• 대한의생명과학회지
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• 제12권3호
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• pp.225-231
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• 2006

Fibrinolytic enzyme was purified from the fruiting bodies of Lepista nuda, using DEAE-Cellulose chromatography, Phenyl Sepharose chromatography, and Mono-S column chromatography. The substance has a molecular weight of 30006.62 Da as measured by MALD-TOF mass spectrometry. The N-terminal amino acid sequence of the enzyme was Tyr-Pro-Ser-Pro-Ser-His-Gln-Thr-Ala-Val-Asn-Ala-Ile-Ile-X. The activity of the enzyme was inhibited by PMSF, indicating that the enzyme is a serine protease. No inhibition was found with E-64, pepstatin, and EDTA. It has broad substrate specificity for synthetic peptides. The enzyme was stable up to $30^{\circ}C$. The enzyme hydrolyzes both Aa and y chains of human fibrinogen but did not show any reactivity for $B{\beta}$ chain of human fibrinogen.

• BMB Reports
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• 제28권2호
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• pp.138-142
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• 1995

An anticoagulant/fibrinolytic protease was purified to homogeneity from the earthworm Lumbricus rubellus. The protein was a single chain glycoprotein of 32 kDa that exhibited strong proteolytic activity on human thrombin and fibrin clots. Proteolytic degradation of these plasma proteins by the purified enzyme occurred at a neutral pH range. Among several human plasma proteins tested as possible substrates for the protease reaction, the 32 kDa enzyme specifically hydrolyzed both thrombin and fibrin polymers without affecting other proteins, such as serum albumin, immunoglobulin, and hemoglobin. Treatment of the purified enzyme at neutral pH with either phenylmethylsulfonylfluoride or soybean trypsin inhibitor resulted in a loss of catalytic activity. The enzyme hydrolyzed the chromogenic substrate H-D-Phe-L-Pipecolyl-L-Arg-p-nitroanilide with a $K_m$ value of 1.1 ${\mu}M$ at a neutral pH. These results suggest that the anticoagulant/fibrinolytic enzyme from Lumbricus rubellus is a member of the serine protease family having a trypsin-like active site, and one of the potential clevage sites for the enzyme is the carbonyl side of arginine residues in polypeptide chains.

• 한국의류산업학회지
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• 제8권2호
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• pp.239-244
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• 2006

For studies of fibrinolytic enzyme strain K-54 was isolated from the Korean traditional food chungkook-jang. Isolated strains K-54 was identified as Bacillus subtilis. The molecular weight of fibrinolytic enzyme from B. subtilis K-54 was 27 kDa. Optimum temperature for fibrinolytic enzyme of B. subtilis K-54 was $50-70^{\circ}C$ and optimum pH for producing the enzyme of this strain was ranging from 8 to 12. Also, it was found out enzyme activity was completely inhibited by 1mM PMSF. The result indicated this enzyme was thermo-stable alkaline serine protease with strong fibrinolytic activity. The wool and silk were treated with protease of B. subtilis K-54. As a result, the property of dyeing of wool fabrics was increased. By the increasing of treatment time became smoothened. But the change of mechanical properties were not changed.

• 생명과학회지
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• 제11권6호
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• pp.561-567
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• 2001

능이버섯[Sarcodon aspratus(Berk.)S.Ito]의 fibrin 분해 활성물질을 분리정제하기 위하여(N $H_4$)$_2$S $O_4$침전법, DE52 anion exchange column chromatography, Sephacryl-S2000 gel filtration chromatography 및 Mono S cation FPLC를 행하였으며 정제된 효소의 특성을 측정하였다. 혈전용해 요소의 활성물질은 DE52 anion exchange colum chroma-tography에 NaCI의 농도가 0.2M 정도에서 용출되었으며 계속된 Sephacryl-S200 gel fitration chromatography 및 Mono S cation EPLC를 실시한 결과 단일 Peak를 얻었고 혈전용해효소의 특이활성은 55.2 U/mg protein으로 조효소액으로 비하여 11.3 배 증가하였으며 수율은 49.5%이었다. 또한 Mono S cation EPLC에서 얻은 활성획분을 12% SDS-PAGE로 전기영동한 결과 단일 band를 얻었으며 gel filtration의 결과와 비교함으로서 정제된 능이의 혈전용해 효소의 분자량은 29.300 Da인 것으로 확인되었다. 능이로 부터 정제한 혈전용해효소는 pH가 높아질수록 효소활성이 증가하였으며 pH 10.5의 알카리성에서도 안정하였으며 6$0^{\circ}C$이상의 온도에서는 효소활성이 급격히 실활하기 시작하였지만 8$0^{\circ}C$에서 25%의 상대활성을 보였다. 또한 본 효소는 C $u^{2+}$이온 $Co^{3+}$ 이온 등 중금속에 의하여 68%및 38%활성이 저해되었으며 $Ca^{2+}$이온 또는 $Mg^{2+}$의 초딤색 인 EDTA및 serine protease inhibitor이 PMSF에 의하여 활성이 저해되었었다. 이러한 결과들은 능이의 혈전용해효소가 Ca.sup 2+/또는M $g^{2+}$에 의하여 활성이 증가하는 serine protease임을 암시해 주고 있다.

• 생명과학회지
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• 제17권5호
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• pp.606-612
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• 2007

혈전(fibrin clot)은 심혈관계 질환을 일으키는 주요 인자로서 전신의 미세동맥이나 모세혈관 내에서 형성되어 주의 조직이나 장기에 혈류의 공급방해가 생겨 허혈, 경색, 나아가 괴사까지도 발생시킨다. 혈전이 원인이 되어 발생한 심혈관계 질환을 예방 혹은 치료할 목적으로 기존으로 사용되고 있는 항혈전제(antithrombolytic drug)나 혈전용해제(thrombolytic drug)의 개발을 위해 많은 연구들이 진행되고 있다. 본 연구에서는 치료목적으로 사용할 혈전용해제를 분리하고자 Protaetia brevitarsis의 maggot로부터 ammonium sulfate 분획과 desalting column을 이용하여 fibrinolytic protease를 분리하고 생화학적 특성을 조사하였다. 활성의 최적 pH는 9.0였고 최적온도는 $50^{\circ}C$였다. pH 7.0-9.0 사이와 온도 $60^{\circ}C$이하에서는 비교적 활성이 안정성을 나타냈다. 효소활성이 phenylmethanesulfonyl fluoride에 의해 강하게 저해되고 있는 것으로 보아 serine protease로 추정되며 금속이온에 의한 영향을 조사해 본 결과 $Ca^{2+}$과 $Zn^{2+}$에 의해서는 저해되지만 $Mg^{2+}$와 $Fe^{2+}$에 의해서는 저해를 받지 않았다.

• Journal of Microbiology
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• 제44권6호
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• pp.622-631
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• 2006

In this study we purified a fibrinolytic enzyme from Cordyceps militaris using a combination of ion-exchange chromatography on a DEAE Sephadex A-50 column, gel filtration chromatography on a Sephadex G-75 column, and FPLC on a HiLoad 16/60 Superdex 75 column. This purification protocol resulted in a 191.8-fold purification of the enzyme and a final yield of 12.9 %. The molecular mass of the purified enzyme was estimated to be 52 kDa by SDS-PAGE, fibrin-zymography, and gel filtration chromatography. The first 19 amino acid residues of the N-terminal sequence were ALTTQSNV THGLATISLRQ, which is similar to the subtilisin-like serine protease PR1J from Metarhizium anisopliae var. anisopliase. This enzyme is a neutral protease with an optimal reaction pH and temperature of 7.4 and $37^{\circ}C$, respectively. Results for the fibrinolysis pattern showed that the enzyme rapidly hydrolyzed the fibrin $\alpha$-chain followed by the $\gamma$-$\gamma$ chains. It also hydrolyzed the $\beta$-chain, but more slowly. The A$\alpha$, B$\beta$, and $\gamma$ chains of fibrinogen were also cleaved very rapidly. We found that enzyme activity was inhibited by $Cu^{2+}$ and $Co^{2+}$, but enhanced by the additions of $Ca^{2+}$ and $Mg^{2+}$ ions. Furthermore, fibrinolytic enzyme activity was potently inhibited by PMSF and APMSF. This enzyme exhibited a high specificity for the chymotrypsin substrate S-2586 indicating it's a chymotrypsin-like serine protease. The data we present suggest that the fibrinolytic enzyme derived from the edible and medicinal mushroom Cordyceps militaris has fibrin binding activity, which allows for the local activation of the fibrin degradation pathway.

• KSBB Journal
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• 제21권1호
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• pp.20-27
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• 2006

미생물 및 동물에 비해 식물에서는 혈전용해효소에 대한 연구가 부족한 실정이며, 기존의 혈전용해효소가 가지는 혈전에 대한 비특이적, 부작용, 고가 등의 단점을 해결할 수 있는 새로운 혈전용해효소의 개발을 위하여 율무, 홍화, 아욱의 종자로부터 추출된 수용성 단백질의 혈전용해 활성을 조사하였다. 각각의 식물들로부터 추출된 조효소 용액은 기존 혈전 용해효소인 plasmin과 양성 대조군으로 하여 비교하여 fibrin 평판법으로 확인한 결과 피브린 응집을 효과적으로 분해하였다. 그 중 율무종자의 수용성 추출물의 혈전용해 활성은 양성 대조군인 plasmin과 비교하여 1.3배의 높은 활성을 나타내었다. 전체 수용성 단백질은 50-75% 에탄올을 이용하여 농축하였으며 율무의 혈전용해효소는 fibrin zymography를 수행하여 확인하고 직접 추출하였다. SDS-PAGE에 의하여 추출효소의 분자량을 측정한 결과 7.8 kDa으로 단일 polypeptide임을 확인하였으며, 효소 활성에 미치는 온도의 효과는 $50^{\circ}C$ 이상에서는 비교적 안정하였으나 더 낮은 온도에서는 급격히 효소활성이 감소하였다. 또한, 각종 단백질분해효소 저해제에 의한 영향을 조사한 결과 APMSF, PMSF, pepstatin A 그리고 TPCK에 강력하게 저해되는 것으로 보아 추출효소는 chymotrypsin과 유사한 serine protease의 하나로 생각되었다. 그러나 EGTA와 EDTA 처리에 의해서는 효소활성의 저해가 두드러지게 나타나지 않았다. 더욱이, 종자저장 중에 미생물에 의한 부패, 활력저하, 생리활성물질의 감소와 장기저장에 따른 에너지소비 증가 등이 문제가 되고 있어 저선량의 감마선 조사를 통해 율무, 홍화, 아욱의 종자로부터 혈전용해 효소활성에 미치는 효과 및 선량에 따른 차이를 조사하였는데 비조사 종자인 대조구와 비교하여 1 Gy, 4 Gy, 16 Gy, 32 Gy선량에서는 낮은 활성을 보였으면 반면에 8 Gy와 64 Gy의 선량에서는 더 높은 활성을 나타내었다. 이러한 결과는 Y선 조사가 종자의 혈전용해 활성을 향상시킬 가능성이 있을 것으로 생각된다. 이상의 모든 결과로 볼 때 율무의 추출 효소는 chymotrypsin-like serine protease에 속하는 혈전용해효소임을 확인할 수 있었다.

• 생명과학회지
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• 제20권2호
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• pp.183-189
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• 2010

혈전용해효소를 생산하는 Bacillus sp. J-19가 보편적인 한국의 조미제인 멸치젓갈에서 분리되었다. 그 혈전용해 효소는 에탄올 침전, Sephadex G-50을 이용한 젤 여과법 등을 포함하는 일련의 크로마토그래피 방법으로 순수분리 되었다. 조효소 추출액과 비교해서, 그 효소의 비활성은 1021배 증가하였고, 수율은 23%이었다. 순수분리한 효소의 분자량은 SDS-PAGE 상 약 45 kDa이었다. 특히, 합성기질인 serine protease (H-D-Ile-Pro-Arg-pNA,S-2288)에 대한 아미드활성은 약 17 U/mg이었다. 또한, 그 45 kDa 혈전용해효소의 60% 이상의 활성이 30%(w/v) sodium chloride 의 존재 하에서도 유지되었다. 이러한 발견들은 특이한 혈전용해효소를 제공해서, 실용적인 혈전용해제 개발을 유도할 수 있다.

• Journal of Microbiology and Biotechnology
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• 제23권7호
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• pp.974-983
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• 2013

Bacillus amyloliquefaciens CB1 was isolated from cheonggukjang, a Korean fermented soy food. B. amyloliquefaciens CB1 secretes proteases with fibrinolytic activities. A gene homologous to aprE of Bacillus subtilis, aprECB1, was cloned from B. amyloliquefaciens CB1, and DNA sequencing showed that aprECB1 can encode a prepro-type serine protease consisting of 382 amino acids. When aprECB1 was introduced into B. subtilis WB600 using an E. coli-Bacillus shuttle vector, pHY300PLK, transformants showed fibrinolytic activity and produced a 28 kDa protein, the size expected for the mature enzyme. The 28 kDa fibrinolytic enzyme was purified from the culture supernatant of B. subtilis WB600 transformant. AprECB1 was completely inhibited by phenylmethylsulfonyl fluoride and almost completely inhibited by EDTA and EGTA, indicating that it is a serine metalloprotease. AprECB1 exhibited the highest specificity for N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide, a known substrate for ${\alpha}$-chymotrypsin. $A{\alpha}$ and $B{\beta}$ chains of fibrinogen were quickly degraded by AprECB1, but the ${\gamma}$-chain was resistant.

• Journal of Microbiology and Biotechnology
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• 제16권2호
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• pp.264-271
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• 2006

Two fibrinolytic enzymes were purified from the culture supernatant of Fomitella fraxinea mycelia by ion-exchange and gel filtration chromatographies, and were designated as F. fraxenia proteases 1 and 2 (FFP1 and FFP2). The apparent molecular masses of the enzymes were estimated to be 32 kDa and 42 kDa, respectively, by SDS-PAGE and gel filtration chromatography. Both enzymes had the same optimal temperature ($40^{\circ}C$), but different pH optima (10.0 and 5.0 for FFP1 and FFP2, respectively). FFP1 was relatively stable at pH 7.0-9.0 and temperature below $30^{\circ}C$, whereas FFP2 was very stable in the pH range of 4-11 and temperature below $40^{\circ}C$. FFPI activity was completely inhibited by phenylmethylsulfonyl fluoride (PMSF) and aprotinin, indicating that this enzyme is a serine protease. The activity of FFP2 was enhanced by the addition of $CO^{2+}$ and $Zn^{2+}$ and inhibited by $Cu^{2+},\;Ni^{2+}$, and $Hg^{2+}$. Furthermore, FFP2 activity was strongly inhibited by EDTA and 1,10-phenanthroline, implying that the enzyme is a metalloprotease. Both enzymes readily hydrolyzed fibrinogen, preferentially digesting the $A{\alpha}$- and $B{\beta}$-chains of fibrinogen over ${\gamma}$-chain. FFP1 showed broad substrate specificity for synthetic substrates, but FFP2 did not. $K_{m}$ and $V_{max}$ values of FFP1 for a synthetic substrate, N-succinyl-Ala-Ala-Pro-Phe-pNA, were 0.213 mM and 39.68 units/ml, respectively. The first 15 amino acids of the N-terminal sequences of both enzymes were APXXPXGPWGPQRIS and ARPP(G)VDGQ(R,I)SK(L)ETLPE, respectively.