KR101106507B1 - Fibrinolytic protease derived from Formitella fracinea - Google Patents
Fibrinolytic protease derived from Formitella fracinea Download PDFInfo
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- KR101106507B1 KR101106507B1 KR1020050066115A KR20050066115A KR101106507B1 KR 101106507 B1 KR101106507 B1 KR 101106507B1 KR 1020050066115 A KR1020050066115 A KR 1020050066115A KR 20050066115 A KR20050066115 A KR 20050066115A KR 101106507 B1 KR101106507 B1 KR 101106507B1
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- Prior art keywords
- fibrin
- activity
- thrombolytic
- enzyme
- longevity
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Abstract
본 발명은 장수버섯 균사체 유래의 혈전분해효소에 관한 것으로, 더욱 구체적으로는 장수버섯 유래의 혈전분해효소 및 그의 정제 방법 및 이들의 혈전 치료 및 예방에 있어서 용도에 관한 것이다. 본 발명의 장수버섯 균사체 유래의 혈전분해 효소는 종래의 혈전분해제와는 상이한 기질 특이성을 가지며 뛰어난 피브린 및 피브리노겐 용해 활성을 나타내므로 혈전증의 치료 및 예방에 효과적으로 사용될 수 있다. The present invention relates to thrombolytic enzymes derived from longevity mushroom mycelium, and more particularly, to thrombolytic enzymes derived from longevity mushrooms and their purification methods and their use in the treatment and prevention of thrombosis thereof. The thrombolytic enzyme derived from the longevity mushroom mycelium of the present invention can be effectively used for the treatment and prevention of thrombosis because it has a different substrate specificity and excellent fibrin and fibrinogen lytic activity than the conventional thrombolytic agent.
장수버섯, 혈전분해효소 Longevity Mushroom, Thrombolytic Enzyme
Description
도 1은 혈전용해 기전의 개략도.1 is a schematic diagram of a thrombolytic mechanism.
도 2는 다양한 배지에서 장수 버섯 균사체를 배양한 결과를 나타내는 도면.2 is a view showing the results of culturing longevity mushroom mycelium in various media.
도 3은 장수버섯으로부터 피브린 분해 효소의 생산을 나타내는 도면.Figure 3 shows the production of fibrin degrading enzymes from longevity mushrooms.
도 4는 장수버섯 탈지유 플레이트로부터 프로테아제 분해 활성을 나타내는 도면.Figure 4 shows the protease degrading activity from longevity skim milk plate.
도 5는 피브린 플레이트에서 장수버섯 균사체로부터의 프로테아제의 피브린 분해 활성을 나타내는 도면.Fig. 5 shows the fibrin degrading activity of protease from longevity mushroom mycelium on fibrin plates.
도 6은 CM-셀룰로오스를 사용한 장수버섯 균사체로부터 피브린분해 효소의 음이온 교환 크로마토그래피도.6 is an anion exchange chromatography diagram of fibrinase from longevity mushroom mycelium using CM-cellulose.
도 7은 DEAE-Sepharose CL6B를 사용한 장수버섯 균사체로부터 피브린 분해 효소의 음이온 교환 크로마토그래피도. 7 is an anion exchange chromatography diagram of fibrin degrading enzyme from longevity mushroom mycelium using DEAE-Sepharose CL6B.
도 8은 Sephadex G-75를 사용한 장수버섯 균사체로부터 피브린 분해 효소의 겔-여과 크로마토그래피도.8 is a gel-filtration chromatography diagram of fibrin degrading enzymes from longevity mushroom mycelium using Sephadex G-75.
도 9는 HiLoadTM SuperdexTM 75를 사용한 장수버섯 균사체로부터 피브린 분해 효소의 FPLC 결과도. Fig. 9 shows FPLC results of fibrin degrading enzymes from longevity mushroom mycelium using HiLoad ™ Superdex ™ 75.
도 10은 정제된 피브린분해 프로테아제 FFP의 SDS-PAGE 및 SDS-피브린 자이모그래프도.10 is an SDS-PAGE and SDS-fibrin zymograph diagram of purified fibrinated protease FFP.
도 11은 아조카제인 에세이에 의해 측정한 피브린 분해효소에 대한 다양한 pH의 영향을 나타내는 도면.FIG. 11 shows the effects of various pH on fibrin degrading enzymes measured by azocaine assays.
도 12는 장수버섯으로부터의 피브린 분해 효소에 대한 다양한 온도의 영향을 나타내는 도면. FIG. 12 shows the effect of various temperatures on fibrin degrading enzymes from longevity mushrooms.
도 13은 정제된 혈전분해 효소 활성에 미치는 다양한 금속 이온의 영향을 나타낸 그래프도.Figure 13 is a graph showing the effect of various metal ions on purified thrombolytic enzyme activity.
도 14는 정제된 피브린 분해 효소 활성에 미치는 여러 가지 프로테아제 억제제의 영향을 나타내는 도면.14 shows the effect of various protease inhibitors on purified fibrin degrading enzyme activity.
도 15는 정제된 피브린분해 효소의 피브린 용해(A) 및 피브리노겐용해(B) 패턴을 나타내는 도면.Fig. 15 shows the fibrin dissolution (A) and fibrinogen dissolution (B) patterns of purified fibrinase.
본 발명은 장수버섯 유래의 혈전분해효소에 관한 것으로, 더욱 구체적으로는 장수버섯 유래의 혈전분해효소 및 그의 정제 방법 및 이들의 혈전 치료 및 예방에 있어서 용도에 관한 것이다. TECHNICAL FIELD The present invention relates to thrombolytic enzymes derived from longevity mushrooms, and more particularly, to thrombolytic enzymes derived from longevity mushrooms and their purification methods, and their use in the treatment and prevention of thrombosis thereof.
전 세계 사망원인의 약 39%를 차지하고 있는 혈전질환관련의 질병은 최근 우 리나라에서도 식생활의 서구화, 과도한 스트레스, 인구 고령화 등으로 인하여 더욱 증가되고 있는 추세이다. Thrombosis-related diseases, which account for about 39% of the world's deaths, are increasing in recent years due to westernization, excessive stress, and aging population.
혈전용해 기전에는 도 1에 나타낸 바와 같이 요약할 수 있다. 현재 사용 중인 혈전분해효소제로는 스트렙토키나아제(streptokinase, SK), 유로키나아제(urokinase, UK), 조직플라스미노겐 활성화제(t-PA) 등이 알려져 있으며, 주로 생체 내 플라스미노겐(plasminogen)을 플라스민(plasmin)으로 활성화하는 간접적인 방법으로 혈전을 용해시킨다. 위와 같은 플라스미노겐 활성화제의 경우 알러지, 발열, 국소출혈, 짧은 반감기 및 고가의 의약품으로 분류되어 혈전치료제에 국한되어 이용된다는 문제점이 있다. 또한, 단백질 분해효소제는 brinase, papain, ochrase, trypsin, plasmin 등이 있으나 정맥주사로 주입시 무작위적인 단백질분해로 인한 혈전외 혈전생성 인자들까지 파괴하여 출혈유발 및 독성이 강하여 거의 사용하고 있지 않고 있다. 스트렙토키나아제 및 유로키나아제는 플라스미노겐을 플라스민으로 전환시키는 외래성 효소 물질로써 발열, 알러지, 국소출혈, 저혈압유발 등의 부작용 내포하고 있을 뿐만 아니라 가격이 매우 비싼 의약치료제로 분류되어있어 치료제에 국한되어 있으며, 체내 혈전 형성예방에는 사용되지 못하고 있는 실정이다.The thrombolytic mechanism can be summarized as shown in FIG. 1. The thrombolytic agents currently in use include streptokinase (SK), urokinase (UK), tissue plasminogen activator (t-PA), and mainly plasminogen in vivo. The blood clots are dissolved by an indirect method of activation with plasmin. In the case of the plasminogen activator as described above is classified as an allergy, fever, local bleeding, short half-life, and expensive medicines, there is a problem that is limited to the use of thrombolytic agents. Proteolytic enzymes include brinase, papain, ochrase, trypsin, and plasmin, but they are rarely used because they cause bleeding and toxicity because they destroy extra-thrombotic thrombogenic factors due to random proteolysis when injected by intravenous injection. . Streptokinase and urokinase are exotic enzymes that convert plasminogen into plasmin and are classified as very expensive medicines because they have side effects such as fever, allergy, local bleeding and hypotension. It is not used to prevent the formation of blood clots in the body.
따라서 직접 혈전에 작용하고 부작용을 최소화시킬 수 있으며 저렴한 가격의 혈전분해제를 개발할 필요성이 대두되고 있다. Therefore, there is a need to develop a low-cost thrombolytic agent that can act directly on the thrombus and minimize side effects.
본 발명자들은 상기한 바의 문제점들을 해결하고, 가격이 저렴하고 반감기가 길며 직접적 분해 기작을 이용한 혈전용해제를 얻기 위하여 다양한 식용 버섯의 균 사체로부터 예의 연구한 결과, 장수버섯 균사체 배양액에서 혈전분해효소를 검출하고 분리하여 그 특성을 시험해 본 결과, 이것이 부작용이 없을 뿐만 아니라 뛰어난 혈전용해 활성을 가지고 있음을 확인하고 본 발명을 완성하기에 이르렀다. The present inventors have solved the problems described above, and as a result of intensive studies on the mycelia of various edible mushrooms to obtain thrombolytics using low-cost, long-life, and direct degradation mechanisms, thrombolytic enzymes in longevity mushroom mycelium cultures As a result of detecting and separating and testing the properties, it was confirmed that it not only has no side effects but also has excellent thrombolytic activity and came to complete the present invention.
따라서, 본 발명의 목적은 장수버섯 균사체으로부터 분리한 혈전용해 효소 및 이를 포함한 배양액, 이들의 제조 방법을 제공하는데 있다.Accordingly, it is an object of the present invention to provide a thrombolytic enzyme isolated from longevity mushroom mycelium, a culture medium containing the same, and a method of preparing the same.
본 발명의 추가의 목적은 장수버섯 균사체의 배양물 또는 이로부터 유래한 혈전용해효소를 포함한 제약조성물을 제공하는데 있다.It is a further object of the present invention to provide a pharmaceutical composition comprising a culture of longevity mushroom mycelium or a thrombolytic enzyme derived therefrom.
본 발명의 추가의 목적 및 이점은 이후의 명세서의 기재 사항으로부터 명백할 것이다.Further objects and advantages of the invention will be apparent from the description in the following specification.
본 발명은, 일면에 있어서, 장수버섯 균사체로부터 분리한 혈전분해 효소, 동일한 기능을 가진 효소변이체 또는 돌연변이 효소를 제공한다.In one aspect, the present invention provides a thrombolytic enzyme, an enzyme variant having a similar function, or a mutant enzyme isolated from a longevity mushroom mycelium.
장수버섯은(Formitella fracinea)은 민주름 버섯목 구멍장이 버섯과에 속하는 담자균류로서 아카시재목버섯이라고도 명명되며 오래 전부터 민간약재로 사용되어 왔다. 북한에서는 만년버섯, 일명 장수버섯을 이용해 간염주사약을 개발해 치료에 이용하고 있는 것으로 알려졌다. 이것은 특히 북한에서는 백두산 불로초라고도 하는 약용버섯으로 스테로이드 화합물 3.5%, 플라보노이드 1.5%, 배당체 0.2%. 쿠마린 0.8%, 알칼로이드 0.5%가 함유되어 있다. 특히 장수버섯은 만성 B형 간염과 암, 신장염, 관절염을 비롯한 여러 가지 면역성 질병에 특효가 있으며 성욕을 높여주고 항산화 효과가 강한 것으로 밝혀졌다. 또한 장수버섯으로부터 유래된 항생제 의 경우 부작용을 덜어주며 핵산합성을 촉진시켜 인체의 원상회복을 돕고 암환자를 치료한 결과 잔존 암세포의 전이를 억제해 성숙된 암조직의 생장도 현저히 억제시키는 것으로 밝혀졌다. Formitella fracinea is a fungus belonging to the Democratic Fungus Perforated Mushroom family, also known as Akashi lumber mushroom, and has been used as a folk medicine for a long time. In North Korea, it is known that hepatitis medicines are developed and used for treatment by using mushrooms, also known as longevity mushrooms. This is a medicinal mushroom, also known as Baekdusan Bulchocho, in North Korea, with 3.5% steroid compounds, 1.5% flavonoids and 0.2% glycosides. It contains 0.8% coumarin and 0.5% alkaloids. In particular, longevity mushrooms have been shown to be effective against chronic hepatitis B and various immune diseases including cancer, nephritis and arthritis. In addition, antibiotics derived from longevity mushrooms have been shown to reduce side effects, promote nucleic acid synthesis, help the human body recover, and treat cancer patients, which significantly inhibit the growth of mature cancer tissues. .
본 발명에서는 임상에서 사용되고 있는 혈전분해제의 단점을 보완할 수 있으면서 대량생산이 가능한 장수버섯 균사체로부터 혈전분해효소를 분리 정제하여 체내 혈전형성 예방 효과 및 혈전분해 효과를 나타내는 혈전분해제를 제공한다.The present invention provides a thrombolytic agent that exhibits a thrombolytic effect and a thrombolytic effect in the body by separating and purifying thrombolytic enzymes from longevity mushroom mycelium capable of mass production while supplementing the disadvantages of thrombolytic agents used in the clinic.
본 발명에 따른 장수버섯(Formitella fracinea) 유래의 혈전분해효소FFP(Purified Formitella fracinea protease), 그의 효소변이체 또는 돌연변이 효소는 이후 자세히 설명하는 바와 같이, 분자량이 약 42 kDa이며 EDTA, EGTA에 의해 활성이 저해되는 메탈로프로테아제(metalloprotease)이고, 최적 온도는 20 ~ 45℃의 범위이고, 최적 활성 pH는 5.0 ~ 7.0의 범위이며, Cu2 +, Fe3 +, Zn2 + 등의 금속이온에 의해 활성이 저해 받으며, 피브린 및 피브리노오겐을 1시간 이내에 분해하는 능력을 지닌 것을 특징으로 한다.Thrombolytic enzyme FFP (Purified Formitella ) derived from Formitella fracinea according to the present invention fracinea protease, its enzymatic variant or mutant enzyme, is a metalloprotease whose molecular weight is about 42 kDa and whose activity is inhibited by EDTA, EGTA, and the optimum temperature is in the range of 20-45 ° C. The optimum activity pH is in the range of 5.0 to 7.0, the activity is inhibited by metal ions such as Cu 2 + , Fe 3 + , Zn 2 + , and has the ability to decompose fibrin and fibrinogen within 1 hour. It is characterized by.
이러한 연구 결과에 더하여, 혈전분해효소에 관련된 유전자 연구와 새로운 혈전분해효소에 대한 앞으로의 연구를 통하여 기능성 버섯류 개발과 인체 부작용을 최소화할 수 있는 새로운 혈전용해제 개발이 가능하리라 사료되어지며, 이와 같은 실험 결과에 의하여 장수버섯의 균사체로부터 분리정제된 혈전분해효소는 혈전증 질환을 예방 및 치료할 수 있을 것이라고 사료된다. In addition to these findings, further research on thrombolytic enzymes and new thrombolytic enzymes will enable the development of functional fungi and new thrombolytic agents that can minimize human side effects. The thrombolytic enzyme isolated from mycelium of longevity mushrooms could be used to prevent and treat thrombosis disease.
본 발명에서 사용된 용어 "혈전분해효소"는 피브린에 직접 작용하여 피브린 을 분해하는 효소를 의미한 것으로 의도한 것이며, 본질적으로는 기능상에 차이가 없으나 "혈전용해효소","피브린 분해효소" 또는 "단백질 분해효소"라는 표현과 상호 교환적으로 사용하며 이들 모두 상기 개념에 포함되는 것으로 의도하였다.The term "thrombolytic enzyme" used in the present invention is intended to mean an enzyme that directly acts on fibrin to decompose fibrin, and there is essentially no difference in function, but "thrombolytic enzyme", "fibrin degrading enzyme" or It is used interchangeably with the expression "proteinase" and all of them are intended to be included in the concept.
또한, 본 명세서에서 사용된 용어, "효소변이체 또는 돌연변이 효소"는 하나 또는 복수의 아미노산이 모유전자 또는 그것의 유도체의 DNA 뉴클레오티드 서열의 변경에 의하여 수식되거나 변경된, 상기 피브린분해효소와 기능적으로 동일한 효소를 의미한다.Also, as used herein, the term “enzyme variant or mutant enzyme” refers to an enzyme that is functionally identical to the fibrinase, in which one or more amino acids are modified or altered by alteration of the DNA nucleotide sequence of the parent gene or derivative thereof. Means.
혈전분해 관련 유전자 연구와 새로운 혈전분해물질에 대한 앞으로의 연구를 통하여 기능성 버섯류 개발과 인체 부작용을 최소화할 수 있는 새로운 혈전용해제 개발이 가능하리라 사료되며, 결과적으로 장수버섯(FF) 균사체 배양액으로부터 분리 정제된 혈전분해효소는 혈전증질환을 예방, 치료용 제약조성물의 주성분으로서 사용될 수 있다.Through research into genes related to thrombosis and future studies on new thrombolytic substances, it is possible to develop functional mushrooms and develop new thrombolytic agents that can minimize human side effects.As a result, they are separated and purified from longevity mushroom (FF) mycelia. The thrombolytic enzyme can be used as a main component of the pharmaceutical composition for preventing and treating thrombosis diseases.
본 발명에 따른 혈전분해효소는 조 추출물을 그대로 사용할 수도 있고, 이를 정제하여 순도 높게 사용해도 좋다. Thrombolytic enzyme according to the present invention may be used as it is crude extract, it may be purified and used high purity.
조 추출물은 배양된 장수버섯 균사체의 배양 여액을 원심분리한 후 그 상층액을 취하여 이를 동결건조하여 용적을 줄인 후 에탄올 침전법으로 단백질을 분리함으로써 조단백질을 조제하여 필요한 용도에 사용할 수 있다. 균사체로부터 조단백질을 조제할 때는 완충액으로 깨끗이 세척하여 현탁시킨 다음 이를 초음파 파쇄기(Sonics & Materials Inc.)를 이용하여 파쇄한 다음 여과기로 여과하여 조단백질을 제조할 수 있다. 조단백질을 자실체에서 조제하는 경우에는 먼저 자실체에 증 류수를 가하여 마쇄한 후 진공원심분리기로 원심 분리하여 그 상층액을 취하고 에탄올 침전법을 이용하여 50∼75%의 에탄올로 추출하여 조제할 수 있다.The crude extract is centrifuged from the culture filtrate of the cultured longevity mushroom mycelium, and then the supernatant is taken and lyophilized to reduce the volume, and the crude protein is prepared by separating the protein by ethanol precipitation. When the crude protein is prepared from the mycelium, the crude protein may be prepared by washing and suspending it with a buffer solution, then crushing it by using an ultrasonic crusher (Sonics & Materials Inc.) and then filtering it with a filter. When the crude protein is prepared in the fruiting body, first, distilled water is added to the fruiting body, and then ground. The crude protein is centrifuged with a vacuum centrifuge, and the supernatant is taken and extracted with 50-75% ethanol using ethanol precipitation.
위와 같이 분리된 조단백질은 필요에 따라 원하는 용도로 사용할 수 있으나 더욱 순도가 높은 것이 필요한 경우에는 통상의 방식으로 추가로 정제하여 사용하여도 좋다. 예를 들면 CM-Cellulose column을 완충액으로 평형화시키고 조 단백질을 주입하여 액체 크로마토그래피를 수행하여 분획을 회수하고, 피브린 아가로오스 플레이트 확인법과 아조카제인 에세이를 진행하여 혈전분해 활성이 있는 부분만을 취한 다음, 예를 들면, DEAE Sephadex A-50 column을 완충액으로 평형화시킨 후 단백질 분획들을 음이온 교환 크로마토그래피(Anion-exchange chromatography)를 진행하고 액체크로마토크래피를 이용하여 분획을 회수한다. 피브린 분해 활성을 가진 것으로 확인되면 이들 분획을 추가의 정제에 사용할 수 있다. 활성단백질 분획은 모아서 투석하고 동결건조를 이용하여 농축시킨 후, 예를 들면, Sephadex G-75 column 등을 사용하여 완충액으로 평형화시킨 후 단백질 분획을 주입하여 겔여과 크로마토그래피 및 액체 크로마토그래피에 의해 활성 분획을 회수하여 더욱 정제할 수 있다.Crude protein separated as described above can be used for the desired use as needed, but if it is required to have a higher purity may be further purified and used in a conventional manner. For example, the CM-Cellulose column is equilibrated with buffer, crude protein is injected, liquid chromatography is performed, the fractions are recovered, fibrin agarose plate identification and azocaine assays are performed, and only thrombolytic activity is taken. For example, after equilibrating the DEAE Sephadex A-50 column with a buffer, the protein fractions are subjected to anion-exchange chromatography, and the fractions are recovered using liquid chromatography. Once identified as having fibrin degrading activity, these fractions can be used for further purification. The active protein fractions are collected, dialyzed, concentrated using lyophilization, equilibrated with a buffer solution using, for example, a Sephadex G-75 column, etc., followed by injection of protein fractions, followed by activity by gel filtration chromatography and liquid chromatography. The fractions can be recovered and further purified.
또한, 본 발명은, 추가의 일면에 있어서, 유효 성분으로서 상기 혈전분해효소, 그의 효소 변이체 또는 돌연변이 효소 및 제약학상 허용되는 담체를 포함하는 혈전에 직접 작용하여 피브린을 용해시키는 혈전 용해제를 제공한다.In still another aspect, the present invention provides a thrombolytic agent that directly acts on a blood clot to dissolve fibrin, including the thrombolytic enzyme, an enzyme variant or mutant thereof, and a pharmaceutically acceptable carrier as an active ingredient.
본 발명의 제약 조성물은 혈전에 직접 작용하여 피브린을 용해시키는 혈전 용해제로서 사용될 수 있으며, 당업자는 그러한 질병, 상태 및 이상으로부터 고통 을 받는 것으로 추정되는 개인들을 표준 진단 기술을 사용하여 용이하게 파악할 수 있다. 본 발명의 혈전분해제를 적용할 수 있는 질환으로는, 이들에 한정되지 않고, 뇌혈전, 뇌색전증 등의 뇌질환, 폐색전, 폐경색증 등의 폐질환, 하지심부정맥 혈전증, 보행장애, 혈류장해성빈혈, 동맥경화성괴저증, 신경통, 고지혈증 등의 말초신경계질환, 신성고혈합, 신장병, 신부전 등의 신장질환, 협심증, 허혈성심장질환, 심근경색 등의 심장계 질환 등을 포함한다.The pharmaceutical composition of the present invention can be used as a thrombolytic agent that directly acts on a thrombus to dissolve fibrin, and those skilled in the art can readily identify, using standard diagnostic techniques, individuals suspected of suffering from such diseases, conditions and abnormalities. . Diseases to which the thrombolytic agent of the present invention can be applied include, but are not limited to, brain diseases such as cerebral thrombosis and embolism, lung diseases such as pulmonary embolism and pulmonary infarction, lower extremity arrhythmia thrombosis, gait disorder, and thrombopathy Peripheral nervous system diseases such as anemia, atherosclerotic necrosis, neuralgia, hyperlipidemia, renal diseases such as nephrotic hypertension, kidney disease, renal failure, cardiac diseases such as angina pectoris, ischemic heart disease, and myocardial infarction.
본 발명의 제약 조성물은 공지의 의약용 담체와 조합하여 제제화할 수 있다. 일반적으로는, 유효 성분을 약학적으로 허용할 수 있는 액상 또는 고체상의 담체와 배합하고, 또한 필요에 따라 용제, 분산제, 유화제, 완충제, 안정제, 부형제, 결합제, 붕괴제, 활택제 등를 가하고, 정제, 과립제, 산제, 분말제, 캡슐제 등의 고형제, 통상액제, 현탁제, 유제 등의 액제로 할 수 있다. 또한 이것을 사용 전에 적당한 담체의 첨가에 의해 액상으로 만들 수 있는 건조품으로 할 수도 있다.The pharmaceutical composition of the present invention can be formulated in combination with a known medical carrier. Generally, the active ingredient is blended with a pharmaceutically acceptable liquid or solid carrier, and a solvent, a dispersant, an emulsifier, a buffer, a stabilizer, an excipient, a binder, a disintegrant, a lubricant and the like are added as necessary and purified. And solid solutions such as granules, powders, powders, and capsules, liquids such as ordinary liquids, suspensions, and emulsions. Moreover, it can also be set as the dry product which can be made into a liquid state by addition of a suitable carrier before use.
본 발명의 제약 조성물은 경구제나, 주사제, 점적용제 등의 비경구제 중의 어떠한 것에 의해서도 투여할 수 있다.The pharmaceutical composition of the present invention can be administered by any of parenteral agents such as oral preparations, injections, and drops.
의약용 담체는, 상기 투여형태 및 제형에 따라서 선택할 수 있고, 경구제의 경우는, 예를 들어 전분, 유당, 백당, 만니톨, 카복시메틸셀룰로스, 콘 스타치, 무기염 등이 이용된다. 또한 경구제의 조제에 있어서는 추가로 결합제, 붕괴제, 계면활성제, 윤택제, 유동성 촉진제, 교미제, 착색제, 향료 등을 배합할 수도 있다.A medical carrier can be selected according to the said dosage form and formulation, and, in the case of an oral preparation, starch, lactose, white sugar, mannitol, carboxymethylcellulose, corn starch, an inorganic salt, etc. are used, for example. Moreover, in preparation of an oral preparation, a binder, a disintegrating agent, surfactant, a lubricating agent, a fluidity promoter, a copper, a coloring agent, a fragrance | flavor, etc. can also be mix | blended.
한편, 비경구제의 경우는 통상의 방법에 따라서 본 발명의 유효성분인 단백분해효소를 포함하는 조성물을 희석제로서의 주사용 증류수, 생리식염수, 포도당 수용액, 주사용 식물유, 참기름, 낙화생유, 대두유, 옥수수유, 프로필렌글리콜, 폴리에틸렌글리콜 등에 용해 내지 현탁시켜서 필요에 따라 살균제, 안정제, 등장화제, 무통화제 등을 가함으로써 조제된다.On the other hand, in the case of parenterals, distilled water for injection as a diluent, saline solution, glucose aqueous solution, vegetable oil for injection, sesame oil, peanut oil, soybean oil, corn according to a conventional method It is melt | dissolved or suspended in oil, propylene glycol, polyethyleneglycol, etc., and it is prepared by adding a disinfectant, a stabilizer, an isotonicity agent, a non-fatting agent etc. as needed.
본 발명의 의약 조성물은 제제 형태에 따른 적당한 투여경로로 투여된다. 투여방법은 특히 한정할 필요는 없고, 내용, 외용 및 주사에 의해 투여할 수 있다. 주사제는, 예를 들어 정맥내, 근육내, 피하, 피내 등에 투여할 수 있다.The pharmaceutical composition of the present invention is administered by a suitable route of administration according to the formulation form. The administration method need not be particularly limited and can be administered by internal content, external application, and injection. Injections can be administered, for example, intravenously, intramuscularly, subcutaneously, intradermally or the like.
본 발명의 의약 조성물의 투여량은, 그 제제형태, 투여방법, 사용 목적 및 이것에 적용되는 환자의 연령, 체중, 증상에 따라서 적절히 설정되고, 일정하지 않지만 일반적으로는 제제중에 함유되는 유효성분의 양은 성인 1일당 예컨대 10㎍∼200 ㎎/㎏이다. 그러나, 당업자는 특정 시약의 약물동력학적인 특성 및 그의 투여 모드 및 경로; 수여자의 나이, 건강, 체중; 증상의 성질 및 정도, 동시 치료의 종류, 치료의 빈도 및 목적하는 효과 등의 공지의 요인에 따라서 투여량이 변화함은 이해할 것이다. The dosage of the pharmaceutical composition of the present invention is appropriately set depending on the form of the preparation, the method of administration, the purpose of use, and the age, weight, and symptoms of the patient to be applied thereto. Amounts are, for example, 10 μg-200 mg / kg per adult. However, those skilled in the art will appreciate the pharmacokinetic properties of certain reagents and their mode and route of administration; Age, health and weight of the recipient; It will be appreciated that the dosage will vary depending on known factors such as the nature and extent of the symptoms, the type of concurrent treatment, the frequency of treatment and the desired effect.
<실시예><Examples>
이하, 본 발명을 실시예에 의해 더욱 구체적으로 설명한다. 그러나 이들은 단지 예시적인 것이며, 본원 발명을 이들에 한정하고자 의도한 것은 아니다. Hereinafter, the present invention will be described in more detail with reference to Examples. However, these are merely exemplary and are not intended to limit the present invention to them.
본 발명의 실시예에서 사용된 장수버섯 균사체(Formitella fracinea 이하, "FF"라고도 한다.)는 국립익산대학에서 얻은 것을 사용하였으나, 특별한 제한없이 모든 장수버섯이 사용될 수 있다. 소 피브리노겐, 인간 피브리노겐, 트롬빈, 플라스민, 아조카제인, 아크릴아마이드, 스트레이트 모노하이드레이트, 트리즈마 염기, 염산 트리즈마, 카르복시메틸(CM)-셀룰로오스, 페닐메틸술포닐플루오라이드(PMSF), 토실라이신 클로로메틸케톤(TLCK), 4-(아미디노페닐)메탄술포닐 플루오라이드(APMSF), 아프로티닌, 펩스타틴 A, 토실페닐알라닌 클로로메틸 케톤(TPCK), 에틸렌글리콜-비스-(2-(아미노에틸)-N,N,N,N'-테트라아세트산(EGTA), 소듐 클로라이드, 카제인 밀크 및 디에틸아미노에틸(DEAE)-세파로스 CL6B는 Sigma Co.제품을 사용하였다. Agarose는 Promega Co.제품을 사용하였다. 예비염색 단백질 마아커 및 컬러 마아커는 Cambrex Co. 제품을 사용하였다. Sephadex G-75 및 HiLoad™ Superdex™ 75 FPLC 컬럼은 Pharmacia Biotech로부터 구입하였다.Longevity mushroom mycelium ( formitella fracinea hereinafter, also referred to as "FF") used in the embodiment of the present invention was obtained from the National University of Iksan, but all longevity mushrooms can be used without particular limitation. Bovine fibrinogen, human fibrinogen, thrombin, plasmin, azocaine, acrylamide, straight monohydrate, trisma base, trisma hydrochloride, carboxymethyl (CM) -cellulose, phenylmethylsulfonylfluoride (PMSF), tosylysine chloro Methylketone (TLCK), 4- (amidinophenyl) methanesulfonyl fluoride (APMSF), aprotinin, pepstatin A, tosylphenylalanine chloromethyl ketone (TPCK), ethylene glycol-bis- (2- (aminoethyl) -N, N, N, N'-tetraacetic acid (EGTA), sodium chloride, casein milk and diethylaminoethyl (DEAE) -Sepharose CL6B used Sigma Co. Agarose used Promega Co. Prestained protein markers and color markers were from Cambrex Co. Sephadex G-75 and HiLoad
실시예 1: 장수버섯 균사체(FF)의 배양 Example 1 Culture of Longevity Mushroom Mycelium (FF)
장수버섯(F. fraxinea)의 최적의 균사체 생산을 위한 배지조건을 확립하기 위하여 표 1에 나타낸 다양한 배지를 사용하여 장수버섯을 배양하였다. 장수버섯 균사체를 PDA 배지(Potato extract 20%, Dextrose 2%)에 접종하여 25 ℃에서 일주일 동안 배양하였다. 균사체는 멸균된 메스와 핀셋을 사용하여 약 5 mm의 크기로 도려내어 그 중 5 조각을 액체 배지 100ml을 함유하는 플라스크에 접종시켰다. 배양물은 회전 조건(120 rpm, 직경 20 mm)에서 10일 동안 25 ℃에서 배양시켰다. 최적 배양 배지를 스크리닝한 후, 높은 생산량을 위한 최적 온도 및 pH를 각각 5℃ 간격의 다양한 온도 범위 및 4.0~8.0 사이의 pH에서 배양시킴으로써 결정하였다. 모든 시험은 3회 반복하였다.Longevity mushrooms were cultured using various media shown in Table 1 to establish medium conditions for optimal mycelium production of F. fraxinea . Longevity mushroom mycelium was inoculated in PDA medium (Potato extract 20%, Dextrose 2%) and incubated for one week at 25 ℃. The mycelium was cut out to about 5 mm in size using sterile scalpels and tweezers, and 5 pieces were inoculated into flasks containing 100 ml of liquid medium. Cultures were incubated at 25 ° C. for 10 days in rotating conditions (120 rpm, 20 mm diameter). After screening the optimal culture medium, the optimum temperature and pH for high yield were determined by incubating at various temperature ranges between 5 ° C. and pH between 4.0 and 8.0, respectively. All tests were repeated three times.
KH2PO4 0.046%, K2HPO4 0.1%, MgSO4·7H2O 0.05%Dextrose 2%, peptone 0.4%, yeast x 0.6%,
KH 2 PO 4 0.046%, K 2 HPO 4 0.1%, MgSO 4 7H 2 O 0.05%
그 결과는 도 2에 나타낸 바와 같다. 도 2는 다양한 배지에서 장수 버섯 균사체를 25℃에서 10일 동안 배양한 결과를 나타내는 도면이다. GMEB; 발아 맥아즙 브로쓰 (11 Brix°), YEPD; 효모 추출 펩톤 덱스트로스, CVM; 장수버섯 배지(Coriolus versicolor medium), MCM ; 버섯 완전 배지, PDM; 감자 덱스트로스 배지. 그 결과 GMEB 배지에서 균사체 생산수율이 가장 높은 것을 확인하였다(건조 중량 1.8 g/200 ㎖). 이 결과는 천연 배지가 화학 합성 배지 보다 우수하였고, 효율적인 균사체 생산을 나타냄을 알 있다.The result is as shown in FIG. Figure 2 is a view showing the result of incubating the longevity mushroom mycelium in various media for 10 days at 25 ℃. GMEB; Germinated wort broth (11 Brix °), YEPD; Yeast extract peptone dextrose, CVM; Longevity mushroom medium ( Coriolus versicolor medium), MCM; Mushroom complete medium, PDM; Potato dextrose badge. As a result, it was confirmed that the mycelial production yield was the highest in GMEB medium (dry weight 1.8 g / 200 ml). This result shows that the natural medium was superior to the chemical synthetic medium, showing efficient mycelial production.
배양 시간에 따른 피브린 분해 활성을 검사하기 위하여 장수버섯 균사체를 다양한 배지에서 10일 동안 성장시킨 후 피브린 분해 활성을 매일 분석하고 그 결과를 도 3에 나타냈다. 도 3은 장수버섯으로부터 피브린 분해 효소의 생산을 나타내는 도면이다. GMEB 배지에서 배양한 균사체가 접종한지 8일째에 최대 피브린 분해 활성을 나타냈다.To examine the fibrin degradation activity according to the incubation time, longevity mushroom mycelium was grown in various media for 10 days, and then the fibrin degradation activity was analyzed daily and the results are shown in FIG. 3. Fig. 3 shows the production of fibrin degrading enzymes from longevity mushrooms. Maximum fibrin degrading activity was shown on
최적 온도 및 pH의 결정의 결과는 표 2에 나타냈다. 표 2는 GMEB 배지에서 균사체 생산을 위한 온도 및 pH의 영향을 나타낸다. 최대 균사체 생산(wet weight 12 g/200 ㎖)은 각각 25℃의 온도 및 pH 5.0에서 발견되었다.The results of the determination of the optimum temperature and pH are shown in Table 2. Table 2 shows the effect of temperature and pH for mycelium production in GMEB medium. Maximum mycelium production (wet weight 12 g / 200 mL) was found at a temperature of 25 ° C. and pH 5.0, respectively.
실시예 2: 단백질 및 피브린 가수순해 활성의 스크리닝Example 2: Screening of Protein and Fibrin Hydrolytic Activity
(2-1) 균사체로부터 프로테아제(피브린분해 효소)의 제조(2-1) Preparation of Protease (Fibrinase) from Mycelium
균사체를 여과에 의해 배양 브로쓰로부터 분리하였다. 이어서 3차 증류수로 3회 세척하였다. 균사체(건조 중량 1 g)를 초음파기(Sonics & Materials Inc., USA)에서 5초 간격으로 10분 동안 2 부피의 물(2 ㎖)로 균질화시켰다. 균질화물을 진공 원심분리기(Model J2-21, BECKMAN)를 사용하여 5,000 x g로 4 ℃에서 15 분 동안 원심분리시켰다. 미리 얼린(-70 ℃) 에탄올을 지속적으로 교반하면서 각각의 0 ~ 90%에 10% 단위로 떨어 뜨려 주었다. 이어서 용액을 추가로 1 시간 동안 교반하에 보관하였다. 침전 단백질은 4 ℃에서 30 분 동안 10,000 x g로 원심분리에 의해 제거하였다. 상층액을 제거한 후, 펠릿들을 건조시키고 단백질을 멸균 3차 증류수에서 재현탁시켰다.Mycelium was isolated from the culture broth by filtration. Then washed three times with distilled
(2-2) 프로테아제 활성 에세이(2-2) Protease Activity Essay
페트리 디쉬에 0.3% 탈지유 5 ㎖과 2% 아가로오스 용액 5 ㎖을 첨가하여 탈지 플레이트를 만들었다. 플레이트를 실온에서 1 시간 동안 정치시키고 굳어진 플레이트에 직경 5㎜로 구멍을 낸 후 이어서 샘플 용액 20 ㎕을 플레이트 상에 조심스럽게 넣어주었다. 상이한 에탄올 함량을 지닌 각 시료 10 ㎕을 2% 탈지유 플레이트 상에 조심스럽게 위치시켰다. 트립신을 양성 대조군으로 사용하였다. 플레이트를 37 ℃에서 14 시간 동안 인큐베이션시키고, 용해된 원의 직경을 측정하였다. 프로테아제 활성의 1 유니트는 37℃에서 14시간 동안 인큐베이션시킨 후 10 ㎟의 용해된 원을 생성하는데 필요한 양으로 정의하였다. 그 결과를 도 4에 나타냈다. 도 4는 장수버섯 탈지유 플레이트로부터 프로테아제 분해 활성을 나타내는 도면이다. (1. 양성 대조군으로서 트립신, 2. 음성 대조군으로서 3차 증류수, 3. 0%, 4. 10%, 5. 20%, 6. 30%, 7. 40%, 8. 50%, 9. 60%, 10. 70%, 11. 80%, 12. 90%). 에탄올 농도가 높을수록 더 큰 용해 원이 관찰되었고, 70% 이상의 에탄올을 함유한 시료는 거의 동등한 용해 원을 나타냈다.A degreasing plate was made by adding 5 ml of 0.3% skim milk and 5 ml of 2% agarose solution to a Petri dish. The plate was allowed to stand at room temperature for 1 hour, punctured into a hardened plate with a diameter of 5 mm, and then 20 μl of sample solution was carefully placed on the plate. Ten μl of each sample with different ethanol content was carefully placed on a 2% skim milk plate. Trypsin was used as a positive control. Plates were incubated at 37 ° C. for 14 hours and the diameter of the dissolved circles was measured. One unit of protease activity was defined as the amount required to produce 10
(2-3) 피브린 분해 활성 에세이(2-3) Fibrin Degradation Activity Essay
피브린 분해 활성 에세이는 Mullertz(Mullertz, S. and Lassen, M., An activator system in blood indispensable for formation of plasmin by streptokinase. Proc. Soc. Exp. Biol. Med., 82 : 264, 1953)의 방법을 약간 변형하여 측정하였다. 0.1 M NaCl을 포함한 50 mM Tris-HCl 완충액(pH 7.8) 중의 0.4% 인간 피브리노겐 5 ㎖을 2% 아가로스 용액 5 ㎖에 첨가하고, 0.1 ㎖ 트롬빈 (100 NIH units/ml)과 혼합하였다. 혼합물을 10 ㎝ 페트리 디쉬 내에 붓고, 실온에서 1 시간 동안 정치시켰다. 이어서, 시료 용액 20 ㎕(각 시료 용액 10 ㎕ 및 양성 대조군으로서 플라스민 10 ㎍)을 조심스럽게 피브린 플레이트 상에 위치시켰다. 플레이트를 37℃에서 14 시간 동안 인큐베이션 시키고, 용해 원의 직경을 측정하였다. 그 결과를 도 5에 나타냈다. 도 5는 피브린 플레이트에서 장수버섯 균사체로부터의 프로테아제의 피브린 분해 활성을 나타내는 도면이다(1. 양성 대조군으로서 플라스민, 2. 음성 대조군으로서 3차 증류수, 3. 0%, 4. 10%, 5. 20%, 6. 30%, 7. 40%, 8. 50%, 9. 60%, 10. 70%, 11. 80%, 12. 90%). 14 시간 반응 후, 더 높은 에탄올 농도는 더 큰 용해 원을 나타내었고 70% 이상의 에탄올을 함유한 시료는 거의 동등한 용해 원을 나타냈다. 따라서, 장수 버섯은 혈전 예방을 위한 기능성 (건강) 식품으로써 개발 및 사용될 수 있다.Fibrin degrading activity assays were performed by Mullertz (Mullertz, S. and Lassen, M., An activator system in blood indispensable for formation of plasmin by streptokinase.Proc. Soc.Exp. Biol.Med ., 82: 264, 1953). Slightly deformed and measured. 5 ml of 0.4% human fibrinogen in 50 mM Tris-HCl buffer (pH 7.8) with 0.1 M NaCl was added to 5 ml of 2% agarose solution and mixed with 0.1 ml thrombin (100 NIH units / ml). The mixture was poured into 10 cm Petri dishes and left at room temperature for 1 hour. 20 μl of sample solution (10 μl of each sample solution and 10 μg of plasmin as positive control) were then carefully placed on the fibrin plate. The plate was incubated at 37 ° C. for 14 hours and the diameter of the dissolution circle was measured. The result is shown in FIG. FIG. 5 shows fibrin degrading activity of protease from longevity mycelium on fibrin plates (1. plasmin as positive control, 2. tertiary distilled water as negative control, 3. 0%, 4. 10%, 5. 20%, 6. 30%, 7. 40%, 8. 50%, 9. 60%, 10. 70%, 11. 80%, 12. 90%). After 14 hours of reaction, higher ethanol concentrations showed larger sources of dissolution and samples containing more than 70% ethanol showed nearly equivalent dissolution sources. Thus, longevity mushrooms can be developed and used as functional (healthy) foods for preventing blood clots.
실시예 3: 피브린 분해 효소의 정제Example 3: Purification of Fibrinase
(3-1) 조 추출 단백질의 농축(3-1) Concentration of Crude Extracted Protein
달리 언급하지 않는 한, 모든 절차는 피브린 분해 효소 활성 및 생산 수율을 높이기 위해 4 ℃에서 수행하였다. 단백질 농도는 BCA 단백질 에세이 키트(Pierce co.)를 사용하여 결정하였다. 배양한 장수 버섯 균사체(F. fraxinea mycelia)를 3차 증류수로 3회 세척하였다. 균사체(100 g)를 동일 부피의 물(100 ㎖)로 균질화기내에서 최대 속도로 2 분 동안 균질화시키고, 액체 질소를 사용하여 동결 및 해동시켰다. 이를 수차례 반복한 후, 균질화물을 4 ℃에서 30 분 동안 5,000 x g로 원심분리 시켰다. 상층액을 식염 얼음조(salt-ice bath)에 함유된 1 리터 스테인레스강 부켓(bucket) 내에 위치시켰다. 동일한 부피의 미리 얼린(-70 ℃) 에탄올을 지속적으로 교반하면서 적하하였다. 이어서 용액을 추가로 1 시간 동안 교반하에 보관하였다. 침전 단백질은 4 ℃에서 30 분 동안 10,000 x g로 원심분리에 의해 제거하였다. 정화된 에탄올 가용성 분획은 식염-얼음 조 내에 위치된 강철 부켓으로 되돌아 왔고, 그의 에탄올 농도는 지속적인 혼합에 의해 75%까지 적하하여 증가시켰다. 교반을 1 시간 동안 계속하고 이어서 침전된 단백질을 4 ℃에서 30 분 동안 10,000 x g로 원심분리시켜 회수하였다. 상층액을 제거한 후, 펠릿들을 건조시키고 단백질을 멸균 3차 증류수 내에 재현탁시켰다. 불용성 물질들은 10,000 x g로 4℃에서 10분 동안 원심분리시켜서 제거하였다.Unless otherwise noted, all procedures were performed at 4 ° C. to increase fibrinase activity and production yield. Protein concentration was determined using the BCA protein assay kit (Pierce co.). The cultured longevity mushroom mycelium ( F. fraxinea mycelia) was washed three times with tertiary distilled water. Mycelium (100 g) was homogenized with equal volume of water (100 mL) at maximum speed for 2 minutes in the homogenizer, and frozen and thawed with liquid nitrogen. After repeating this several times, the homogenate was centrifuged at 5,000 xg for 30 minutes at 4 ° C. The supernatant was placed in a 1 liter stainless steel bucket contained in a salt-ice bath. An equal volume of pre-frozen (-70 ° C.) ethanol was added dropwise with continuous stirring. The solution was then stored under stirring for an additional hour. Precipitated protein was removed by centrifugation at 10,000 xg for 30 minutes at 4 ° C. The purified ethanol soluble fraction returned to the steel bucket located in the salt-ice bath and its ethanol concentration was increased by drop to 75% by continuous mixing. Stirring was continued for 1 hour and then the precipitated protein was recovered by centrifugation at 10,000 xg for 30 minutes at 4 ° C. After removing the supernatant, the pellets were dried and the protein was resuspended in sterile tertiary distilled water. Insoluble materials were removed by centrifugation at 4O < 0 > C for 10 minutes at 10,000 xg.
(3-2) 단백질 측정(3-2) Protein Measurement
단백질 농축물은 BCA 또는 Micro BCA 단백질 에세이 리어전트 키트(Pierce, Rockford, IL, USA) (Lowry, O.H., Rosebrough, N.J., Farr, A.L., and Randall, R.T., Protein measurement with the folin reagent. J. Biol. Chem., 193 : 265-275, 1951)를 사용하여 측정하였다. 단백질 표준으로 소 혈청 알부민(BSA)을 사용하였다. Protein concentrates include BCA or Micro BCA protein assay regent kits (Pierce, Rockford, IL, USA) (Lowry, OH, Rosebrough, NJ, Farr, AL, and Randall, RT, Protein measurement with the folin reagent.J. Biol Chem., 193: 265-275, 1951). Bovine serum albumin (BSA) was used as the protein standard.
도 4 및 5에서 보는 바와 같이, 피브린 분해 활성은 에탄올의 농도가 증가함에 따라 증가되었고, 70% 이상의 에탄올 농도에서는 유사한 피브린 분해 활성을 나타내었다. 단백질은 장수버섯 (F. fraxinea)의 50 ~ 75% 농도의 에탄올을 사용하여 배양 균사체로부터 농축하고, 펠릿들을 추가의 정제에 사용하였다.As shown in Figures 4 and 5, fibrin degrading activity increased with increasing ethanol concentration, showing similar fibrin degrading activity at ethanol concentrations of 70% or more. Protein was concentrated from the culture mycelium using 50-75% concentration of ethanol ( F. fraxinea ) of the mushroom, and the pellets were used for further purification.
(3-3) 이온 교환 크로마토 그래피(3-3) Ion Exchange Chromatography
(1) 양이온 교환 컬럼 크로마토그래피(1) cation exchange column chromatography
상기 재현탁된 펠릿들은 20 mM Tris-HCl(pH 7.4) buffer로 예비평형화시킨, CM-cellulose column(7 x 15 ㎝)에 가하고, Tris-HCl(pH 7.4) 20 ~ 500 mM의 선형 구배로, 4 ℃에서 1.5 ㎖/fr의 유속으로 용출시켰다. 각 분획 1.5 ㎖을 수집하여 아조카제인 및 피브린 플레이트 에세이를 사용하여 피브린 분해 활성을 측정하고 그 결과를 도 6에 나타냈다. 높은 프로테아제 활성을 나타내는 분획들을 음이온 교환 크로마토그래피에 적재하였다. 도 6은 CM-셀룰로오스를 사용한 장수버섯 균사체로부터 피브린분해 효소의 음이온 교환 크로마토그래프도이다. 단백질 농도는 UV 분광광도계를 사용하여 계산하였다. 피브린 분해 활성은 피브린 플레이트(데이터에 나타내지 않음)를 사용하여 결정하였다. 화살로 나타낸 프로테아제 분획들을 수집한 것이고 이를 DEAE-Sepharose CL6B column에 가하였다. 도 6에서 보는 바와 같이, 선두부분의 분획들이 피브린분해 활성을 나타냈고, 그러한 분획들이 뉴터 젠더(neuter gender) 또는 음성 하전 단백질로서 여겨진다.The resuspended pellets were added to a CM-cellulose column (7 × 15 cm), pre-equilibrated with 20 mM Tris-HCl (pH 7.4) buffer, with a linear gradient of 20-500 mM Tris-HCl (pH 7.4), It was eluted at 4 ° C. at a flow rate of 1.5 mL / fr. 1.5 ml of each fraction was collected and fibrin degradation activity was measured using azocaine and fibrin plate assays and the results are shown in FIG. 6. Fractions showing high protease activity were loaded on anion exchange chromatography. 6 is an anion exchange chromatogram of fibrinase from longevity mushroom mycelium using CM-cellulose. Protein concentration was calculated using a UV spectrophotometer. Fibrin degradation activity was determined using fibrin plates (not shown in data). Protease fractions indicated by arrows were collected and added to the DEAE-Sepharose CL6B column. As shown in FIG. 6, the leading fractions showed fibrinogenic activity, and such fractions are considered to be Neuter gender or negatively charged protein.
(2) 음이온 교환 컬럼 크로마토그래피(2) anion exchange column chromatography
용출된 CM-셀룰로오스 컬럼 분획들은 20 mM Tris-HCl(pH 7.4) buffer로 예비평형시킨 DEAE-Sepharose CL6B column(5 x 20 ㎝)에 가하고, 0 ~ 2 M의 NaCl 선형 구배로 4 ℃에서 1.5 ㎖/fr의 유속으로 용출시켰다. 각 분획을 수집하고 피브린 분해활성을 아조카제인 및 피브린 플레이트 에세이에 의해 측정하였다. 그 결과를 도 7에 나타냈다. 도 7은 DEAE-Sepharose CL6B를 사용한 장수버섯 균사체로부터 피브린 분해 효소의 음이온 교환 크로마토 그래피도이다. 도 7에서 보는 바와 같이, 0.6 M NaCl을 함유한 분획들이 높은 프로테아제 활성을 나타냈으며, 이들 단백질은 음하전을 띠었다. 이들 모든 활성 분획들을 수집하여 동결건조 전에 투석시켰다. 높은 프로테아제 할성을 나타내는 분획들을 Sephadex G-75 컬럼에 부하하였다.Eluted CM-cellulose column fractions were added to a DEAE-Sepharose CL6B column (5 × 20 cm) pre-equilibrated with 20 mM Tris-HCl (pH 7.4) buffer and 1.5 mL at 4 ° C. with a NaCl linear gradient of 0-2 M. eluted at a flow rate of / fr. Each fraction was collected and fibrin degrading activity was measured by azocaine and fibrin plate assays. The result is shown in FIG. 7 is an anion exchange chromatography diagram of fibrin degrading enzyme from longevity mushroom mycelium using DEAE-Sepharose CL6B. As shown in FIG. 7, fractions containing 0.6 M NaCl showed high protease activity, and these proteins were negatively charged. All these active fractions were collected and dialyzed before lyophilization. Fractions showing high protease activity were loaded on a Sephadex G-75 column.
(3-4) 프로테아제 활성 분획의 분리(3-4) Isolation of Protease Active Fraction
프로테아제 활성은 아조카제인(Sigma co.)으로부터 산-가용성 물질의 방출을 측정함으로써 결정하였다. 효소 시료/컬럼 분획(50 ㎕)을 0.1%(w/v) 아조카제인(50 mM Tris-HCl 중에 제조함, pH 7.0) 300 ㎕에 첨가하였다. 37 ℃에서 20 분 동안 인큐베이션 시킨 후, 빙냉 10% (w/v) 트리클로로아세트산(TCA) 600 ㎕을 즉시 섞어 첨가하였다. 시료를 10 분 동안 얼음 상에 위치시킨 후, 15,000 x g로 15분 동안 원심분리를 하였다. 상층액 중의 산-가용성 물질의 양은 366 ㎚에서 흡광도에 의해 측정하였다. 프로테아제 활성의 1 유닛은 37 ℃에서 1 시간 동안 인큐베이션 시킨 후, 아조카제인으로부터 366 ㎚에서 0.1의 흡광도를 나타내기에 충분한 산-가용성 물질을 생성하는데 필요한 양으로 정의한다. 또한, 효소 시료/컬럼 분획은 피브린 분해 활성을 위해 플레이트에 조심스럽게 위치시켰다.Protease activity was determined by measuring the release of acid-soluble material from azocaine (Sigma co.). Enzyme sample / column fractions (50 μl) were added to 300 μl 0.1% (w / v) azocasein (prepared in 50 mM Tris-HCl, pH 7.0). After incubation at 37 ° C. for 20 minutes, 600 μl of ice-cold 10% (w / v) trichloroacetic acid (TCA) was added immediately and mixed. The sample was placed on ice for 10 minutes and then centrifuged at 15,000 x g for 15 minutes. The amount of acid-soluble material in the supernatant was measured by absorbance at 366 nm. One unit of protease activity is defined as the amount necessary to produce an acid-soluble substance sufficient to exhibit an absorbance of 0.1 at 366 nm from incubation at 37 ° C. for 1 hour. In addition, enzyme sample / column fractions were carefully placed on plates for fibrin degradation activity.
(3-5) 겔 여과(3-5) gel filtration
음이온 교환 크로마토그래피의 용출된 DEAE-Sepharose CL6B 컬럼 분획들을 0.15 M NaCl을 함유한, 10 mM Tris-HCl(pH 7.4) 완충액으로 예비 평형시킨 Sephadex G-75 column(1.5 x 130 ㎝) 상에 가하고 동일한 완충액으로 4 ℃에서 0.1 ㎖/min의 유속으로 용출하였다. 각 분획을 수집하고 피브린 분해활성을 아조카제인 및 피브린 플레이트 에세이에 의해 측정하였다. 그 결과를 도 8에 나타냈다. 활성 분획들을 수집하고 동결 건조에 의해 농축시켰다. 도 8은 Sephadex G-75를 사용한 장수버섯 균사체로부터 피브린 분해 효소의 겔-여과 크로마토그래피도이다. 화살로 나타낸 프로테아제 분획들을 수집하여 HiLoadTM SuperdexTM 75 column에 가하였다.The eluted DEAE-Sepharose CL6B column fractions of anion exchange chromatography were added onto a Sephadex G-75 column (1.5 × 130 cm) pre-equilibrated with 10 mM Tris-HCl, pH 7.4, containing 0.15 M NaCl and the same The buffer was eluted at 4 ° C. at a flow rate of 0.1 mL / min. Each fraction was collected and fibrin degrading activity was measured by azocaine and fibrin plate assays. The result is shown in FIG. Active fractions were collected and concentrated by lyophilization. 8 is a gel-filtration chromatography diagram of fibrin degrading enzyme from longevity mushroom mycelium using Sephadex G-75. Protease fractions indicated by arrows were collected and added to the HiLoad ™ Superdex ™ 75 column.
(3-6) 고성능액체 크로마토그래피 (FPLC) (3-6) High Performance Liquid Chromatography (FPLC)
용출된 Sephadex G-75 컬럼 분획들은 20 mM Tris-HCl(pH 7.4) 완충액으로 예비 평형시킨 HiLoadTM SuperdexTM 75 column 상에 가하고, 동일한 완충액으로 1.5 ㎖/min의 유속으로 용출시켰다. 각 분획을 수집하고 피브린 분해활성을 아조카제인 및 피브린 플레이트 에세이에 의해 측정하였다. 그 결과를 도 9 및 표 3에 나타냈다. Eluted Sephadex G-75 column fractions were pre-equilibrated with HiLoad ™ in 20 mM Tris-HCl (pH 7.4) buffer. Superdex TM It was added on 75 columns and eluted with the same buffer at a flow rate of 1.5 ml / min. Each fraction was collected and fibrin degrading activity was measured by azocaine and fibrin plate assays. The results are shown in Figure 9 and Table 3.
(㎎)Total protein
(Mg)
(U/㎎)Singular mars
(U / mg)
(%)Recovery
(%)
도 9는 HiLoadTM SuperdexTM 75를 사용한 장수버섯 균사체로부터 피브린 분해 효소의 FPLC 결과도이다. 표 3으로부터 알 수 있는 바와 같이, 피브린 분해 효소 1.1 ㎎은 HiLoadTM SuperdexTM 75 column에 의해 0.8%의 수율로 14.2 배로 정제되었다. 화살로 나타낸 프로테아제 분획들을 수집하고 SDS-PAGE 및 피브린 자이모그래피에 의해 측정하였다.9 is HiLoad TM Superdex TM Figure FPLC results of fibrin degrading enzyme from longevity mushroom mycelium using 75. As can be seen from Table 3, 1.1 mg of fibrin degrading enzyme was purified 14.2 times with 0.8% yield by HiLoad ™ Superdex ™ 75 column. Protease fractions indicated by arrows were collected and measured by SDS-PAGE and fibrin zymography.
실시예 4: 분자량 측정Example 4: Molecular Weight Measurement
(4-1) SDS-PAGE(4-1) SDS-PAGE
정제된 효소의 분자량은 Laemmli(1970)의 방법에 따라 SDS-polyacrylamide gel elecrophoresis(PAGE)에 의해 결정하였다(Laemmli, U.K. Nature, 227 : 680-685, 1970). 단백질 시료 20 ㎍을 5 x 시료 완충액 (60 mM Tris-HCl, pH 6.8, 25% glycerol, 2% SDS, 14.4 mM β-mercaptoethanol, 0.1% bromophenol blue)과 혼합하였다. 이 혼합물을 100 ℃에서 5 분 동안 비등시키고, 12% 폴리아크릴아마이드 겔 중에서 전기영동시켰다. 겔을 Coomassie Brilliant Blue R-250으로 염색시킨 후 효소의 분자량을 표준 마아커(ProSieve™ protein marker, Cambrex Co.)를 사용하여 결정하였다.The molecular weight of the purified enzyme was determined by SDS-polyacrylamide gel elecrophoresis (PAGE) according to the method of Laemmli (1970) (Laemmli, UK Nature, 227: 680-685, 1970). 20 μg of protein samples were mixed with 5 × sample buffer (60 mM Tris-HCl, pH 6.8, 25% glycerol, 2% SDS, 14.4 mM β-mercaptoethanol, 0.1% bromophenol blue). The mixture was boiled at 100 ° C. for 5 minutes and electrophoresed in 12% polyacrylamide gel. The gel was stained with Coomassie Brilliant Blue R-250 and the molecular weight of the enzyme was determined using standard markers (ProSieve ™ protein marker, Cambrex Co.).
(4-2) 피브린-자이모그래피(4-2) Fibrin-Zymography
피브린 자이모그래피는 Leber et al.(1997) and Kim et al.(1998)의 방법에 따라 수행하였다(Leber, T.M. and Balkwill, F.R., Zymography: A Single-step Staining Method for Quantitation of Proteolytic activity on Substrate gels. Anal. Biochem ., 249 : 24-28, 1997; Kim, J.H., and Kim, Y.S., Purification and characterization of fibrinolytic enzyme from Armillaria mellea. Kor. J. Mycology, 26(4) : 583-588, 1998). 0.12%(w/v)). 피브리노겐을 함유한 해상겔 (resolving gel) 용액 (12%) 전체 용적 10 ㎖로 준비하여 원심분리시켜 SDS 원액을 혼합하였을때 유도되는 불용성 불순물을 제거하였다. 트롬빈(1 unit/㎖) 용액 및 TEMED(N,N,N',N'-tetramethyl ethylenediamine)을 각각 최종 농도 0.1 unit/㎖ 및 0.028%(v/v)로 겔 용액에 첨가하였다. 정제된 FFP 효소는 피브린 젤 내로 전기영동시키고, 이어서 2.5% Triton X-100 solution 중에서 세척하고 피브린에 대한 반응 완충액(20 mM Tris-HCl로 제조함, pH 7.5, 0.15 M NaCl, 0.02% NaN3 함유)을 함유한 통에서 37 ℃에서 인큐베이션 시켰다. 피브린 자이모그래피에서, 피브린 가수분해 효소는 비가수분해된 피브린 기질의 푸른 배경에 비하여 피브린 분해의 단일 맑은 밴드로 나타났다. 그 결과를 도 10에 나타낸다. 도 10은 정제된 피브린분해 프로테아제 FFP의 SDS-PAGE 및 SDS-피브린 자이모그래프도이다. Lane 1, 단백질 표준 마아커; lane 2, 정제 FFP(SDS-PAGE); lane 3, 정제 FFP(SDS-피브린 자이모그래피).Fibrin zymography is described in Leber et al. (1997) and Kim et al. (1998) (Leber, TM and Balkwill, FR, Zymography: A Single-step Staining Method for Quantitation of Proteolytic activity on Substrate gels.Anal . Biochem ., 249: 24-28, 1997; Kim, JH, and Kim, YS, Purification and characterization of fibrinolytic enzyme from Armillaria mellea.Kor . J. Mycology, 26 (4): 583-588, 1998). 0.12% (w / v)). A total volume of 10 ml of a resolving gel solution (12%) containing fibrinogen was prepared and centrifuged to remove insoluble impurities induced when the SDS stock solution was mixed. Thrombin (1 unit / ml) solution and TEMED (N, N, N ', N'-tetramethyl ethylenediamine) were added to the gel solution at a final concentration of 0.1 unit / ml and 0.028% (v / v), respectively. Purified FFP enzyme is electrophoresed into fibrin gel, followed by washing in 2.5% Triton X-100 solution and reaction buffer for fibrin (prepared with 20 mM Tris-HCl, pH 7.5, 0.15 M NaCl, 0.02% NaN 3) ) Was incubated at 37 ° C. in a vat. In fibrin zymography, the fibrin hydrolase appeared as a single clear band of fibrin degradation compared to the blue background of the non-hydrolyzed fibrin substrate. The result is shown in FIG. 10 is an SDS-PAGE and SDS-fibrin zymograph diagram of purified fibrinated protease FFP.
그의 겉보기 분자량은 SDS-PAGE 및 SDS-피브린 자이모그래피 모두에 의해 약 42 kDa으로 계산되었다. 이 결과는 정제된 피브린분해 효소가 단량체임을 의미한다. 이는 기존에 일본의 전통발표식품인 natto로부터 분리된 효소는 35 kDa로 발효미생물에서 분리된 혈전분해효소와는 다른 효소임을 확인할 수 있었다. 또한 현재 임상에서 사용 중인 sPA는 47 kDa, uPA는 55 kDa 그리고 tPA는 70 kDa로 분리 정제된 효소는 완전히 다른 크기의 혈전분해효소로 측정된다. 그리고 약용버섯인 CM에서는 52 kDa, PT에서는 65kDa 그리고 AM에서는 22.5 kDa의 혈전분해효소가 분리되었는데 이들과 크기가 전혀 다른 혈전분해효소임이 확인되었다. 이들 결과는 장수버섯으로부터 혈전분해 효소가 다른 혈전분해 효소와는 상이한 것임을 나타낸다.Its apparent molecular weight was calculated to be about 42 kDa by both SDS-PAGE and SDS-fibrin zymography. This result means that the purified fibrinase is a monomer. It was confirmed that the enzyme isolated from the traditional Japanese food, natto, is 35 kDa, which is different from the thrombolytic enzyme isolated from fermented microorganisms. In addition, sPA is 47 kDa, uPA is 55 kDa and tPA is 70 kDa. The medicinal mushroom, CM, 52 kDa, PT, 65kDa, and AM 22.5 kDa were isolated from thrombolytic enzymes, and their size was completely different. These results indicate that thrombolytic enzymes from longevity mushrooms are different from other thrombolytic enzymes.
실시예 5: 정제 효소의 특성화Example 5: Characterization of Purifying Enzymes
(5-1) 피브린 분해 활성에 대한 pH의 영향(5-1) Effect of pH on Fibrin Degradation Activity
효소의 피브린 분해 활성에 대한 pH의 영향은 아조카세인 에세이를 사용하여 검사하였다. 효소의 피브린 분해 활성에 대한 최적 pH는 2.0 ~ 10.0의 pH 범위 내에서 결정하였다. 효소 용액 10 ㎕을 각각 0.5 M Glycine-HCl (pH 2.0 ~ 3.0), 0.5 M Acetate(pH 4.0 ~ 5.0), 0.5 M Tris-HCl(pH 6.0 ~ 8.0), 및 0.5 M Glycine-NaOH(pH 9.0 ~ 10.0) 완충액 90 ㎕에 첨가하였다. 37 ℃에서 1 시간 동안 인큐베이션 시킨 후, 잔여 프로테아제 활성을 0.1% 아조카제인을 사용하여 측정하였다. 그 결과를 도 11에 나타낸다. 도 11은 아조카제인 에세이에 의해 측정한 장수버섯 균사체로부터의 피브린 분해효소에 대한 다양한 pH의 영향을 나타내는 도면이다. 다음의 완충액을 상이한 pH에서 사용하였다; 0.5 M Glycine-HCl (pH 2~3), 0.5 M Acetate-NaOH (pH 4∼pH 5), 0.5 M Tris-HCl (pH 6∼pH 8), 0.5 M Glycine-NaOH (pH 9∼pH 10)를 사용하였다. 모든 시험은 3회 반복하였다.The effect of pH on the fibrin degrading activity of the enzyme was examined using an azocasein assay. The optimum pH for fibrin degrading activity of the enzyme was determined within a pH range of 2.0-10.0. 10 μl of enzyme solution was added to 0.5 M Glycine-HCl (pH 2.0 to 3.0), 0.5 M Acetate (pH 4.0 to 5.0), 0.5 M Tris-HCl (pH 6.0 to 8.0), and 0.5 M Glycine-NaOH (pH 9.0 to 10.0) was added to 90 μl of buffer. After incubation at 37 ° C. for 1 hour, residual protease activity was measured using 0.1% azocaine. The result is shown in FIG. FIG. 11 shows the effects of various pH on fibrin degrading enzymes from longevity mushroom mycelium measured by azocaine assays. The following buffer was used at different pHs; 0.5 M Glycine-HCl (pH 2-3), 0.5 M Acetate-NaOH (pH 4-pH 5), 0.5 M Tris-HCl (pH 6-pH 8), 0.5 M Glycine-NaOH (pH 9-pH 10) Was used. All tests were repeated three times.
그 결과 피브린 분해 효소는 넓은 범위의 pH(2.0 ~ 9.0)에 걸쳐서 활성을 나타냈으나, 최적 활성은 pH 5.7에서 나타났다. 효소는 pH 5.0 ~ 7.0의 범위에서 37 ℃에서 1시간 동안 안정하였고, pH 8.0 까지 50% 이상의 활성을 나타냈다. 활성은 pH 8.0 이상에서 급격히 감소하였다.As a result, fibrin degrading enzyme showed activity over a wide range of pH (2.0-9.0), but the optimum activity was found at pH 5.7. The enzyme was stable for 1 hour at 37 ℃ in the range of pH 5.0 ~ 7.0, and showed at least 50% activity up to pH 8.0. Activity sharply decreased above pH 8.0.
(5-2) 피브린 분해 활성에 대한 온도의 영향(5-2) Effect of Temperature on Fibrin Degradation Activity
효소 활성의 최적 온도는 피브린 분해 효소 0.1 ㎖을 다양한 온도(20 ~ 80 ℃)에서 1시간 동안 인큐베이션 시킨 후 잔류 활성의 측정에 의해 결정하였다. 인큐베이션 후, 잔류 프로테아제 활성은 366 ㎚에서 흡광도 및 0.1% 아조카제인으로 측정하였다. 도 12는 장수버섯으로부터의 피브린 분해 효소에 대한 다양한 온도의 영향을 나타내는 도면이다. 피브린분해 활성에 관한 온도의 영향은 효소가 20 ~ 40 ℃ 사이에서 활성을 나타낸 것으로 밝혀졌다. 최적 활성은 35 ~ 40 ℃ 이었으나, 45 ℃ 이상에서는 이상에서 효소 활성은 급격히 감소하였다. The optimum temperature of enzyme activity was determined by measuring the residual activity after incubating 0.1 ml of fibrin degrading enzyme at various temperatures (20-80 ° C.) for 1 hour. After incubation, residual protease activity was measured at 366 nm with absorbance and 0.1% azocaine. 12 shows the effect of various temperatures on fibrin degrading enzymes from longevity mushrooms. The effect of temperature on fibrinogenic activity was found to be the enzyme exhibiting activity between 20 and 40 ° C. Optimal activity was 35 ~ 40 ℃, the enzyme activity was sharply reduced above 45 ℃.
미생물로부터 분리정제된 혈전분해효소와 할미송이버섯(Tricholoma asponaceum)으로부터 분리 정제된 혈전분해효소는 최적 활성 온도가 50∼60 ℃로 보고되었다. 상기 최적 pH 및 온도를 고려할 때, 장수버섯으로부터 정제된 혈전분해효소는 인체에 투여되었을 때 안정된 효소적 활성을 나타낼 수 있는 것으로 보인다. 장수버섯은 혈전생성 제어 및 혈전분해용의 기능성 식품으로서 사용가능할 것으로 기대된다.The thrombolytic enzyme isolated from microorganisms and purified from thrombolytic enzymes and Tricholoma asponaceum has been reported to have an optimum activity temperature of 50-60 ° C. In view of the optimal pH and temperature, it seems that thrombolytic enzymes purified from longevity mushrooms can exhibit stable enzymatic activity when administered to humans. Longevity mushrooms are expected to be used as functional foods for controlling thrombus formation and thrombosis.
(5-3) 피브린분해 활성에 대한 금속이온의 영향(5-3) Effect of Metal Ions on Fibrin Degradation Activity
금속 이온의 영향은 CuSO4, CoCl2, CaCl2, ZnCl2, MgCl2, MnCl2, KCl, FeSO4, Fe2(SO4)3 및 CsCl 이온들을 사용하여 조사하였다. 정제된 효소들은 Cu2+, Co2+, Ca2+, Zn2+, Mg2+, Mn2+, K+, Fe2+, Fe3+ 및 Cs+ 등의 양이온들의 존재 및 부재하에서 10 mM Tris-HCl 중의 1 mM의 최종 농도로 37 ℃에서 1 시간 동안 예비 배양시켰다. 37 ℃에서 1 시간 동안 인큐베이션시킨 후, 잔류 프로테아제 활성은 0.1% 아조카제인으로 측정하였다. 그 결과를 도 13에 요약하였다. 모든 시험은 3회 반복하였고, 나타낸 값은 평균 ± SEM이다; p<0.05. 도 13은 정제된 혈전분해 효소 활성에 미치는 다양한 금속 이온의 영향을 나타낸 그래프도이다.The effect of metal ions was investigated using CuSO 4 , CoCl 2 , CaCl 2 , ZnCl 2 , MgCl 2 , MnCl 2 , KCl, FeSO 4 , Fe 2 (SO 4 ) 3 and CsCl ions. Purified enzymes were prepared in the presence and absence of cations such as Cu 2+ , Co 2+ , Ca 2+ , Zn 2+ , Mg 2+ , Mn 2+ , K + , Fe 2+ , Fe 3+ and Cs + . Pre-incubation at 37 ° C. for 1 hour at a final concentration of 1 mM in mM Tris-HCl. After incubation at 37 ° C. for 1 hour, residual protease activity was measured with 0.1% azocaine. The results are summarized in FIG. 13. All tests were repeated three times and the values shown are mean ± SEM; p <0.05. 13 is a graph showing the effect of various metal ions on purified thrombolytic activity.
피브린 분해 활성의 50% 이상이 Cu2 + 및 Fe3 +에 의해 감소되었고, Zn2 +은 40% 이상으로 감소시켰다. 한편, 거의 20%의 활성이 Ca2 +, Mn2 + 및 Mg2 + 이온에 의해 증가되었다.At least 50% of the fibrin-decomposing activity was decreased by the Cu + 2 and Fe + 3, Zn 2 + was decreased by 40% or more. On the other hand, almost 20% of the activity was increased by the Ca 2 +, Mn 2 + and Mg 2 + ions.
(5-4) 피브린분해 활성에 대한 프로테아제 억제제의 영향(5-4) Effect of Protease Inhibitors on Fibrinolytic Activity
프로테아제 억제제의 영향은 PMSF, APMSF, TLCK, TPCK, EDTA, EGTA, 아프로티닌 및 펩스타틴 A를 사용하여 조사하였다. 정제된 효소를 10 mM Tris-HCl 중의 100 ㎍/㎖ PMSF, APMSF 및 TPCK, 50 ㎍/㎖ TLCK, 1 mM EDTA 및 EGTA, 1 ㎍/㎖ 아프로티닌 및 펩스타틴 A의 최종 농도로 PMSF, APMSF, TLCK, TPCK, EDTA, EGTA, 아프로티닌 및 펩스타닌 A 중에 37 ℃에서 1 시간 동안 예비 배양시켰다. 37 ℃에서 1 시간 동안 인큐베이션 시킨 후, 잔류 프로테아제 활성을 0.1% 아조카제인으로측정하였다. PMSF, APMSF, TLCK 및 TPCK는 세린프로테아제 억제제이고, 아프로티닌은 트립신-유사 세린프로테아제 억제제이다. EDTA 및 EGTA는 메탈로프로테아제 억제제인 반면, 펩스타틴 A는 산 프로테아제 억제제에 속한다. 그 결과를 도 14에 나타낸다. 도 14는 정제된 피브린 분해 효소 활성에 미치는 여러 가지 프로테아제 억제제의 영향을 나타낸다. 모든 시험은 3회 반복하였고, 나타낸 값은 평균 ±SEM이다.The effects of protease inhibitors were investigated using PMSF, APMSF, TLCK, TPCK, EDTA, EGTA, aprotinin and pepstatin A. The purified enzyme was purified at 100 μg / ml PMSF, APMSF and TPCK, 50 μg / ml TLCK, 1 mM EDTA and EGTA, 1 μg / ml aprotinin and pepstatin A at 10 mM Tris-HCl at a final concentration of PMSF, APMSF, Pre-incubated for 1 hour at 37 ° C. in TLCK, TPCK, EDTA, EGTA, aprotinin and pepstanin A. After incubation at 37 ° C. for 1 hour, residual protease activity was measured by 0.1% azocaine. PMSF, APMSF, TLCK and TPCK are serine protease inhibitors, and aprotinin is a trypsin-like serine protease inhibitor. EDTA and EGTA are metalloprotease inhibitors, while pepstatin A belongs to acid protease inhibitors. The result is shown in FIG. 14 shows the effect of various protease inhibitors on purified fibrin degrading enzyme activity. All tests were repeated three times and the values shown are mean ± SEM.
도 14에서 보는 바와 같이, PMSF, TLCK, 아프로티닌 및 펩스타닌 A에 의해서는 의미있는 영향이 관찰되지 않았다. APMSF 및 TPCK에 의해서는 대략 10%의 활성이 감소되었고, EDTA, EGTA는 활성을 약 40% 감소시켰다. 이들 결과는 정제된 효소가 메탈로프로테아제임을 나타낸다. 버섯유래의 혈전분해효소들이 상당수 metalloprotease로 알려져 왔다. 식물체를 기주로 하는 할미송이버섯(Tricholoma asponaceum)으로부터 분리된 혈전분해효소와 PT 자실체와 AM의 자실체로부터 분리된 효소는 모두 EDTA 처리시 불활성화되는 metalloprotease로 보고되었다(참조: Doonan S., Healy V., O'Connell J., and McCarthy T.V., 1999. The Lysine-Specific Proteinase from Armillaria mellea Is a Member of a Novel Class of Metalloendopeptidases Located in Basidiomycetes. Biochem. Biophysi. Com., 262(1): 60∼63.). As shown in FIG. 14, no significant effects were observed with PMSF, TLCK, aprotinin and pepstanin A. APMSF and TPCK reduced approximately 10% of their activity, while EDTA and EGTA reduced their activity by about 40%. These results indicate that the purified enzyme is a metalloprotease. Mushroom-derived thrombolytic enzymes have been known for many metalloproteases. Thrombolytic enzymes isolated from the plant-based Tricholoma asponaceum and enzymes from the fruiting bodies of PT and fruiting bodies have been reported as metalloprotease that is inactivated upon EDTA treatment (see Doonan S., Healy V). O'Connell J., and McCarthy TV, 1999.The Lysine-Specific Proteinase from Armillaria mellea Is a Member of a Novel Class of Metalloendopeptidases Located in Basidiomycetes.Biochem.Biophysi.Com., 262 (1): 60-63. .).
(5-5) 시간에 따른 피브린 및 피브리노겐 분해의 분석(5-5) Analysis of Fibrin and Fibrinogen Degradation Over Time
피브린분해 활성은 Datta et al.(1995)의 방법을 약간 변형하여 수행하였다(Datta, G., Dong, A., Witt, J., and Tu, A. T., Biochemical characterization of basilase, a fibrinolytic enzyme from Crotalus basiliscus basiliscus. Archives of Biochemistry and Biophysics, 317(2) : 365-373, 1995). 구체적으로, 0.1 M NaCl을 함유하는 50 mM Tris-HCl 완충액(pH 7.8) 중의 1% 인간 피브리노겐 용액 30 ㎕을 동일한 완충액 중의 트롬빈 용액 (10 NIH units/㎖) 30 ㎕과 혼합하였다. 피브린 클롯을 실온에서 1 시간 정도 정치시켰다. 총 30 ㎕의 정제된 알갈(algal) 프로테아제 용액 (20 ㎍/㎖)을 클롯 표면 상에 위치시키고, 다양한 시간 간격으로 37 ℃에서 인큐베이션시켰다. 플라스민 (0.1 U/㎖)을 양성 대조군으로 사용하였다. 반응은 변성 용액(denaturing solution, 10 M urea, 4% SDS, 및 4% β-mercaptoethanol) 90 ㎕ 을 첨가함으로써 정지시켰다. 결과의 펩티드들은 12% 젤 상의 SDS-PAGE에 의해 분석하였다(Laemmli, 1970, 상기함). Fibrinolytic activity was performed by slightly modifying the method of Datta et al . (1995) (Datta, G., Dong, A., Witt, J., and Tu, AT, Biochemical characterization of basilase, a fibrinolytic enzyme from Crotalus basiliscus basiliscus.Archives of Biochemistry and Biophysics, 317 (2): 365-373, 1995). Specifically, 30 μl of a 1% human fibrinogen solution in 50 mM Tris-HCl buffer (pH 7.8) containing 0.1 M NaCl was mixed with 30 μl of thrombin solution (10 NIH units / ml) in the same buffer. Fibrin clots were allowed to stand at room temperature for 1 hour. A total of 30 μl of purified algal protease solution (20 μg / ml) was placed on the clot surface and incubated at 37 ° C. at various time intervals. Plasmin (0.1 U / mL) was used as a positive control. The reaction was stopped by adding 90 μl of denaturing solution (10 M urea, 4% SDS, and 4% β-mercaptoethanol). The resulting peptides were analyzed by SDS-PAGE on 12% gels (Laemmli, 1970, supra).
피브리노겐 분해 활성은 상기한 바와 같이 측정하였다(Matsubara, K., Hori, K. Matsuura, Y. and Miyazawa, K., Purification and characterization of a fibrinolytic enzyme and fibrinogen clotting enzyme in a Marine Green Alga, Codium divaricatum. Biochem. Mole. Biol., 125(1) : 137-143, 2000). 구체적으로, 0.1 M NaCl을 함유한 50 mM Tris-HCl 완충액 (pH 7.8) 중의 1% 인간 피브리노겐 80 ㎕을 4 ㎍의 정제 프로테아제와 37 ℃에서 인큐베이션시켰다. 다양한 간격으로, 반응 용액의 부분을 철회하고, Laemmli(1970)의 방법에 따라 SDS-PAGE에 의해 분석하였다.Fibrinogen degradation activity was measured as described above (Matsubara, K., Hori, K. Matsuura, Y. and Miyazawa, K., Purification and characterization of a fibrinolytic enzyme and fibrinogen clotting enzyme in a Marine Green Alga, Codium divaricatum . Biochem.Mole. Biol., 125 (1): 137-143, 2000). Specifically, 80 μl of 1% human fibrinogen in 50 mM Tris-HCl buffer (pH 7.8) containing 0.1 M NaCl was incubated at 37 ° C. with 4 μg purified protease. At various intervals, portions of the reaction solution were withdrawn and analyzed by SDS-PAGE according to the method of Laemmli (1970).
정제된 효소에 의한 가수분해는 SDS-PAGE에 의해 나타내고 그 결과를 도 15에 나타냈다. 도 15는 장수버섯으로부터 정제된 피브린분해 효소의 피브린 용해(A) 및 피브리노겐 용해(B) 패턴을 나타내는 도면이다. Lane C, 대조군으로서 플라스만; lane 1, 1 분 반응후; lane 2, 2분 반응후; lane 3, 3분 반응후; lane 4, 4분 반응후; lane 5, 5분 반응후; lane 6, 10분 반응후; lane 7, 15 반응후; lane 8, 30분 반응후; lane 9, 1시간 반응후. 도 15(A)에서 보는 바와 같이, 정제 효소는 α 및 γ-γ chain을 급속히 가수분해 시켰고, β chain을 서서히 가수분해시켰다.Hydrolysis by purified enzyme was shown by SDS-PAGE and the results are shown in FIG. 15. Fig. 15 shows the fibrin dissolution (A) and fibrinogen dissolution (B) patterns of fibrinase purified from longevity mushrooms. Lane C, Plasman as a control;
또한, 정제 효소는 피브리노겐 용해 활성을 가졌다. 도 15(B)는 Aα 및 γ 체인을 급속히 가수분해시키는 반면, Bβ 체인은 인큐베이션한지 30분 내에 가수분해되지 않았음을 나타낸다. 그러나, 모든 체인들은 1 시간 이내에 가수분해되었다. 정제효소에 의한 피브린의 가수분해 패턴의 분석 결과는 직접 작용을 명백히 나타내며, 대부분의 통상적으로 사용되는 혈전 용해제, 예컨대 SK, UK 및 tPA는 간접적으로 작용하는 것을 나타낸다. 따라서, 정제된 피브린 분해 효소는 혈전 치료용의약으로 사용될 수 있고, 경구 투여될 수 있다.In addition, the purified enzyme had fibrinogen lytic activity. FIG. 15 (B) shows rapid hydrolysis of Aα and γ chains, whereas Bβ chains did not hydrolyze within 30 minutes of incubation. However, all chains were hydrolyzed within 1 hour. The results of analysis of the hydrolysis pattern of fibrin by purified enzymes clearly show direct action, and most commonly used thrombolytic agents such as SK, UK and tPA act indirectly. Thus, the purified fibrin degrading enzyme can be used as a thrombosis medicament and orally administered.
실시예 6: 제약학적 제제Example 6: Pharmaceutical Formulations
제형의 형태(정제)Form of Formulation (Tablet)
본 발명 피브린분해효소 50 mg50 mg of the present fibrinase
락토오스 80 mg
전분 17 mgStarch 17 mg
스테아르산 마그네슘 3 mg
결정성 셀룰로오스 10 mg10 mg of crystalline cellulose
본 발명의 한 실시 형태는 그 예를 상기에 나타낸 정제의 유효 성분으로써 상기 피브린분해효소의 용도이다. 정제는 통상의 방법에 의해 제조될 수 있으며, 한 바람직한 실시형태는 통상의 장용성 피복(예를 들면, 히드록시프로필메틸 셀룰로오스 프탈레이트), 당코팅 또는 피막 코팅을 갖는 정제 또는 연질캅셀 형태이다.One embodiment of the present invention is the use of the fibrinase as an active ingredient of the tablet shown above. Tablets may be prepared by conventional methods, and one preferred embodiment is in the form of tablets or soft capsules with conventional enteric coatings (eg hydroxypropylmethyl cellulose phthalate), sugar coatings or film coatings.
이상 살펴본 바와 같이, 본 발명에 따른 장수버섯 균사체 유래의 혈전분해 효소는 플라스민과는 다른 기질 특이성을 갖는, 피브린 및 피브리노겐을 특이적으로 분해하는 신규의 혈전분해효소임을 확인할 수 있었다. 따라서, 본 발명에 따른 피브린 분해 효소 및 이를 포함하는 추출물은 혈전증 또는 유사 질환의 치료 및 예방에 효과적으로 이용될 수 있다.As described above, the thrombolytic enzyme derived from the longevity mushroom mycelium according to the present invention was found to be a novel thrombolytic enzyme that specifically degrades fibrin and fibrinogen having different substrate specificities from plasmin. Therefore, the fibrin degrading enzyme and extract containing the same according to the present invention can be effectively used for the treatment and prevention of thrombosis or similar disease.
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