KR20050052418A - Bacillus subtilis bb-1 - Google Patents
Bacillus subtilis bb-1 Download PDFInfo
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- KR20050052418A KR20050052418A KR1020050020068A KR20050020068A KR20050052418A KR 20050052418 A KR20050052418 A KR 20050052418A KR 1020050020068 A KR1020050020068 A KR 1020050020068A KR 20050020068 A KR20050020068 A KR 20050020068A KR 20050052418 A KR20050052418 A KR 20050052418A
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- thrombolytic
- bacillus subtilis
- fibrin
- blood
- kfcc
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- 235000014469 Bacillus subtilis Nutrition 0.000 title claims abstract description 20
- 230000002537 thrombolytic effect Effects 0.000 claims abstract description 38
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- DBMJMQXJHONAFJ-UHFFFAOYSA-M Sodium laurylsulphate Chemical compound [Na+].CCCCCCCCCCCCOS([O-])(=O)=O DBMJMQXJHONAFJ-UHFFFAOYSA-M 0.000 description 1
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Classifications
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K91/00—Lines
- A01K91/03—Connecting devices
- A01K91/04—Connecting devices for connecting lines to hooks or lures
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K91/00—Lines
- A01K91/03—Connecting devices
- A01K91/053—Fishing booms, i.e. connecting devices spreading out the leaders, e.g. to avoid tangling thereof
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K97/00—Accessories for angling
- A01K97/06—Containers or holders for hooks, lines, sinkers, flies or the like
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Environmental Sciences (AREA)
- Animal Husbandry (AREA)
- Biodiversity & Conservation Biology (AREA)
- Marine Sciences & Fisheries (AREA)
- Enzymes And Modification Thereof (AREA)
Abstract
본 발명은 국내산 흑두를 이용하여 제조한 청국에서 분리된 혈전용해효소 생산능이 우수한 바실러스 서브틸리스(Bacillus subtilis)BB-1(KFCC 11344P) 균주로서 기질특이적인 면에서 스킴밀크(skim milk)에서만 아주 약한 분해를 보인 반면 혈액내의 다른 단백질류(casein, gelatin, blood agar plate)들을 전혀 분해하지 않음으로서 장관출혈, 적혈구 파괴 등 부작용을 배제할 수 있으며, 5종류의 강력한 혈전용해동위효소를 생산하고 피브린(fibrin)분해능이 특이적으로 우수한 미생물에 관한 것이다.The present invention is Bacillus subtilis BB-1 (KFCC 11344P) strain having excellent thrombolytic enzyme production ability isolated from Cheongguk prepared using domestic black bean, and only in skim milk in terms of substrate specificity. While it shows mild degradation, it does not break down other proteins in the blood (casein, gelatin, blood agar plates), thereby eliminating side effects such as intestinal bleeding and red blood cell destruction.It produces five kinds of powerful thrombolytic isotope enzymes and fibrin (fibrin) relates to a microorganism having a particularly excellent resolution.
Description
본 발명은 국내산 흑두를 이용하여 제조한 청국에서 분리된 혈전용해효소 생산능이 우수한 바실러스 서브틸리스(Bacillus subtilis)BB-1(KFCC 11344P) 균주로서 기질특이적인 면에서 스킴밀크에서만 아주 약한 분해능을 보이며 혈액내의 다른 단백질류(casein, gelatin, blood agar plate)들을 전혀 분해하지 않음으로서 장관출혈 등 많은 부작용에 대한 배제가 있으며, 5종류의 강력한 혈전분해동위효소를 생산하고 피브린(fibrin)분해능이 특이적으로 우수한 것에 관한 것이다.The present invention is Bacillus subtilis BB-1 (KFCC 11344P) strain having excellent thrombolytic enzyme production ability in Cheongguk prepared using domestic black bean, showing very weak resolution only in skim milk in terms of substrate specificity. By not degrading other proteins in the blood (casein, gelatin, blood agar plates), there is no exclusion of many side effects such as intestinal bleeding, and it produces five kinds of powerful thrombolytic isozymes and has specific fibrin degradability. It is about being excellent.
우리몸의 혈액은 혈관을 통하여 각 조직 구석구석까지 영양물질이나 산소를 공급함으로서 생명을 유지해 나가는데 이러한 순환기계의 질환으로 인한 사망율의 빈도가 매우 높은 편이다. 순환계 질환은 모두 혈액내에서 형성되는 혈전에 기인하는 경우가 대부분이며, 이혈전이 원인이 되어 질병을 유발시키는 것을 혈전성 성인병이나 혈전증이라고 한다.The blood of our body keeps life by supplying nutrients or oxygen to every corner of each tissue through blood vessels, and the frequency of death due to diseases of the circulatory system is very high. Most of the circulatory diseases are caused by blood clots formed in the blood, and these diseases are caused by thrombosis, which is called thrombotic adult disease or thrombosis.
혈관벽이 손상되면 혈소판에서 트롬빈(thrombin)이 생성되어 섬유소를 섬유소원으로 만들어 섬유소혈관을 형성하게 되면서 출혈을 막게 된다. 이러한 지혈과정을 거친 후 조직이 재생되고 생성되었던 혈전은 혈관내 플라스미노젠 (plasmi -nogen)활성인자에 의해서 활성화된 플라스민(plasmin)이 작용하여 용해되지만, 완전한 용해는 일어나지 않아 혈관을 따라 흐르면서 혈액의 흐름을 방해하여 혈관계 질환을 초래하는데, 이 혈전에 의한 질환이 인간의 가장 큰 사망원인으로 알려져 큰 관심을 불러 일으키고 있다. 이들 질환의 예방 및 치료를 위한 연구개발의 추세는 혈전의 생성을 억제하는 항 혈전제제의 개발과 생성된 혈전을 용해시키는 혈전용해제의 개발등에 초점이 맞추어져 있다.When the blood vessel wall is damaged, thrombin is produced in the platelets, which makes the fibrin a source of fibrin, forming fibrin blood vessels, thereby preventing bleeding. After the hemostasis process, the tissue was regenerated and the thrombus generated was lysed by the plasmin activated by the plasmi-nogen activator. However, complete lysis does not occur and flows along the blood vessel. Interfering with the flow of blood, resulting in vascular disease, the disease caused by the thrombus is known as the largest cause of death in humans, causing great interest. The trend of research and development for the prevention and treatment of these diseases is focused on the development of antithrombotic agents that inhibit the production of thrombi and the development of thrombolytic agents that dissolve the generated thrombi.
현재 임상에서 일반적으로 사용하고 있는 항 혈전제제로서는 유기합성제인 쿠마린(coumarin)과 외판(warfarin)제제가 있고, 천연물질로는 소의 심장이나 돼지의 소장에서 추출한 헤파린(heparin)제제, 거머리에서 생성되는 히루딘(hirudin)제제가 있으며, 혈전용해제제로는 스트렙토기나아제(streptokinase), 유로키나아제(urokinase), 그리고 1987년 미국의 제네텍(Genetech)사에 의해 유전자 재조합기술로 개발된 tPA(tissue type plasminogen activator)가 있다. 그리고 최근에는 뱀독과 지렁이에서 혈전분해효소를 분리 및 정제하였다고 보고되고 있다. 그러나 이러한 혈전용해제들은 혈액내에서 반감기가 짧고 기질특이적인 면에서 혈액내의 다른 단백질류들을 분해함으로서 장관출혈 등 많은 부작용을 일으키는 것으로 알려져 문제점으로 되고 있다. 이러한 문제점들을 극복하기 위하여 최근에 식품에서 혈전용해제에 대한 개발이 많이 이루어 지고 있으며, 예로서 일본의 수미(sumi)등[Fibrinolysis, 2(1), 67. 1988]이 natto를 발효하여 그 점질물에 혈전용해효소가 존재하는 것을 발견하고 기능성 식품으로서의 개발을 시도한 바 있으며, 이를 사람에게 경구투여하여 혈액으로부터 혈전용해작용의 활성을 확인한 바 있으나, 현재까지 식품을 섭취하여 혈전용해능이 증가된다는 보고는 natto이외에는 거의 없는 실정이다.Antithrombotic agents commonly used in clinical practice include organic synthetic coumarin and warfarin. Natural substances include heparin extracted from bovine heart, swine intestine, and leech. There are hirudin preparations, and thrombolytic agents are streptokinase, urokinase, and tPA (tissue type plasminogen) developed by genetic recombination technology by Genetech in the United States in 1987. activator). Recently, thrombolytic enzymes have been reported to be isolated and purified from snake venom and earthworms. However, these thrombolytic agents have been known to cause many side effects such as intestinal bleeding by degrading other proteins in the blood in terms of short half-life and substrate specificity in the blood. In order to overcome these problems, the development of thrombolysis in foods has been made in recent years. For example, Sumi et al [ Fibrinolysis , 2 (1), 67. 1988] in Japan have fermented natto, The discovery of the presence of thrombolytic enzymes and the development of functional foods have been attempted, and they have been orally administered to humans to confirm the activity of thrombolytic activity from the blood. There is almost no other situation.
따라서 본 발명자는 우리나라에도 일본의 나토와 마찬가지로 콩을 원료로하여 발효 숙성시켜 섭취해온 전통장류인 청국장이 있으므로, 우리나라의 청국장으로 부터도 일본의 나토키나아제와 유사한 혈전용해효소가 있을 것이라는 점을 착안하여 흑두를 이용하여 청국을 제조하고 연구, 노력한 결과 혈전에 대한 기질특이적이며 혈전용해능이 우수한 균주를 분리함으로서 본 발명을 완성시켰다.Therefore, the inventors of the present invention, like Korea's natto as a raw material, soybean fermented and fermented aged chunggukjang so that the intake, so we thought that there will be a thrombolytic enzyme similar to the nattokinase of Japan from Korea's Cheonggukjang As a result of preparing, studying, and working with Cheongguk using a black bean, the present invention was completed by separating a strain specific for thrombosis and having excellent thrombolytic ability.
따라서, 본 발명의 목적은 기질특이적인 면에서 스킴밀크(skim milk)에서만 아주 약한 분해능을 보인반면 혈액내의 다른 단백질류 (casein, gelatin, blood agar plate)들을 전혀 분해하지 않음으로서 장관출혈 등 많은 부작용에 대한 배제가 있으며, 5종류의 강력한 혈전동위효소를 생산하고 피브린 (fibrin)분해능이 특이적인 것으로서 혈전용해효소 생산능이 우수한 바실러스 서브틸리스(Bacillus subtilis)BB-1(KFCC 11344P) 균주를 제공하는데 그 목적이 있다.Therefore, the object of the present invention is to show a very weak resolution only in the skim milk (skim milk) in the substrate-specific aspect, while not decomposing other proteins (casein, gelatin, blood agar plates) in the blood at all, many side effects such as bleeding It provides the Bacillus subtilis BB-1 (KFCC 11344P) strain, which produces five kinds of powerful thrombosis enzymes and has a specific fibrin degrading ability, and is excellent in thrombolytic enzyme production. The purpose is.
본 발명자는 흑두를 이용하여 청국을 제조하고 혈전에 대한 기질특이적이며 혈전용해능이 우수한 균주를 분리함으로서 본 발명을 완성시켰다.The present inventors have completed the present invention by preparing a cheonguk using black bean and isolating substrate-specific strains excellent for thrombosis and excellent thrombolytic ability.
따라서 본 발명은 기질특이적인 면에서 스킴밀크(skim milk)에서 아주 약한 분해능을 보인반면 혈액내의 다른 단백질류 (casein, gelatin, blood agar plate)들을 전혀 분해하지 않음으로서 장관출혈 등 많은 부작용에 대한 배제가 있으며, 5종류의 강력한 혈전용해동위효소를 생산하고 피브린(fibrin)분해능이 특이적인것으로서 혈전용해효소 생산능이 우수한 바실러스 서브틸리스(Bacillus subtilis)BB-1(KFCC 11344P) 균주를 제공하는 것이다.Therefore, the present invention shows very weak resolution in skim milk in substrate-specific terms, while eliminating other side effects such as intestinal bleeding by not degrading other proteins (casein, gelatin, blood agar plates) in the blood at all. In addition, it produces five kinds of strong thrombolytic isotope enzymes, and fibrin (fibrin) degrading ability to provide a Bacillus subtilis BB-1 (KFCC 11344P) strain excellent in thrombolytic enzyme production ability.
상기 미생물을 배양하여 혈전용해효소를 생산하는 것은 통상의 배양법에 의해 행해질수 있으며, 미생물의 배양법에 대한 특별한 제한은 없다.The production of thrombolytic enzymes by culturing the microorganism may be performed by a conventional culture method, and there is no particular limitation on the culture method of the microorganism.
한편, 본 발명에서 채택한 각종 실험법을 아래에 설명한다.On the other hand, various test methods adopted in the present invention will be described below.
혈전용해는 아스트럽과 뮐러츠의 방법(Astrup and Muellertz, Arch. Biochem. Biophys., 40. 346, 1952)을 활용하였다. 즉 40mM되도록 염(NaCl)을 첨가한 10mM 보레이트(borate. pH 7.2)용액에 피브리노젠(fibrinogen)을 0.4%되게 용해시켜 얻은 피브리노젠(fibrinogen)용액 10ml을 페트리 접시에 분주하여 트롬빈(thrombin. 100NIH/ml) 10unit를 첨가하여 고형화 하여 제조하였다. 제조된 피브린배지(fibrin plate)에 적당량의 시료를 적하하여 37℃에서 2시간 반응시킨 후 형성되는 투명환의 지름을 측정하였다.Thrombolysis was performed using Astrup and Muellertz's method (Astrup and Muellertz, Arch. Biochem. Biophys ., 40. 346, 1952). In other words, 10 ml fibrinogen solution obtained by dissolving fibrinogen 0.4% in 10 mM borate (pH 7.2) added with salt (NaCl) to 40 mM was dispensed in a petri dish to thrombin. 100NIH / ml) was prepared by solidification by adding 10 units. An appropriate amount of the sample was added dropwise to the manufactured fibrin plate, and the diameter of the transparent ring formed after reacting at 37 ° C. for 2 hours was measured.
대조군으로 농도별로 희석한 플라스민(plasmin, 시그마)을 상기와 동일한 방법으로 피브린배지(fibrin plate)에서 반응시켜 플라스민(plasmin)의 투명환 면적을 측정, 비교하였다.Plasmin (plasmin, sigma) diluted by concentration as a control was reacted in a fibrin plate in the same manner as described above to measure and compare the clear ring area of plasmin.
이하 실시예를 통하여 본 발명을 보다 구체적으로 설명하지만, 본 발명의 개념이 실시예에만 국한되는 것은 아니다.Hereinafter, the present invention will be described in more detail with reference to Examples, but the concept of the present invention is not limited to Examples.
[실시예 1. 발명에 사용된 청국]Example 1. Cheongguk used in the invention
본 발명에 있어서의 흑두를 이용한 청국의 제조는 연등의[J. Korean Soc. Food Sci. Nutr. 31(2), 204-210]방법을 변형하여 도 1과 같이 제조하였다.The production of chungkuk using black bean in the present invention is described in Yeondeung et al. Food Sci. Nutr . 31 (2), 204-210] was modified to prepare as shown in FIG. 1.
[실시예 2.제조된 청국에서의 미생물 분리]Example 2 Microbial Separation in Prepared Cheongguk
혈전용해능이 우수한 미생물을 분리하기 위하여 제조된 청국 1g을 멸균생리식염수에 현탁, 희석하고 이를 영양한천배지에 0.1ml 도말한 후 단일 균락(colony)를 유도한 다음 단일 균락을 영양액체배지에 접종하여 37℃에서 배양한 후 피브린배지(fibrin plate)에 적당량 적하하여 혈전용해능이 가장 우수한 미생물을 선발하였다.To isolate microorganisms with excellent thrombolytic ability, 1 g of Cheongguk prepared is suspended and diluted in sterile physiological saline solution, 0.1 ml of nutrient agar medium is inoculated, and a single colony is induced. After culturing at 37 ° C., an appropriate amount was added to fibrin plates to select microorganisms having the best thrombolytic ability.
[실시예 3. 미생물의 동정]Example 3. Identification of Microorganisms
실시예 2에서 분리된 미생물에 대해 16에스 알 디엔에이 시퀀스(16s rDNA sequence)에 의한 유전자간의 상동성비교로 동정하였던 바 그 유전자 상동성이 바실러스 서브틸리스(Bacillus subtilis)와 99% 유사성을 보였다The homology between genes by 16s rDNA sequence was identified for the microorganism isolated in Example 2, which showed 99% similarity to Bacillus subtilis.
이상에서 분리된 미생물은 바실러스 서브틸리스(Bacillus subtilis)로 동정하였고, BB-1으로 명명하고 미생물을 2005년 2월 19일자로 한국미생물보존센터(KCCM)에 기탁하여 수탁번호 KFCC 11344P를 부여받았다.The microorganism isolated from above was identified as Bacillus subtilis , named BB-1, and the microorganism was deposited with the Korea Microorganism Conservation Center (KCCM) on February 19, 2005 and received accession number KFCC 11344P. .
[실시예 4. 혈전용해능이 있는 조효소액의 조제]Example 4 Preparation of Coenzyme Solution with Thrombolytic Activity
실시예 2의 바실러스 서브틸리스(Bacillus subtilis) BB-1(KFCC 11334P)를 영양배지 100ml에서 37℃, 18시간 배양하고, 원심분리하여 균체를 제거하고, 0.2㎛ 필터(filter)를 통과시켰다. 이 배양액을 위에서 기술한 혈전용해활성측정법으로 혈전용해활성을 측정하여 도 2에 나타내었다. 그 결과 대조구인 대장균(JM109)에서는 활성을 보이지 않은 반면 본 효소는 지름 약 1.5cm의 둥근환을 나타내면서 아주 높은 혈전용해 활성을 나타내었다. 이 배양액에 79% 암모니움 설페이트(ammonium sulfate)를 부여한 후 완전히 용해 시킨다음 4℃에서 12시간 정치하여 배양액의 단백질을 침전시켰다. 그 후 원심분리하여 상등액을 제거하고 남은 침전물을 20mM 소디움 아세테이트(sodium acetate)로 재 용해하여 멤브레인 솔팅 아웃 컬럼(membrane salting out column)을 이용하여 12시간 동안 4℃에서 교반하면서 염을 제거하여 단백질을 추출하였다. 이와 같은 방법으로 혈전용해능을 갖는 조효소를 바실러스 서브틸리스(Bacillus subtilis) BB-1(KFCC11334P)의 배양액으로부터 손쉽게 제조할 수 있었다. Bacillus subtilis BB-1 (KFCC 11334P) of Example 2 was incubated for 18 hours at 37 ° C. in 100 ml of nutrient medium, centrifuged to remove cells, and passed through a 0.2 μm filter. The culture solution was measured by thrombolytic activity as described above, and is shown in FIG. 2. As a result, the control E. coli (JM109) showed no activity while the enzyme showed a round ring of about 1.5cm in diameter and showed very high thrombolytic activity. 79% ammonium sulfate was added to the culture solution, completely dissolved, and left to stand at 4 ° C for 12 hours to precipitate protein in the culture solution. Subsequently, the supernatant was removed by centrifugation, and the remaining precipitate was re-dissolved with 20 mM sodium acetate, and salt was removed while stirring at 4 ° C. for 12 hours using a membrane salting out column. Extracted. In this way, coenzymes having thrombolytic ability could be easily prepared from the culture of Bacillus subtilis BB-1 (KFCC11334P).
[실시예 5. 혈전용해효소 생산능이 우수한 바실러스 서브틸리스(Bacillus subtilis) BB-1(KFCC 11344P)조효소의 기질특이성][Example 5. Substrate specificity of Bacillus subtilis BB-1 (KFCC 11344P) coenzyme having excellent thrombolytic enzyme production ability]
실시예 4에서와 같이 제조된 조효소의 기질특이성에 사용된 것은 혈액내의 다른 단백질류인 스킴밀크(skim milk), 카세인(casein), 젤라틴(gelatin), 적혈구 배지(blood agar plate)을 제조하여 사용하였으며, 각 단백질 배지에 조효소 0.1ml씩을 페이퍼 디스크(paper disc)를 이용하여 흡착시킨 후 37℃ 항온기에서 24시간까지 반응시켜 분해여부를 확인하였다. 그 결과를 도 3에 나타내었다.As used in the substrate specificity of the coenzyme prepared in Example 4 was used to prepare other proteins in the blood, such as skim milk (casekim), casein (gelatin), gelatin (gelatin), blood agar plate (blood agar plate) In addition, 0.1 ml of coenzyme was adsorbed onto each protein medium using a paper disc, followed by reaction at 37 ° C. for 24 hours to confirm degradation. The results are shown in FIG.
[실시예 6. 혈전용해효소 생산능이 우수한 바실러스 서브틸리스(Bacillus subtilis) BB-1(KFCC 11344P)의 혈전용해동위효소 측정][Example 6. Measurement of thrombolytic isotope of Bacillus subtilis BB-1 (KFCC 11344P) excellent in thrombolytic enzyme production ability]
실시예 4에서와 같이 제조된 바실러스 서브틸리스(Bacillus subtilis) BB-1(KFCC 11344P)조효소의 혈전용해동위효소검출을 위해 최등의 방법(nack-shick choi, J. Biochemistry and Molecular Biology, 34:6, 531-536)을 활용하였다. 즉 ,피브리노젠(fibrinogen)농도가 0.12%(w/w)가 되게 폴리아크릴아마이드(polyacrylamide)용액에 혼합한 후 즉시 트롬빈(thrombin)(1NIH unit/ml)을 첨가하여 제조한 12% 피브린-폴리아크릴아마이드 젤(fibrin-polyacryamide gel)에서 수행하였다. 레인(lane)에 실시예 4에서 제조된 조효소를 적하한 후 150V의 일정한 전압을 걸어 전기영동을 실시한 다음 소디움 도데실 설페이트(Sodium Dodecyl Sulfate)에 의해 불활성화된 효소를 재활성화 시키기 위하여 젤(gel)을 2.5% 트리톤 엑스 100(triton X-100)을 포함한 완충용액(Tris-HCl. pH 7.4)에 3시간 동안 침적하여 단백질을 활성화 시킨 후 트리톤 엑스 100(Triton X-100)을 제거하기 위하여 멸균생리식염수로 15분간 2회 세척하였다. 분획된 단백질의 혈전분해활성은 활성반응 완충용액(200mM NaCl, 10mM CaCl2, 0.02% NaN3. 30mM Tris-HCl. pH 7.4)에 젤(gel)을 침적하여 37℃ 항온기에서 12에서16시간 반응을 시켰다. 피브린(fibrin)분해능을 지닌 단백질 분획은 활성염색법에 의해 젤(gel)상의 피브린(fibrin)을 분해하여 코마시 블루 알 250(comassie blue R 250) 염색결과 투명대로서 도 4와 같이 나타내었다.For the detection of Bacillus subtilis BB-1 (KFCC 11344P) coenzyme prepared as in Example 4, the best method (nack-shick choi, J. Biochemistry and Molecular Biology, 34: 6, 531-536). That is, 12% fibrin-prepared by adding thrombin (1 NIH unit / ml) immediately after mixing in a polyacrylamide solution to a fibrinogen concentration of 0.12% (w / w) Performed on a polyacrylamide gel (fibrin-polyacryamide gel). After dropping the coenzyme prepared in Example 4 into a lane, electrophoresis was performed at a constant voltage of 150 V, and then gel was used to reactivate the enzyme inactivated by sodium dodecyl sulfate. ) Was immersed in a buffer solution containing 2.5% triton X-100 (Tris-HCl. PH 7.4) for 3 hours to activate the protein and then sterilized to remove Triton X-100. Washed twice with physiological saline for 15 minutes. The thrombolytic activity of the fractionated proteins was obtained by immersing a gel in an active reaction buffer solution (200 mM NaCl, 10 mM CaCl2, 0.02% NaN3.30 mM Tris-HCl. . The protein fraction having fibrin resolution was shown as FIG. 4 as a result of comase blue R 250 staining by decomposing fibrin on gel by active staining.
[실시예 7. 나토키나아제 유전자와 본 바실러스 서브틸리스(Bacillus subtilis) BB-1(KFCC 11344P)에서 크로닝된 유전자의 상동성][Example 7] Homology of Natokinase Gene and Gene Cropped by B. Bacillus subtilis BB-1 (KFCC 11344P)]
혈전용해효소를 강력하게 생산하는 바실러스 서브틸리스(Bacillus subtilis) BB-1(KFCC 11344P)의 염색체를 분리한 후 기존의 바실러스 서브틸리스((Bacillus subtilis)로부터 크로닝된 공통적인 혈전용해효소 유전자들의 연결순서 영역으로부터 프라이머(primer)를 표 1과 같이 제작하여 PCR법에 의한 유전자 증폭후 염기서열을 T7, T3프라이머(primer)를 이용하여 양방향으로 확인하고 최종 중복되는 연결순서에 의해 전체적인 염기서열을 결정하였다. 이를 유전자 은행에서 검색한 결과 도 5과 같이 나토기나아제 유전자와 99% 일치성을 나타내었다.Common thrombolytic enzyme gene cloned from Bacillus subtilis after isolation of chromosomes of Bacillus subtilis BB-1 (KFCC 11344P), which produces thrombolytic enzymes Primers are prepared from the linking sequence of the cells as shown in Table 1, and the base sequences after gene amplification by the PCR method are confirmed bidirectionally using T7 and T3 primers. The gene bank search showed 99% concordance with the nattoginase gene as shown in FIG. 5.
표1 PCR에 사용된 프라이머(primer)Table 1 Primers used for PCR
상기 설명한 바와 같이, 본 발명에 의한 균주에 의하여 혈전용해효소를 다량 생산할 수 있으며, 상기 균주에서의 혈전용해효소는 스킴밀크(skim milk)에서만 아주 약한 분해능을 보이고 일반적인 단백질에서는 전혀분해능을 보이지 않는 혈전만을 특이적으로 분해하는 기질특이성이 있는 혈전분해효소로서의 이용가능성이 충분할 것으로 고 기능성 식품개발에 적용가능할 것으로 판단된다.As described above, thrombolytic enzymes can be produced in large quantities by the strain according to the present invention, and the thrombolytic enzymes in the strains show very weak resolution in only skim milk and no resolution in general protein. Applicability as a substrate-specific thrombolytic enzyme that specifically breaks down the bay will be sufficient, and it is considered to be applicable to the development of high functional foods.
도 1은 본 발명에 있어서의 흑두청국의 간략한 제조 공정도1 is a simplified manufacturing process diagram of the black bean soup in the present invention
도 2는 제조된 청국에서 분리한 바실러스 서브틸리스(Bacillus subtilis) BB-1(KFCC 11344P) 균주의 혈전용해능을 나타내는 도면2 is a diagram showing the thrombolytic activity of Bacillus subtilis BB-1 (KFCC 11344P) strain isolated from the prepared Cheongguk.
도 3은 혈전용해효소 생산능이 우수한 바실러스 서브틸리스(Bacillus subtilis) BB-1(KFCC 11344P) 균주의 16s rDNA sequence에 의한 균주동정을 나타내는 도면Figure 3 shows the strain identification by 16s rDNA sequence of Bacillus subtilis BB-1 (KFCC 11344P) strain having excellent thrombolytic enzyme production capacity
도 4은 혈전용해효소 생산능이 우수한 바실러스 서브틸리스(Bacillus subtilis) BB-1(KFCC 11344P) 균주의 기질특이성을 보여주는 도면4 is a diagram showing the substrate specificity of Bacillus subtilis BB-1 (KFCC 11344P) strain having excellent thrombolytic enzyme production ability
도 5는 혈전용해능이 우수한 바실러스 서브틸리스(Bacillus subtilis) BB-1(KFCC 11344P) 균주의 혈전용해동위효소를 보여주는 도면Figure 5 shows the thrombolytic isotope of Bacillus subtilis BB-1 (KFCC 11344P) strain excellent in thrombolytic activity
도 6는 혈전용해효소인 Nattokinase 유전자 연결순서와의 상동성 비교를 나타내는 도면.6 is a diagram showing a homology comparison with Nattokinase gene linking sequence which is a thrombolytic enzyme.
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| WO2008062930A1 (en) * | 2006-11-22 | 2008-05-29 | Industry-Academic Cooperation Foundation, Keimyung University | Bacillus subtilis ha producing fibrinolytic enzyme and mucilage highly, method of preparing fermented soybeans using the same strain, and soybeans prepared by the method |
| CN109706092A (en) * | 2018-10-08 | 2019-05-03 | 四川农业大学 | The preparation method of one plant of bacillus coagulans for producing fibrinolysin and fibrinolysin and viable bacteria tablet |
| KR20190083011A (en) | 2017-12-30 | 2019-07-11 | 전북대학교산학협력단 | Cheonggukjang for improving blood circulation disorder induced by high-cholesterol diet and manufacturing method thereof |
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| WO2008062930A1 (en) * | 2006-11-22 | 2008-05-29 | Industry-Academic Cooperation Foundation, Keimyung University | Bacillus subtilis ha producing fibrinolytic enzyme and mucilage highly, method of preparing fermented soybeans using the same strain, and soybeans prepared by the method |
| KR20190083011A (en) | 2017-12-30 | 2019-07-11 | 전북대학교산학협력단 | Cheonggukjang for improving blood circulation disorder induced by high-cholesterol diet and manufacturing method thereof |
| CN109706092A (en) * | 2018-10-08 | 2019-05-03 | 四川农业大学 | The preparation method of one plant of bacillus coagulans for producing fibrinolysin and fibrinolysin and viable bacteria tablet |
| CN109706092B (en) * | 2018-10-08 | 2020-11-06 | 四川农业大学 | Preparation method of plasmin-producing bacillus coagulans, plasmin and live bacterium tablet |
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