fermentum ) J2 strain producing the cinnamoyl esterase (Cinnamoyl esterase) of the present application, at least one selected from the group consisting of the crushed material and the strain culture Provided is a feed composition comprising as an active ingredient.
본 발명의 다른 측면에 의하면, 본원의 시나모일 에스터라제를 유효성분으로 포함하는 사료용 조성물이 제공된다.According to another aspect of the present invention, there is provided a feed composition comprising the cinnamoyl esterase of the present application as an active ingredient.
일 실시예에 의하면, 본원의 락토바실러스 퍼멘텀 J2 균주(KACC 92245P)는 타 균주의 시나모일 에스터라아제에 비하여, 활성이 우수한 시나모일 에스터라아제를 고효율로 생산할 수 있다.According to an embodiment, the Lactobacillus performance J2 strain (KACC 92245P) of the present application can produce cinnamoyl esterase having excellent activity, with high efficiency, compared to cinnamoyl esterase of other strains.
일 실시예에 의하면, 본원의 락토바실러스 퍼멘텀 J2 균주(KACC 92245P)로부터 유래한, 에틸 페룰레이트에 대해 기질특이성 및 분해율이 개선되어 활성이 우수한 시나모일 에스터라아제를 제공할 수 있다. 본원의 시나모일 에스터라아제는 에틸 페룰레이트에 85% 이상의 높은 분해율을 나타낸다. According to an embodiment, the substrate specificity and decomposition rate of ethyl ferulate derived from the Lactobacillus performance J2 strain (KACC 92245P) of the present application may be improved to provide a cinnamoyl esterase having excellent activity. The cinnamoyl esterase herein exhibits a high decomposition rate of at least 85% in ethyl ferulate.
일 실시예에 의하면, 에틸 페룰레이트에서 페룰산으로의 전환율이 개선된 시나모일 에스터라아제를 제공할 수 있다. 본원의 시나모일 에스터라아제는 에틸 페룰레이트에서 페룰산으로의 전환율이 90% 이상을 나타낸다. According to an embodiment, it is possible to provide a cinnamoyl esterase having an improved conversion rate from ethyl ferulate to ferulic acid. The cinnamoyl esterase herein has a conversion rate of ethyl ferulate to ferulic acid of 90% or more.
일 실시예에 의하면, 락토바실러스 퍼멘텀(Lactobacillus fermentum) J2 균주(KACC 92245P)에서 유래하는 시나모일 에스터라아제(Cinnamoyl esterase)를 포함하는 기능성 식품 조성물을 제공할 수 있다.According to one embodiment, it can provide a functional food composition comprising cinnamoyl esterase derived from Lactobacillus fermentum J2 strain (KACC 92245P).
일 실시예에 의하면, 락토바실러스 퍼멘텀(Lactobacillus fermentum) J2 균주(KACC 92245P)에서 유래하는 시나모일 에스터라아제(Cinnamoyl esterase)를 포함하는 사료용 조성물을 제공할 수 있다.According to one embodiment, it is possible to provide a feed composition comprising cinnamoyl esterase derived from Lactobacillus fermentum J2 strain (KACC 92245P).
본 발명의 락토바실러스 퍼멘텀(Lactobacillus fermentum) J2 균주(KACC 92245P)에서 유래한 시나모일 에스터라아제(Cinnamoyl esterase)는 과채류 및 식품 속에 다량으로 존재하는 고흡수성 기능성 항산화 물질인 에틸 페룰레이트의 에스테르 결합을 한 형태를 인체에 흡수가 용이한 페룰산 형태로 효율적으로 전환시킬 수 있는 바, 항산화 물질의 흡수를 강화하여 인간 및 동물의 건강에 기여할 수 있다.Cinnamoyl esterase derived from Lactobacillus fermentum J2 strain (KACC 92245P) of the present invention is ester bond of ethyl ferulate, a superabsorbent and functional antioxidant present in large amounts in fruits and vegetables. Since it can efficiently convert one form into ferulic acid form, which is easily absorbed by the human body, it can contribute to the health of humans and animals by enhancing the absorption of antioxidants.
도 1은 에틸 페룰레이트(Ethyl ferulate)가 함유된 MRS plate를 이용하여 시나모일 에스테라아제 활성 유산균을 선별하기 위하여, 투명환(Clear zone or halo)을 보인 균주를 나타낸 사진이다.
도 2a는 에틸 페룰레이트와 페룰산을 UPLC를 이용하여, 분석한 크로마토그램이다.
도 2b는 페룰산의 표준곡선을 나타내는 그래프이다.
도 2c는 에틸 페룰레이트의 표준곡선을 나타내는 그래프이다.
도 3a는 UPLC를 이용한 시나모일 에스터라아제 활성을 정량적으로 분석한 결과를 나타낸 그래프이다.
도 3b는 락토바실러스 퍼멘텀 J2 및 L. helveticus KCCM 11223의 시나모일 에스터라아제의 활성을 UPLC 이용하여 분석한 크로마토그램이다.
도 4는 선발된 J2의 16s rDNA 염기서열을 이용한 계통도이다.
도 5a는 락토바실러스 퍼멘텀 J2에서 유래된 시나모일 에스터라아제 유전자의 전기영동 결과 사진이다.
도 5b는 락토바실러스 퍼멘텀 J2에서 유래된 시나모일 에스터라아제 유전자의 염기서열을 나타낸다.
도 6a는 락토바실러스 퍼멘텀 J2의 시나모일 에스터라아제 및 종래의 락토바실러스 퍼멘텀(AGW21364.1)의 시나모일 에스터라아제의 아미노산 서열을 비교한 도면이다.
도 6b는 락토바실러스 퍼멘텀 J2의 시나모일 에스터라아제 및 종래의 락토바실러스 퍼멘텀(AGW21364.1)의 시나모일 에스터라아제의 상동성을 비교한 도면이다.
도 7은 락토바실러스 퍼멘텀 J2의 시나모일 에스터라아제의 기질 특이성을 보여주는 그래프이다.
도 8는 락토바실러스 퍼멘텀 J2의 생육곡선과 페룰산의 생산 곡선을 나타낸 그래프이다.
도 9은 초기 배양 시의 pH에 따른 시나모일 에스터라아제 활성을 나타낸 그래프이다.
도 10은 배양온도에 따른 시나모일 에스터라아제 활성을 나타낸 그래프이다.1 is a photograph showing a strain showing a clear ring (Clear zone or halo), in order to screen for cinnamoyl esterase-activated lactic acid bacteria using MRS plate containing ethyl ferulate (Ethyl ferulate).
Figure 2a is a chromatogram of ethyl ferulate and ferulic acid analyzed using UPLC.
2B is a graph showing the standard curve of ferulic acid.
2C is a graph showing the standard curve of ethyl ferulate.
Figure 3a is a graph showing the results of quantitative analysis of cinnamoyl esterase activity using UPLC.
Figure 3b is a chromatogram of the activity of the cinnamoyl esterase of Lactobacillus performance J2 and L. helveticus KCCM 11223 using UPLC.
Figure 4 is a schematic diagram using the selected 16 s rDNA sequence of J2.
Figure 5a is a photo of the results of electrophoresis of the cinnamoyl esterase gene derived from Lactobacillus performance J2.
Figure 5b shows the nucleotide sequence of the cinnamoyl esterase gene derived from Lactobacillus performance J2.
Figure 6a is a diagram comparing the amino acid sequence of the cinnamoyl esterase of Lactobacillus performance J2 and the cinnamoyl esterase of the conventional Lactobacillus performance (AGW21364.1).
6B is a diagram comparing the homology of cinamoyl esterase of Lactobacillus performance J2 and cinnamoyl esterase of conventional Lactobacillus performance (AGW21364.1).
7 is a graph showing the substrate specificity of cinnamoyl esterase of Lactobacillus performance J2.
8 is a graph showing the growth curve of Lactobacillus performance J2 and the production curve of ferulic acid.
9 is a graph showing cinnamoyl esterase activity according to pH during initial culture.
10 is a graph showing cinnamoyl esterase activity according to the culture temperature.
본원에서 달리 정의하지 않는 한, 본 발명과 관련하여 사용된 과학 용어 및 기술 용어들은 통상의 기술자에 의해 일반적으로 이해되는 의미를 가질 것이다. 뿐만 아니라, 문맥상 특별히 지정하지 않는 한, 단수 형태의 용어는 그것의 복수 형태도 포함하는 것이며, 복수 형태의 용어는 그것의 단수 형태도 포함할 것이다. 일반적으로, 세포 및 조직 배양, 분자 생물학, 면역학, 미생물학, 유전학 및 단백질과 핵산 화학, 그리고 혼성화와 관련되어 사용되는 명명법과 이와 관련된 기술들은 통상의 기술자에게 널리 공지되어 있는 것이며, 또한 본 발명이 속하는 기술분야에서 일반적으로 사용되고 있는 것이다.Unless defined otherwise herein, scientific and technical terms used in connection with the present invention will have the meanings generally understood by one of ordinary skill in the art. In addition, unless otherwise specified in context, singular terms shall include their plural forms, and plural terms shall also include their singular forms. In general, the nomenclature used in connection with cell and tissue culture, molecular biology, immunology, microbiology, genetics and protein and nucleic acid chemistry, and hybridization and related techniques are well known to those skilled in the art, and the invention also belongs to It is generally used in the technical field.
이하, 본 발명에서 사용한 용어를 설명한다.Hereinafter, terms used in the present invention will be described.
본 발명에서 사용되는 용어 "배양물"이란 미생물을 공지의 액체 배지 또는 고체 배지에서 배양시켜 수득한 산물을 의미하며, 미생물이 포함되는 개념이다.The term "culture" as used in the present invention means a product obtained by culturing a microorganism in a known liquid medium or solid medium, and is a concept in which the microorganism is included.
본 발명에서 "사료"란 개체의 생명을 유지하고 상기 개체를 사육하는데 필요한 유기 또는 무기 영양소를 공급하는 물질을 의미한다. 상기 사료는 사료를 섭취하는 개체가 필요로 하는 에너지, 단백질, 지질, 비타민, 광물질 등 영양소를 포함할 수 있으나, 특별히 이에 한정되지 않으며, 상기 개체는 사육 대상을 의미하는 것으로 본 발명의 사료를 섭취할 수 있는 생명체라면 제한 없이 포함되며, 본 발명의 목적상 특히 곤충 및 가축을 포함한다. In the present invention, "feed" refers to a substance that maintains the life of an individual and supplies organic or inorganic nutrients necessary for rearing the individual. The feed may include nutrients such as energy, protein, lipids, vitamins, and minerals required by the individual who consumes the feed, but is not particularly limited thereto, and the individual ingests the feed of the present invention as a breeding target. As long as it is possible, it is included without limitation, and for the purpose of the present invention, especially insects and livestock.
이하, 본 발명을 상세하게 설명한다.Hereinafter, the present invention will be described in detail.
본 발명자들은 시나모일 에스터라아제를 고효율로 생산하는 새로운 유산균 및 이로부터 유래한 활성이 우수한 시나모일 에스터라아제를 찾고자 연구한 결과, 락토바실러스 퍼멘텀(Lactobacillus fermentum) J2 균주(KACC 92245P)에서 유래하는 시나모일 에스터라아제(Cinnamoyl esterase)와 이를 암호화하는 유전자를 분리함으로써 본 발명을 완성하였다.The present inventors researched to find a new lactic acid bacterium that produces cinnamoyl esterase with high efficiency and a cinnamoyl esterase having excellent activity derived therefrom, as a result of Lactobacillus fermentum J2 strain (KACC 92245P) The present invention was completed by separating the cinnamoyl esterase and the gene encoding the same.
본 발명의 일 측면에 의하면, 시나모일 에스터라아제(Cinnamoyl esterase)를 생산하는 락토바실러스 퍼멘텀(Lactobacillus fermentum) J2 균주(KACC 92245P)가 제공된다. According to an aspect of the present invention, a Lactobacillus fermentum J2 strain (KACC 92245P) producing cinnamoyl esterase is provided.
본 발명에 있어서, "락토바실러스 퍼멘텀(Lactobacillus fermentum)"은 그람 양성균으로 락토바실러스속에 속하며, 젖산을 생산할 수 있는 간균이다.In the present invention, " Lactobacillus fermentum ( Lactobacillus fermentum )" is a Gram-positive bacterium belonging to the genus Lactobacillus, a bacterium capable of producing lactic acid.
생후 2주 이내의 신생아 분변에서 시나모일 에스터라아제를 고효율로 생산하는 균주를 분리 및 선발하였고, 분리한 균주를 16S rRNA 분석을 조사하여 락토바실러스 퍼멘텀 J2 균주(KACC 92245P)로 동정하였다. 상기 동정된 균주를 2018. 10. 19.자로 농업생명공학연구원에 기탁번호 KACC 92245P로 기탁하였다.Strains producing cinnamoyl esterase with high efficiency were isolated and selected from newborn feces within 2 weeks of age, and the isolated strains were identified by Lactobacillus performance J2 strain (KACC 92245P) by examining 16S rRNA analysis. The identified strains were deposited with the Accession No. KACC 92245P to the Institute of Agricultural Biotechnology on October 19, 2018.
본 발명의 일 실시예에 의하면, 상기 락토바실러스 퍼멘텀(Lactobacillus fermentum) J2 균주(KACC 92245P)는 서열번호 1로 기재되는 16s rRNA로 구성되는 것을 특징으로 하는 락토바실러스 퍼멘텀(Lactobacillus fermentum) J2 균주(KACC 92245P)가 제공된다.According to an embodiment of the present invention, the Lactobacillus fermentum ( Lactobacillus fermentum ) J2 strain (KACC 92245P) is composed of 16s rRNA of SEQ ID NO: 1, Lactobacillus fermentum ( Lactobacillus fermentum ) J2 strain (KACC 92245P) is provided.
상기 락토바실러스 퍼멘텀(Lactobacillus fermentum) J2 균주(KACC 92245P)는 시나모일 에스터라아제를 타 균주에 비하여 고효율로 생산할 수 있다.The Lactobacillus fermentum J2 strain (KACC 92245P) can produce cinnamoyl esterase with higher efficiency than other strains.
상기 락토바실러스 퍼멘텀 J2 균주(KACC 92245P)로부터 유래한 시나모일 에스터라아제는 에틸 페룰레이트를 페룰산으로 90% 이상 전환할 수 있다. 이는 종래의 공지의 락토바실러스 헬베티커스(L. heveticus KCCM 11223) 균주로부터 유래한 시나모일 에스테라아제가 에틸 페룰레이트를 60% 정도 전환할 수 있는 것에 비해, 페룰산으로의 전환율을 매우 높은 것이다(도 3a). 따라서, 상기 락토바실러스 퍼멘텀 J2 균주(KACC 92245P)로부터 유래한 시나모일 에스터라아제는 타 균주에 비하여, 시나모일 에스터라아제의 농도가 높으며, 에틸 페룰레이트를 페룰산으로의 전환 활성이 우수하다(도 3a).Cinnamoyl esterase derived from the Lactobacillus performance J2 strain (KACC 92245P) can convert ethyl ferulate to ferulic acid by 90% or more. This is a very high conversion rate to ferulic acid, compared to 60% conversion of ethyl ferulate by cinnamoyl esterase derived from the conventionally known Lactobacillus helveticus ( L. heveticus KCCM 11223) strain (Fig. 3a). Therefore, the cinnamoyl esterase derived from the Lactobacillus performance J2 strain (KACC 92245P) has a higher concentration of cinnamoyl esterase than other strains, and has excellent conversion activity of ethyl ferulate to ferulic acid. (Figure 3a).
본 발명의 다른 측면에 따르면, 락토바실러스 퍼멘텀(Lactobacillus fermentum) J2 균주(KACC 92245P)로부터 생산된, 시나모일 에스터라아제가 제공된다.According to another aspect of the present invention, there is provided a cinnamoyl esterase produced from Lactobacillus fermentum J2 strain (KACC 92245P).
본 발명에서 시나모일 에스터라아제란 에틸 페룰레이트(ethyl ferulate)를 페룰산(ferulic acid)으로 가수분해시킬 수 있는 효소이다(도 1). 에틸 페룰레이트는 장내 미생물에 의하여 페룰산의 형태로 흡수되지만 그 작용이 약하여 대부분 흡수되지 못하고 배출된다. 그에 반하여 에틸 페룰레이트의 가수분해 형태인 페룰산은 에틸 페룰레이트에 비하여 인체 흡수력이 높고 뛰어난 항산화 효과를 나타낸다.Cinnamoyl esterase in the present invention is an enzyme capable of hydrolyzing ethyl ferulate to ferulic acid (FIG. 1). Ethyl ferulate is absorbed in the form of ferulic acid by microorganisms in the intestine, but its action is weak, so it is mostly absorbed and discharged. On the other hand, ferulic acid, which is a hydrolyzed form of ethyl ferulate, has a higher absorbency in the human body than ethyl ferulate and exhibits an excellent antioxidant effect.
본 발명의 일 실시예에 의하면, 상기 시나모일 에스터라아제는 서열번호 5으로 기재되는 아미노산 서열을 포함하는 것을 특징으로 하는, 시나모일 에스터라아제가 제공된다.According to one embodiment of the present invention, the cinnamoyl esterase is characterized in that it comprises the amino acid sequence shown in SEQ ID NO: 5, cinnamoyl esterase is provided.
본 발명의 일 실시예에 의하면, 상기 시나모일 에스터라아제는 에틸 페룰레이트(ethyl ferulate)에 대한 기질 특이성이 향상되며, 에틸 페룰레이트(ethyl ferulate)에 대한 85% 이상의 분해율을 나타낸다.According to an embodiment of the present invention, the cinnamoyl esterase improves substrate specificity for ethyl ferulate and exhibits a decomposition rate of at least 85% for ethyl ferulate.
본 발명의 다른 측면에 의하면, 시나모일 에스터라아제를 암호화하는 유전자가 제공된다.According to another aspect of the present invention, a gene encoding cinnamoyl esterase is provided.
본 발명의 일 실시예에 의하면, 상기 유전자는 서열번호 2로 기재되는 폴리뉴클레오티드로 구성된다.According to one embodiment of the present invention, the gene is composed of the polynucleotide of SEQ ID NO: 2.
락토바실러스 퍼멘텀 J2 균주(KACC 92245P)는 게놈 DNA에서 예상되는 시나모일 에스터라아제를 PCR로 증폭하여 염기서열 분석을 실시한 결과, 본 발명의 시나모일 에스터라아제는 서열번호 2로 기재되는 폴리뉴클레오티드 서열을 가진다는 것임을 알 수 있었다.The Lactobacillus performance J2 strain (KACC 92245P) was amplified by sequencing by PCR amplifying the cinnamoyl esterase expected in genomic DNA, and as a result, the cinnamoyl esterase of the present invention was a polynucleotide of SEQ ID NO: 2. It can be seen that it has a sequence.
본 발명의 다른 측면에 의하면, 시나모일 에스터라아제를 암호화하는 유전자의 DNA를 증폭하기 위한 서열번호 3 및 4로 이루어진 프라이머 세트가 제공된다.According to another aspect of the present invention, there is provided a primer set consisting of SEQ ID NOs: 3 and 4 for amplifying the DNA of a gene encoding cinnamoyl esterase.
본 발명의 다른 측면에 의하면, (1) 상기 시나모일 에스터라아제(Cinnamoyl esterase)를 생산하는 락토바실러스 퍼멘텀(Lactobacillus fermentum) J2 균주를 배양하는 단계; 및 (2) 상기 (1) 단계의 배양액으로부터 시나모일 에스터라아제(Cinnamoyl esterase)를 분리하는 단계를 포함하는, 시나모일 에스터라아제의 제조 방법이 제공된다. According to another aspect of the invention, (1) culturing the Lactobacillus fermentum ( Lactobacillus fermentum ) J2 strain to produce the cinnamoyl esterase (Cinnamoyl esterase); And (2) comprising the step of separating the cinnamoyl esterase (Cinnamoyl esterase) from the culture of the step (1), a method for producing cinnamoyl esterase is provided.
본 발명의 일 실시예에 의하면, 상기 (1) 단계에서, 시나모일 에스터라아제는 락토바실러스 퍼멘텀 J2 균주를 45시간 내지 96시간 동안 배양하였을 때, 최대 활성을 나타낸다. 이에 한정되는 것은 아니나, 45 내지 50시간 동안 배양하는 것이 더 적합할 수 있다.According to an embodiment of the present invention, in step (1), cinnamoyl esterase exhibits maximum activity when the Lactobacillus performance J2 strain is cultured for 45 to 96 hours. Without being limited thereto, culturing for 45 to 50 hours may be more suitable.
본 발명의 일 실시예에 의하면, 상기 시나모일 에스터라아제는 pH 6.5 내지 7.5에서 최대 활성을 나타낸다. 이에 한정되는 것은 아니나, pH 6.8 내지 pH 7.2으로 배양하는 것이 더 적합할 수 있다. 통상적으로 유산균은 성장과 동시에 젖산을 생성함에 따라 배지의 pH가 4 내지 4.5로 낮아지기 때문에 시나모일 에스터라아제의 경우에 pH 3.0 미만에서는 매우 낮은 페룰산 전환율을 나타내었고, pH 9.0에서는 페룰산으로 전환되지 않았다. 이는 시나모일 에스터라아제 단백질의 변성으로 매우 낮은 pH에서 활성을 나타내지 않은 것으로 보여진다.According to an embodiment of the present invention, the cinnamoyl esterase exhibits maximum activity at pH 6.5 to 7.5. Without being limited thereto, culturing at pH 6.8 to pH 7.2 may be more suitable. Normally, the lactic acid bacteria showed a very low ferulic acid conversion rate below pH 3.0 in the case of cinnamoyl esterase because the pH of the medium was lowered to 4 to 4.5 as it produced lactic acid at the same time as growth, and converted to ferulic acid at pH 9.0. Did not. It is shown that denaturation of the cinnamoyl esterase protein did not show activity at very low pH.
본 발명의 일 실시예에 의하면, 상기 시나모일 에스터라아제는 30℃ 내지 40℃에서 최대 활성을 나타낸다. 이에 한정되는 것은 아니나, 35℃ 내지 40℃의 조건이 더 적합할 수 있다.According to an embodiment of the present invention, the cinnamoyl esterase exhibits maximum activity at 30°C to 40°C. Without being limited thereto, conditions of 35°C to 40°C may be more suitable.
본 발명의 일 실시예에 의하면, 상기 시나모일 에스터라아제는 에틸 페룰레이트(ethyl ferulate)에 대한 기질 특이성이 향상되며, 에틸 페룰레이트(ethyl ferulate)에 대한 85% 이상의 분해율을 나타낸다.According to an embodiment of the present invention, the cinnamoyl esterase has improved substrate specificity for ethyl ferulate and exhibits a decomposition rate of at least 85% for ethyl ferulate.
본 발명의 일 실시예에 의하면, 상기 시나모일 에스터라아제는 에틸 페룰레이트(ethyl ferulate)에서 페룰산으로의 90% 이상의 전환율을 나타낼 수 있다.According to an embodiment of the present invention, the cinnamoyl esterase may exhibit a conversion rate of at least 90% from ethyl ferulate to ferulic acid.
본 발명의 다른 측면에 의하면, 시나모일 에스터라아제(Cinnamoyl esterase)를 생산하는 신규한 락토바실러스 퍼멘텀(Lactobacillus fermentum) J2 균주, 이의 파쇄물 및 상기 균주 배양물로 이루어진 군에서 선택된 하나 이상을 유효성분으로 포함하는 기능성 식품 조성물이 제공된다.According to another aspect of the present invention, a novel Lactobacillus fermentum ( Lactobacillus fermentum ) J2 strain that produces cinnamoyl esterase, one or more selected from the group consisting of the above-described strain culture, and an active ingredient It provides a functional food composition comprising a.
본 발명의 다른 측면에 의하면, 상기 시나모일 에스터라제를 유효성분으로 포함하는 기능성 식품 조성물이 제공된다.According to another aspect of the present invention, there is provided a functional food composition comprising the cinnamoyl esterase as an active ingredient.
또한, 본 발명의 기능성 식품 조성물에는, 필요에 따라서, 예를 들면 하기의 첨가제의 1종 또는 2종 이상을 첨가 배합할 수도 있다. 상기 첨가제에는, 예를 들면 그레이프프루트, 사과, 오렌지, 레몬, 파인애플, 바나나, 배 등의 각종 과즙(농축 과즙, 분말 과즙 등이어도 좋다); 비타민류 및 프로비타민류(팔미트산 레티놀, 비스벤티아민(bisbentiamine), 리보플라빈, 염산 피리독신, 시아노코발아민(cyanocobalamine), 아스코르빈산 나트륨, 니코틴산 아미드, 판토텐산 칼슘, 엽산, 비오틴, 콜레칼시페롤(cholecalciferol), 중주석산 콜린, 토코페롤, β-카로틴 등의 수용성 및 지용성 비타민류); 향미료(레몬플레이버, 오렌지플레이버, 그레이프프루트플레이버, 바닐라 에센스 등); 아미노산, 핵산 및 그들의 염류(글루탐산, 글루탐산나트륨, 글리신, 알라닌, 아스파라긴산, 아스파라긴산 나트륨, 이노신산 등); 식물 섬유(폴리덱스트로오즈, 펙틴, 크산탄 고무, 아라비아 고무검, 알긴산 등); 미네랄 내지 미량 원소(염화 나트륨, 초산 나트륨, 황산 마그네슘, 염화 칼륨, 염화 마그네슘, 탄산 마그네슘, 염화 칼슘, 인산 2칼륨, 인산 1나트륨, 글리세로인산 칼슘, 구연산제1철 나트륨, 구연산철 암모늄, 구연산철, 황산망간, 황산구리, 요오드화나트륨, 솔빈산칼륨, 아연, 망간, 구리, 요오드, 코발트 등) 등이 포함된다.In addition, the functional food composition of the present invention may be blended with one or two or more of the following additives, if necessary. Examples of the additives include various juices such as grapefruit, apple, orange, lemon, pineapple, banana, and pear (concentrated juice, powder juice, etc.); Vitamins and provitamins (retinol palmitate, bisbentiamine, riboflavin, pyridoxine hydrochloride, cyanocobalamine, sodium ascorbate, nicotinic acid amide, calcium pantothenate, folic acid, biotin, cholecalcici Water-soluble and fat-soluble vitamins such as cholecalciferol, choline citrate, tocopherol, and β-carotene); Spices (lemon flavor, orange flavor, grapefruit flavor, vanilla essence, etc.); Amino acids, nucleic acids and their salts (glutamic acid, sodium glutamate, glycine, alanine, aspartic acid, sodium aspartate, inosinic acid, etc.); Plant fiber (polydextrose, pectin, xanthan rubber, gum arabic gum, alginic acid, etc.); Mineral to trace elements (sodium chloride, sodium acetate, magnesium sulfate, potassium chloride, magnesium chloride, magnesium carbonate, calcium chloride, dipotassium phosphate, monosodium phosphate, calcium glycerophosphate, ferric sodium citrate, ammonium citrate, citric acid Iron, manganese sulfate, copper sulfate, sodium iodide, potassium sorbate, zinc, manganese, copper, iodine, cobalt, etc.).
본 발명의 식품조성물은, 상기 각 성분을 혼합하여 조제된다. 그 조제 방법은 특별히 제한되는 것은 아니고, 필요한 성분을 모두 동시에 혼합해도 좋고, 또한 유성 성분과 수성 성분의 양자를 사용하는 경우에는, 미리 유성 성분을 적당한 유제에 용해하고, 이것과 수용성 성분의 수용액을 유화제를 이용하여 유화할 수도 있다. 상기 유화 조작은, 일반적인 방법에 따라서, 통상의 유화 분산기, 예를 들면 호모믹서, 고압 호모디나이저 등을 이용하여, 완전 통과 방식 또는 순환 방식에 따라서 실시할 수 있다. 또한, 상기 각 성분의 혼합 내지 유화는, 통상 상온하에서 실시되지만, 가온 조작을 채용할 수도 있다. 이렇게 해서 조제되는 본 발명 조성물은, 이것을 적당한 용기에 충전 후, 통상적인 방법에 따라서, 가열 멸균, 무균 여과 등에 의해 멸균한 후 제품으로 제공된다.The food composition of the present invention is prepared by mixing the above components. The preparation method is not particularly limited, and all necessary components may be mixed at the same time, and when both oily and aqueous components are used, the oily component is previously dissolved in a suitable oily agent, and an aqueous solution of this and water-soluble components is prepared. It can also be emulsified using an emulsifier. The emulsification operation can be carried out in accordance with a general method, using a conventional emulsifying and dispersing machine, for example, a homomixer, a high pressure homogenizer, or the like, in a full-pass method or a circulating method. In addition, although mixing or emulsification of each component is usually performed at room temperature, a heating operation can also be employed. The composition of the present invention thus prepared is provided as a product after filling it in a suitable container and sterilizing it by heat sterilization, aseptic filtration, or the like according to a conventional method.
본 발명의 다른 측면에 의하면, 상기 시나모일 에스터라아제(Cinnamoyl esterase)를 생산하는 신규한 락토바실러스 퍼멘텀(Lactobacillus fermentum) J2 균주, 이의 파쇄물 및 상기 균주 배양물로 이루어진 군에서 선택된 하나 이상을 유효성분으로 포함하는 사료용 조성물이 제공된다.According to another aspect of the present invention, one or more selected from the group consisting of a novel Lactobacillus fermentum J2 strain producing its cinnamoyl esterase, its lysate and the strain culture is effective Provided is a feed composition comprising as an ingredient.
본 발명의 다른 측면에 의하면, 상기 시나모일 에스터라아제를 유효성분으로 포함하는 사료용 조성물이 제공된다.According to another aspect of the present invention, there is provided a feed composition comprising the cinnamoyl esterase as an active ingredient.
본 발명의 사료용 조성물은, 개 또는 고양이 사료용일 수 있다. 이에 한정되는 것은 아니며, 개, 고양이 등 다양한 반려동물에 적용할 수도 있다.The feed composition of the present invention may be for dog or cat food. The present invention is not limited thereto, and may be applied to various companion animals such as dogs and cats.
본 발명의 사료 조성물은, 기능성 물질을 추가로 더 포함할 수 있다.The feed composition of the present invention may further include a functional substance.
상기 기능성 물질은 DHA, EPA, 알로에, 스쿠알렌, 옥수수수염, 가르시니아캄보지아, 히비스커스, 대나무잎, 클로렐라, 스피루리나, 부추, 녹차, 둥글레, 감잎, 뽕잎, 작설, 현미, 보리, 루이보스, 보이, 및 결명자 중에서 선택된 어느 하나 이상을 사용할 수 있다. 이에 한정되는 것은 아니나, 상기 기능성 물질은 사료조성물 전체 중량 대비 0.1∼10 중량부가 되도록 첨가할 수 있다.The functional substance is DHA, EPA, aloe, squalene, corn beard, garcinia cambogia, hibiscus, bamboo leaf, chlorella, spirulina, leek, green tea, roundle, persimmon leaf, mulberry leaf, rice bran, brown rice, barley, rooibos, boy, and terminator Any one or more selected can be used. Although not limited thereto, the functional material may be added in an amount of 0.1 to 10 parts by weight based on the total weight of the feed composition.
본 발명의 사료조성물은 종래 사료조성물에 첨가되는 성분을 더 포함할 수 있다. 이러한 사료조성물에 첨가되는 성분의 일예로서 곡류분말, 고기분말, 및 두류 등을 포함할 수 있다.The feed composition of the present invention may further include a component added to a conventional feed composition. Examples of ingredients added to the feed composition may include grain powder, meat powder, and soybeans.
상기에서 고기분말은 이에 한정되는 것은 아니나, 닭고기, 소고기, 돼지고기, 및 타조고기 중에서 선택된 어느 하나 이상을 분말화한 고기분말을 사용할 수 있다.In the above, the meat powder is not limited to this, but any one or more selected from chicken, beef, pork, and ostrich meat powder may be used.
상기에서 두류는 이에 한정되는 것은 아니나, 대두, 강낭콩, 완두콩, 및 검정콩 중에서 선택된 어느 하나 이상을 사용할 수 있다.In the above, the beans are not limited thereto, and any one or more selected from soybeans, kidney beans, peas, and black beans may be used.
본 발명의 사료조성물은 상기에서 언급한 종래 사료조성물에 첨가되는 성분인 곡류분말, 고기분말, 및 두류 이외에도 사료의 영양성을 증대시키기 위해 영양제, 및 무기물 중에서 선택된 어느 하나 이상을 첨가할 수 있으며, 사료 품질의 저하를 막기 위해 항곰팡이제, 항산화제, 항응고제, 유화제, 및 결착제 중에서 선택된 어느 하나 이상을 포함할 수 있다.The feed composition of the present invention, in addition to the grain powder, meat powder, and soybean, which are components added to the above-mentioned conventional feed composition, may be added any one or more selected from nutritional agents and minerals to increase the nutritional properties of the feed, In order to prevent the degradation of quality, it may include any one or more selected from antifungal agents, antioxidants, anticoagulants, emulsifiers, and binders.
상기에서 언급한 종래 사료조성물의 성분은 당업자가 적의 선택하여 실시할 수 있고, 본 발명에서 필수 구성성분과는 다소 차이가 있으므로 이하 본 발명에서 자세한 내용은 생략하기로 한다.The above-mentioned components of the conventional feed composition can be selected by a person skilled in the art, and there are some differences from essential components in the present invention.
이하, 본 발명을 실시예에 의거하여 보다 구체적으로 설명한다. 실시예는 오로지 본 발명을 보다 구체적으로 설명하기 위한 것으로, 본 발명의 요지에 따라 발명의 범위가 이들 실시예에 의해 제한되지 않는다는 것은 당업계에서 통상의 지식을 가진 자에 있어서 자명할 것이다.Hereinafter, the present invention will be described in more detail based on examples. The examples are only intended to illustrate the present invention in more detail, and it will be apparent to those skilled in the art that the scope of the invention is not limited by these examples according to the gist of the present invention.
실시예Example
1.One. 락토바실러스 퍼멘텀(Lactobacillus performance ( Lactobacillus fermentumLactobacillus fermentum ) J2 균주(KACC 92245P)의 분리 및 선발) Isolation and selection of J2 strain (KACC 92245P)
총 15개의 신생아 분변 시료 약 1 g을 회수 후 바로 50 ml 의 멸균된 Falcon tube에 옮긴 후, 혐기적 상태에서 9 ml의 펩톤수(peptone water)에 희석하였다. 희석 샘플로부터 시나모일 에스테라아제 활성 유산균을 분리하기 위하여 연속적으로 10배씩 희석액을 준비한 후 에틸 페룰레이트(0.1%, v/v)가 첨가된 유산균 선택배지(BCP: bromocresol purple agar powder)에 100ul씩 도말하였다. 37℃에서 72시간 동안 배양하여 균주의 성장과 동시에 생성되는 효소활성에 의하여 에틸 페룰레이트가 분해되어 형성된 투명환(Clear zone or halo)을 보인 균주를 CE 활성 균주로 추정 분리하였으며, 그 중 환의 크기가 직경 3mm 이상인 균주 5개를 시나모일 에스테라아제 고활성 후보 유산균으로 선발하였다(도 1).Approximately 1 g of 15 newborn fecal samples were immediately transferred to a 50 ml sterile Falcon tube immediately after recovery, and then diluted in 9 ml of peptone water in an anaerobic state. In order to separate the cinnamoyl esterase-activated lactic acid bacteria from the diluted sample, dilutions were prepared 10 times in succession, and then 100 ul were plated on lactic acid bacteria selective medium (BCP: bromocresol purple agar powder) to which ethyl ferulate (0.1%, v/v) was added . A strain showing a clear zone or halo formed by decomposition of ethyl ferulate by enzymatic activity generated at the same time as the growth of the strain by culturing at 37° C. for 72 hours was presumed to be separated as a CE active strain, and the size of the ring Five strains having a diameter of 3 mm or more were selected as cinnamoyl esterase highly active candidate lactic acid bacteria (FIG. 1).
2. 에틸 페룰레이트(Ethyl ferulate)와 페룰산(ferulic acid)의 분석 방법2. Ethyl ferulate and ferulic acid analysis method
시나모일 에스터라아제 효소 활성은 에틸 페룰레이트를 기질로 하여 유산균 발효를 통해 생성된 페룰산(ferulic acid)의 양으로 측정되었다. 이를 위한 분석용 시료는 다음과 같이 준비하였다. Cinnamoyl esterase enzyme activity was measured by the amount of ferulic acid produced through lactic acid bacteria fermentation using ethyl ferulate as a substrate. Samples for analysis for this were prepared as follows.
배양액 5 ml를 0.1% 아스코르브산(w/v, Sigma, USA)을 첨가하여 잘 혼합한 후 3 ml의 에틸 페룰레이트를 첨가하여 10분간 강하게 볼텍싱(votexing)하였다. 그 후 4℃에서 10분간 원심분리(3,000rpm, 1580MGR Gyrozen)하여 에틸 페룰레이트와 페룰산이 포함된 유기용매 층을 회수하였다. 회수된 추출액은 45℃에서 완전히 건조시킨 후 1 ml 메탄올에 녹인 다음 0.20 μm 멤브레인 필터로 여과하여 UPLC/PDA(waters, ACQUITY UPLC SYSTEM)의 분석시료로 사용하였다. 표준물질로서 에틸 페룰레이트와 페룰산은 필요한 농도별로 메탄올에 녹여 -20℃에 냉동보관하여 정량분석에 사용하였다.5 ml of the culture solution was mixed well by adding 0.1% ascorbic acid (w/v, Sigma, USA) and then vortexed strongly for 10 minutes by adding 3 ml of ethyl ferulate. Thereafter, centrifugation was performed at 4°C for 10 minutes (3,000 rpm, 1580 MGR Gyrozen) to recover an organic solvent layer containing ethyl ferulate and ferulic acid. The recovered extract was completely dried at 45° C., dissolved in 1 ml methanol, filtered through a 0.20 μm membrane filter, and used as an analysis sample for UPLC/PDA (waters, ACQUITY UPLC SYSTEM). As standard, ethyl ferulate and ferulic acid were dissolved in methanol at the required concentration and stored frozen at -20°C for use in quantitative analysis.
본원에서는 정성적 또는 정량적으로 에틸 페룰레이트 및 페룰산을 분석하기 위하여 UPLC/PDA(waters, ACQUITY UPLC SYSTEM) 분석법을 확립하였다. 분석 결과 에틸 페룰레이트는 2.1분, 페룰산은 3.45분에서 머무름 시간을 나타내었다(도 2). 도 2a는 페룰산와 에틸 페룰레이트의 UPLC 크로마토그램이고, 도 2b는 페룰산의 표준곡선 그래프이고, 도 2c는 에틸 페룰레이트의 표준곡선 그래프이다. UPLC/PDA(waters, ACQUITY UPLC SYSTEM) 기기의 분석조건은 하기 표 1과 같다.In this application, UPLC/PDA (waters, ACQUITY UPLC SYSTEM) assay was established to qualitatively or quantitatively analyze ethyl ferulate and ferulic acid. As a result of the analysis, ethyl ferulate showed a retention time of 2.1 minutes and ferulic acid at 3.45 minutes (FIG. 2 ). 2A is a UPLC chromatogram of ferulic acid and ethyl ferulate, FIG. 2B is a standard curve graph of ferulic acid, and FIG. 2C is a standard curve graph of ethyl ferulate. The analysis conditions of the UPLC/PDA (waters, ACQUITY UPLC SYSTEM) device are shown in Table 1 below.
3. 분리된 균주 J2와 3. The isolated strain J2 락토바실러스Lactobacillus 헬베티쿠스Helvetica KCCM11223와의With KCCM11223 시나모일Cinnamoyl 에스터라아제의Esterase 활성 비교 Active comparison
에틸 페룰레이트가 함유된 플레이트 어세이(plate assay)법에서 1차 선발된 5개 분리 균주와 기존에 시나모일 에스테라아제 활성 균주로 알려진 표준균주인 락토바실러스 헬베티쿠스(L. helveticus) KCCM11223의 효소 활성능을 비교하기 위해 2 mM의 에틸 페룰레이트가 함유된 MRS broth에서 48시간 배양한 후 UPLC/PDA를 수행하여 전환된 페룰산의 함량을 비교분석하였다. 그 결과, 음성 대조군으로 사용한 균주를 접종하지 않은 배지에서는 페룰산이 검출되지 않았으며, J1을 제외한 4개의 균주는 페룰산의 생산양이 1.4mM 이상으로 락토바실러스 헬베티쿠스 KCCM11223의 1.2 mM 보다 높은 생산량을 보여주었다. 특히, J2 분리균주는 1.8 mM의 생산량을 나타냈으며 이것은 에틸 페룰레이트가 페룰산으로 90% 이상 전환되어질 수 있음을 나타낸다(도 3). 도 3a는 UPLC를 이용한 시나모일 에스터라아제 활성의 정량적 분석 그래프이고, 도 3b는 크로마토그램이다.Enzyme activity of L. helveticus KCCM11223, a standard strain known as the five isolated strains first selected in a plate assay method containing ethyl ferulate, and a strain previously known as cinnamoyl esterase active strain In order to compare the performance, after incubating for 48 hours in MRS broth containing 2 mM ethyl ferulate, UPLC/PDA was performed to compare and analyze the converted ferulic acid content. As a result, ferulic acid was not detected in the medium that was not inoculated with the strain used as the negative control, and the production amount of ferulic acid was 1.4 mM or more in the four strains except J1. Lactobacillus helveticus KCCM11223 showed a yield higher than 1.2 mM. In particular, the J2 isolate showed a production amount of 1.8 mM, indicating that ethyl ferulate can be converted to ferulic acid by 90% or more (FIG. 3). 3A is a quantitative analysis graph of cinnamoyl esterase activity using UPLC, and FIG. 3B is a chromatogram.
4. 선발 미생물의 동정4. Identification of selected microorganisms
시나모일 에스터라아제 활성이 가장 우수한 균주인 J2의 동정을 위하여 16S ribosomal DNA 분석을 실시하였다. 16S rDNA gene 염기서열을 GenBenk 의 BLAST 데이터와 비교하여 분자 생물학적 연관성을 나타내는 계통도를 얻었다(도 4). 이 결과에 따라 선발미생물 J2는 락토바실러스 퍼멘텀과 98%의 상동성으로 가장 가까운 유연관계를 나타내었으며, 락토바실러스 퍼멘텀 J2로 명명하였다. 16S ribosomal DNA analysis was performed for the identification of J2, the strain having the best cinnamoyl esterase activity. A 16S rDNA gene base sequence was compared with GenBenk's BLAST data to obtain a phylogenetic tree showing molecular biological association (FIG. 4). According to these results, the selected microorganism J2 showed the closest flexible relationship with 98% homology to Lactobacillus performance, and was named Lactobacillus performance J2.
5. 5. 시나모일Cinnamoyl 에스테라아제 유전자 확인 Esterase gene identification
락토바실러스 퍼멘텀 J2에서 추출한 게놈 DNA를 주형으로 PCR를 실시한 결과, 목적하는 약 0.7kb의 유전자의 증폭이 확인되었다(도 5a 및 도 5b). 유전자 서열 분석결과 락토바실러스 퍼멘텀 ATCC 14932 시나모일 에스터라아제의 유전자 서열과 DNA 수준에서 약 99.1%, 아미노산 수준에서 약 97.9%의 상동성을 보이는 유전자를 확인하였다(도 6a 및 도 6b).As a result of PCR using the genomic DNA extracted from Lactobacillus performance J2 as a template, amplification of the desired gene of about 0.7 kb was confirmed (FIGS. 5A and 5B ). As a result of the gene sequence analysis, genes showing the homology of Lactobacillus performance ATCC 14932 cinnamoyl esterase at the DNA level and about 99.1% at the DNA level and about 97.9% at the amino acid level were identified (FIGS. 6A and 6B ).
유전자서열을 검색한 결과, 알파/베타 폴드 패밀리 하이드로라아제 로커스에 속해 있었으며, 시나모일 에스터라아제의 염기서열의 중요한 Catalytic site S106, H225, D197을 포함하였다(도 6a). 따라서 락토바실러스 퍼멘텀 균주의 게놈데이터베이스에서 해당하는 유전자를 바탕으로 두가지 CE_FM_F와 CE_FM_R의 프라이머를 설계하였다(표 1).As a result of searching the gene sequence, it belonged to the alpha/beta fold family hydrolase locus, and included important catalytic sites S106, H225, and D197 of the base sequence of cinnamoyl esterase (FIG. 6A). Therefore, Lactobacillus performance Two CE_FM_F and CE_FM_R primers were designed based on the corresponding gene in the genome database of the strain (Table 1).
6. 락토바실러스 퍼멘텀 J2로부터 6. From Lactobacillus performance J2 시나모일Cinnamoyl 에스테라아제의 기질특이성 분석 Analysis of substrate specificity of esterase
락토바실러스 퍼멘텀 J2로부터 시나모일 에스터라아제의 기질특이성 분석을 위해 에틸 페룰레이트, 클로로젠산, 메틸 P-쿠마레이트(Methyl P-coumarate), 메틸-시나페이트(Methyl-sinnapate)를 이용하여 가수분해능을 검토하였다. Lactobacillus performance For the analysis of the substrate specificity of cinnamoyl esterase from J2, the hydrolysis ability was examined using ethyl ferulate, chlorogenic acid, methyl P-coumarate, and methyl-sinnapate. .
그 결과 에틸 페룰레이트에서 85.1%의 가장 높은 분해율을 보여주었고 클로로젠산, 메틸 P-쿠마레이트는 각각 63.2%, 59.8%의 비교적 우수한 분해능을 보인 반면, 메틸-시나페이트는 1.7%로, 매우 낮은 분해능을 나타냈다(도 7). 따라서, 락토바실러스 퍼멘텀 J2로부터 시나모일 에스터라아제는 에틸 페룰레이트에서 85.1%의 가장 높은 분해율을 나타내어, 에틸 페룰레이트에 대한 기질특이성이 높은 것으로 나타났다.The result showed the highest decomposition rate of 85.1% in ethyl ferulate, while chlorogenic acid and methyl P-coumarate showed relatively good resolutions of 63.2% and 59.8%, respectively, while methyl-cinnamate was 1.7%, which was very low. Resolution was shown (Fig. 7). Therefore, from Lactobacillus performance J2, cinnamoyl esterase showed the highest decomposition rate of 85.1% in ethyl ferulate, indicating that the substrate specificity for ethyl ferulate was high.
7. 선발 미생물의 시나모일 에스터라아제 최적 활성 조건 확립7. Establishing optimal active conditions for cinnamoyl esterase of selected microorganisms
선발미생물의 생육에 따른 시나모일 에스테라아제 활성 특성을 조사하였다(도 8). 락토바실러스 퍼멘텀 J2가 MRS 액체배지에서 배양하였을 때, 생육은 37℃에서 배양 48시간까지 대수증식기였고 72시간까지는 정체기이며 그 이후, 사멸기에 도달하였음을 확인하였다. Cinnamoyl esterase activity characteristics according to the growth of the selected microorganisms were investigated (FIG. 8). Lactobacillus performance When J2 was cultivated in MRS liquid medium, the growth was logarithmic growth up to 48 hours incubation at 37° C., and plateaued up to 72 hours, after which time it was confirmed that the death phase was reached.
시나모일 에스터라아제 효소활성은 대수증식기인 배양 12시간부터 생성되어 사멸기인 48시간에 가장 높은 활성(페룰산 생성양 1.69 mM)을 나타내었다. 대부분의 시나모일 에스터라아제 효소 활성이 생육과 동시에 생산되는 것과 마찬가지로 락토바실러스 퍼멘텀 J2의 시나모일 에스터라아제 효소 또한 대수기 시작단계인 9시간 이후부터 급격하게 활성이 증가하는 것을 확인하였다. 또한 항균활성은 가장 높은 배양 48시간(2일)부터 96시간(4일)까지 활성이 유지되는 것으로 확인하였다(도 8).Cinnamoyl esterase enzyme activity was generated from 12 hours of culture, which is a logarithmic growth phase, and showed the highest activity (ferulic acid production amount: 1.69 mM) at the death time, 48 hours. As with most of the cinnamoyl esterase enzyme activity produced at the same time as growth, it was confirmed that the cinnamoyl esterase enzyme of Lactobacillus Performance J2 also rapidly increases from 9 hours after the start of log phase. In addition, it was confirmed that the activity of antibacterial activity was maintained from 48 hours (2 days) to 96 hours (4 days), the highest culture (Fig. 8).
7-1. pH의 영향7-1. Effect of pH
선정된 락토바실러스 퍼멘텀 J2의 시나모일 에스터라아제 활성 균주의 pH에 따른 활성을 검토하기 위하여 2mM 에틸 페룰레이트가 첨가된 MRS broth (Difco)를 1N NaOH와 1N HCl을 사용하여 각각의 배지를 pH3, pH5, pH7 및 pH9로 조정하였다. 전배양한 선택 유산균을 1% (v/v) 접종한 후 37℃에서 48시간 배양한 후 활성 측정은 배양 후 추출액에 대하여 상기와 동일하게 UPLC/PDA방법을 이용하였다. 그 결과 락토바실러스 퍼멘텀 J2는 초기 pH가 7.0 일 때 약 1.8 mM의 페룰산을 생성함으로서 가장 높은 효소활성을 나타낸 반면, 초기 pH 3.0에서는 매우 낮는 페룰산(0.2 mM)를 생성하였고, pH 9.0에서는 페룰산으로 전환되지 못하였다. 이는 균체의 양과 일치하는 결과를 보여주었다. 일반적으로 유산균은 성장과 동시에 젖산을 생성함에 따라 배지의 pH가 4∼4.5로 낮아지기 때문에 일부의 효소의 경우 단백질의 변성으로 매우 낮은 pH에서 활성을 나타내지 않은 것으로 사료된다(도 9). MRS broth (Difco) with 2 mM ethyl ferulate was added to 1C NaOH and 1N HCl to check the activity of the selected Lactobacillus performance J2 cinnamoyl esterase active strain according to pH. , pH5, pH7 and pH9. After inoculating 1% (v/v) of the pre-cultured selected lactic acid bacteria, after culturing at 37°C for 48 hours, the measurement of activity was performed by UPLC/PDA method in the same manner as above for the extract after culture. As a result, Lactobacillus performance J2 showed the highest enzymatic activity by generating about 1.8 mM ferulic acid when the initial pH was 7.0, while producing very low ferulic acid (0.2 mM) at initial pH 3.0 and at pH 9.0. Failed to convert to ferulic acid. This showed results consistent with the amount of cells. In general, as the lactic acid bacteria produce lactic acid at the same time as growth, the pH of the medium is lowered to 4 to 4.5, so it is thought that some enzymes do not exhibit activity at very low pH due to protein denaturation (FIG. 9).
7-2. 온도의 영향7-2. Effect of temperature
선정된 락토바실러스 퍼멘텀 J2의 시나모일 에스터라아제 활성 최적 온도를 검토하기 위하여 2 mM 에틸 페룰레이트가 첨가된 MRS broth (Difco)를 pH 7.0로 조정한 배지에 활성 유산균을 1% (v/v)을 접종한 후 25℃, 30℃, 37℃ 및 45℃의 각기 다른 온도에서 48시간 배양한 후 활성을 비교하였다. 온도를 제외한 나머지 배양 조건은 동일하게 유지하였고 활성 측정은 배양 후 추출액에 대하여 상기와 동일하게 UPLC/PDA방법을 이용하였다. 그 결과, 37℃ 배양 후 약 1.8mM의 페룰산을 생성하면서 최적의 활성을 보인 반면 45℃ 배양 조건에서는 페룰산을 거의 생성하지 못하였다(도 10).In order to examine the optimal temperature of cinnamoyl esterase activity of the selected Lactobacillus performance J2, 1% (v/v) of active lactic acid bacteria was added to the medium in which MRS broth (Difco) with 2 mM ethyl ferulate was adjusted to pH 7.0. ) And inoculated at different temperatures of 25°C, 30°C, 37°C, and 45°C for 48 hours, and then compared activities. The rest of the culture conditions except for the temperature were kept the same, and the measurement of activity was performed using the UPLC/PDA method in the same manner as above for the extract after culture. As a result, after incubation at 37° C., it showed optimal activity while producing ferulic acid of about 1.8 mM, whereas ferulic acid was hardly generated at 45° C. (FIG. 10).
<110> RURAL DEVELOPMENT ADMINISTRATION <120> Cinnamoyl esterase from Lactobacillus fermentum J2 and preparation method thereof <130> NPF-32461 <160> 5 <170> PatentIn version 3.2 <210> 1 <211> 745 <212> RNA <213> Artificial Sequence <220> <223> Lactobacillus fermentum J2 16s rRNA <400> 1 atggaagttg caatcaagag tgccggtctt accttacggg ggctgctcga ggggagcgac 60 caggtgccaa atgaccggat cgccatttta atgcacggtt ttaagggtga cctaggttac 120 accgaggaga acctcttaaa ccaactggcc caccgcttaa acgaccaggg cctggccacg 180 ttacggtttg actttgccgg ttgcggtaag tccgacggtc agtttagcga catgaccgtg 240 ttaagcggac ttcaagacgg catgaagatc atcgactacg cccgccagga ggttcaggca 300 aaggaaatca tcctggttgg ccactcacaa ggcggggtgg tggcttcgat gctcgccgcc 360 tactaccgcg acgtgattga caagttagtt ctcttggccc cagctgccac cctcaaagat 420 gacgccctga tcgggacttg ccgaggaacc acctacgatc ccaaccacat tcccgactac 480 gtaacggttg gtggcttcaa agtcggcggc aactacttca ggaccgccca actgctgcca 540 atttacgaaa ccgcccagca ctacgccggc ccggttttga tgatccacgg cctggccgac 600 acggtggtcg accccaaggc ctcacaaaag tacaacgtta tgtaccaaaa cggggtgatc 660 cacttcctgg agggggccag ccaccagtta cgtggcgacg gcgaccaacg ggaaaccacc 720 cttcaattag tgtccgattt cttaa 745 <210> 2 <211> 747 <212> DNA <213> Artificial Sequence <220> <223> Cinnamoyl esterase nucleotide sequence <400> 2 atggaagttg caatcaagag tgccggtctt accttacggg ggctgctcga ggggagcgac 60 caggtgccaa atgaccggat cgccatttta atgcacggtt ttaagggtga cctaggttac 120 accgaggaga acctcttaaa ccaactggcc caccgcttaa acgaccaggg cctggccacg 180 ttacggtttg actttgccgg ttgcggtaag tccgacggtc agtttagcga catgaccgtg 240 ttaagcggac ttcaagacgg catgaagatc atcgactacg cccgccagga ggttcaggca 300 aaggaaatca tcctggttgg ccactcacaa ggcggggtgg tggcttcgat gctcgccgcc 360 tactaccgcg acgtgattga caagttagtt ctcttggccc cagctgccac cctcaaagat 420 gacgccctga tcgggacttg ccgaggaacc acctacgatc ccaaccacat tcccgactac 480 gtaacggttg gtggcttcaa agtcggcggc aactacttca ggaccgccca actgctgcca 540 atttacgaaa ccgcccagca ctacgccggc ccggttttga tgatccacgg cctggccgac 600 acggtggtcg accccaaggc ctcacaaaag tacaacgtta tgtaccaaaa cggggtgatc 660 cacttcctgg agggggccag ccaccagtta cgtggcgacg gcgaccaacg ggaaaccacc 720 cttcaattag tgtccgattt cttaaac 747 <210> 3 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> CE_FM_F <400> 3 atggaagttg caatcaagag tg 22 <210> 4 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> CE_RM_R <400> 4 ttagtttaag aaatcggaca c 21 <210> 5 <211> 249 <212> PRT <213> Artificial Sequence <220> <223> Cinnamoyl esterase amino acid sequence <400> 5 Met Glu Val Ala Ile Lys Ser Ala Gly Leu Thr Leu Arg Gly Leu Leu 1 5 10 15 Glu Gly Ser Asp Gln Val Pro Asn Asp Arg Ile Ala Ile Leu Met His 20 25 30 Gly Phe Lys Gly Asp Leu Gly Tyr Thr Glu Glu Asn Leu Leu Asn Gln 35 40 45 Leu Ala His Arg Leu Asn Asp Gln Gly Leu Ala Thr Leu Arg Phe Asp 50 55 60 Phe Ala Gly Cys Gly Lys Ser Asp Gly Gln Phe Ser Asp Met Thr Val 65 70 75 80 Leu Ser Gly Leu Gln Asp Gly Met Lys Ile Ile Asp Tyr Ala Arg Gln 85 90 95 Glu Val Gln Ala Lys Glu Ile Ile Leu Val Gly His Ser Gln Gly Gly 100 105 110 Val Val Ala Ser Met Leu Ala Ala Tyr Tyr Arg Asp Val Ile Asp Lys 115 120 125 Leu Val Leu Leu Ala Pro Ala Ala Thr Leu Lys Asp Asp Ala Leu Ile 130 135 140 Gly Thr Cys Arg Gly Thr Thr Tyr Asp Pro Asn His Ile Pro Asp Tyr 145 150 155 160 Val Thr Val Gly Gly Phe Lys Val Gly Gly Asn Tyr Phe Arg Thr Ala 165 170 175 Gln Leu Leu Pro Ile Tyr Glu Thr Ala Gln His Tyr Ala Gly Pro Val 180 185 190 Leu Met Ile His Gly Leu Ala Asp Thr Val Val Asp Pro Lys Ala Ser 195 200 205 Gln Lys Tyr Asn Val Met Tyr Gln Asn Gly Val Ile His Phe Leu Glu 210 215 220 Gly Ala Ser His Gln Leu Arg Gly Asp Gly Asp Gln Arg Glu Thr Thr 225 230 235 240 Leu Gln Leu Val Ser Asp Phe Leu Asn 245 <110> RURAL DEVELOPMENT ADMINISTRATION <120> Cinnamoyl esterase from Lactobacillus fermentum J2 and preparation method thereof <130> NPF-32461 <160> 5 <170> PatentIn version 3.2 <210> 1 <211> 745 <212> RNA <213> Artificial Sequence <220> <223> Lactobacillus fermentum J2 16s rRNA <400> 1 atggaagttg caatcaagag tgccggtctt accttacggg ggctgctcga ggggagcgac 60 caggtgccaa atgaccggat cgccatttta atgcacggtt ttaagggtga cctaggttac 120 accgaggaga acctcttaaa ccaactggcc caccgcttaa acgaccaggg cctggccacg 180 ttacggtttg actttgccgg ttgcggtaag tccgacggtc agtttagcga catgaccgtg 240 ttaagcggac ttcaagacgg catgaagatc atcgactacg cccgccagga ggttcaggca 300 aaggaaatca tcctggttgg ccactcacaa ggcggggtgg tggcttcgat gctcgccgcc 360 tactaccgcg acgtgattga caagttagtt ctcttggccc cagctgccac cctcaaagat 420 gacgccctga tcgggacttg ccgaggaacc acctacgatc ccaaccacat tcccgactac 480 gtaacggttg gtggcttcaa agtcggcggc aactacttca ggaccgccca actgctgcca 540 atttacgaaa ccgcccagca ctacgccggc ccggttttga tgatccacgg cctggccgac 600 acggtggtcg accccaaggc ctcacaaaag tacaacgtta tgtaccaaaa cggggtgatc 660 cacttcctgg agggggccag ccaccagtta cgtggcgacg gcgaccaacg ggaaaccacc 720 cttcaattag tgtccgattt cttaa 745 <210> 2 <211> 747 <212> DNA <213> Artificial Sequence <220> <223> Cinnamoyl esterase nucleotide sequence <400> 2 atggaagttg caatcaagag tgccggtctt accttacggg ggctgctcga ggggagcgac 60 caggtgccaa atgaccggat cgccatttta atgcacggtt ttaagggtga cctaggttac 120 accgaggaga acctcttaaa ccaactggcc caccgcttaa acgaccaggg cctggccacg 180 ttacggtttg actttgccgg ttgcggtaag tccgacggtc agtttagcga catgaccgtg 240 ttaagcggac ttcaagacgg catgaagatc atcgactacg cccgccagga ggttcaggca 300 aaggaaatca tcctggttgg ccactcacaa ggcggggtgg tggcttcgat gctcgccgcc 360 tactaccgcg acgtgattga caagttagtt ctcttggccc cagctgccac cctcaaagat 420 gacgccctga tcgggacttg ccgaggaacc acctacgatc ccaaccacat tcccgactac 480 gtaacggttg gtggcttcaa agtcggcggc aactacttca ggaccgccca actgctgcca 540 atttacgaaa ccgcccagca ctacgccggc ccggttttga tgatccacgg cctggccgac 600 acggtggtcg accccaaggc ctcacaaaag tacaacgtta tgtaccaaaa cggggtgatc 660 cacttcctgg agggggccag ccaccagtta cgtggcgacg gcgaccaacg ggaaaccacc 720 cttcaattag tgtccgattt cttaaac 747 <210> 3 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> CE_FM_F <400> 3 atggaagttg caatcaagag tg 22 <210> 4 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> CE_RM_R <400> 4 ttagtttaag aaatcggaca c 21 <210> 5 <211> 249 <212> PRT <213> Artificial Sequence <220> <223> Cinnamoyl esterase amino acid sequence <400> 5 Met Glu Val Ala Ile Lys Ser Ala Gly Leu Thr Leu Arg Gly Leu Leu 1 5 10 15 Glu Gly Ser Asp Gln Val Pro Asn Asp Arg Ile Ala Ile Leu Met His 20 25 30 Gly Phe Lys Gly Asp Leu Gly Tyr Thr Glu Glu Asn Leu Leu Asn Gln 35 40 45 Leu Ala His Arg Leu Asn Asp Gln Gly Leu Ala Thr Leu Arg Phe Asp 50 55 60 Phe Ala Gly Cys Gly Lys Ser Asp Gly Gln Phe Ser Asp Met Thr Val 65 70 75 80 Leu Ser Gly Leu Gln Asp Gly Met Lys Ile Ile Asp Tyr Ala Arg Gln 85 90 95 Glu Val Gln Ala Lys Glu Ile Ile Leu Val Gly His Ser Gln Gly Gly 100 105 110 Val Val Ala Ser Met Leu Ala Ala Tyr Tyr Arg Asp Val Ile Asp Lys 115 120 125 Leu Val Leu Leu Ala Pro Ala Ala Thr Leu Lys Asp Asp Ala Leu Ile 130 135 140 Gly Thr Cys Arg Gly Thr Thr Tyr Asp Pro Asn His Ile Pro Asp Tyr 145 150 155 160 Val Thr Val Gly Gly Phe Lys Val Gly Gly Asn Tyr Phe Arg Thr Ala 165 170 175 Gln Leu Leu Pro Ile Tyr Glu Thr Ala Gln His Tyr Ala Gly Pro Val 180 185 190 Leu Met Ile His Gly Leu Ala Asp Thr Val Val Asp Pro Lys Ala Ser 195 200 205 Gln Lys Tyr Asn Val Met Tyr Gln Asn Gly Val Ile His Phe Leu Glu 210 215 220 Gly Ala Ser His Gln Leu Arg Gly Asp Gly Asp Gln Arg Glu Thr Thr 225 230 235 240 Leu Gln Leu Val Ser Asp Phe Leu Asn 245
Claims (19)
상기 락토바실러스 퍼멘텀(Lactobacillus fermentum) J2 균주(KACC 92245P)는 서열번호 1로 기재되는 16s rRNA로 구성되는 것을 특징으로 하는 락토바실러스 퍼멘텀(Lactobacillus fermentum) J2 균주(KACC 92245P).According to claim 1,
The momentum spread Lactobacillus (Lactobacillus fermentum) J2 strain (KACC 92245P) is Lactobacillus momentum spread (Lactobacillus fermentum) J2 strain, characterized in that consisting of 16s rRNA represented by SEQ. ID. NO: 1 (KACC 92245P).
상기 시나모일 에스터라아제는 서열번호 5으로 기재되는 아미노산 서열을 포함하는 것을 특징으로 하는, 시나모일 에스터라아제.According to claim 3,
The cinnamoyl esterase is characterized in that it comprises the amino acid sequence shown in SEQ ID NO: 5, cinnamoyl esterase.
상기 시나모일 에스터라아제는 에틸 페룰레이트(ethyl ferulate)에 대한 기질 특이성이 향상된 것을 특징으로 하는, 시나모일 에스터라아제.According to claim 3,
The cinnamoyl esterase is characterized in that the substrate specificity for ethyl ferulate (ethyl ferulate) is improved, cinnamoyl esterase.
상기 시나모일 에스터라아제는 에틸 페룰레이트(ethyl ferulate)에서 페룰산으로 90% 이상의 전환율을 나타내는 것을 특징으로 하는, 시나모일 에스터라아제.According to claim 3,
The cinnamoyl esterase is characterized in that it exhibits a conversion rate of at least 90% from ethyl ferulate to ferulic acid, cinnamoyl esterase.
(2) 상기 (1) 단계의 배양액으로부터 시나모일 에스터라아제(Cinnamoyl esterase)를 분리하는 단계를 포함하는, 시나모일 에스터라아제의 제조 방법.(1) culturing the Lactobacillus fermentum J2 strain producing the cinnamoyl esterase of claim 1 or 2; And
(2) separating the cinnamoyl esterase (Cinnamoyl esterase) from the culture of the step (1), a method for producing cinnamoyl esterase.
상기 (1) 단계에서, 락토바실러스 퍼멘텀 J2 균주를 45시간 내지 96시간 동안 배양하는, 시나모일 에스터라아제의 제조방법.The method of claim 10,
In the step (1), the Lactobacillus performance J2 strain is cultured for 45 hours to 96 hours, a method for producing cinnamoyl esterase.
상기 (1) 단계에서, 락토바실러스 퍼멘텀 J2 균주를 pH 6.5 내지 7.5에서 배양하는, 시나모일 에스터라아제의 제조방법.The method of claim 10,
In the step (1), the Lactobacillus performance J2 strain is cultured at pH 6.5 to 7.5, a method for producing cinnamoyl esterase.
상기 (1) 단계에서, 락토바실러스 퍼멘텀 J2 균주를 30℃ 내지 40℃에서 배양하는, 시나모일 에스터라아제의 제조방법.The method of claim 10,
In the step (1), Lactobacillus performance J2 strain is cultured at 30 ℃ to 40 ℃, a method of producing cinnamoyl esterase.
상기 시나모일 에스터라아제는 에틸 페룰레이트(ethyl ferulate)에 대한 기질 특이성이 향상된 것을 특징으로 하는, 시나모일 에스터라아제의 제조방법.The method of claim 10,
The cinnamoyl esterase is characterized in that the substrate specificity for ethyl ferulate (ethyl ferulate) is improved, a method for producing cinnamoyl esterase.
상기 시나모일 에스터라아제는 에틸 페룰레이트(ethyl ferulate)에서 페룰산으로 90% 이상의 전환율을 나타내는 것을 특징으로 하는, 시나모일 에스터라아제의 제조방법.The method of claim 10,
The cinnamoyl esterase is characterized in that it exhibits a conversion rate of at least 90% from ethyl ferulate (ethyl ferulate) to ferulic acid, a method for producing cinnamoyl esterase.
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| US20160244734A1 (en) * | 2015-02-24 | 2016-08-25 | The United States Of America, As Represented By The Secretary Of Agriculture | Novel ferulate esterase isolated from lactobaccillus fermentum |
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