• 한국생명과학회:학술대회논문집
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• 한국생명과학회 2001년도 제32회 학술심포지움
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• pp.67-86
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• 2001

A strain producing strongly fibrinolytic enzyme was isolated from soil and was identified to be Bacillus subtilis by biochemical and physiological characterization. The optimal culture conditions for the production of fibrinolytic enzyme was determined to be 1.0% tryptone, 1.5% soluble starch, 0.5% Peptone, 0.5% NaCl, $(NH_{4})_{3}PO_4.3H_{2}O, and MgSO_{4}.7H_{2}O.$ Initial pH and temperature were pH 8.0 and $30^{\circ}C$ , respectively, The highest enzyme production was observed at 30 hours of cultivation at $30^{\circ}C$ The fibrinolytic enzyme was purified to homogeneity by DEAE Sephadex A-50 ion exchange column chromatography, 70% ammonium sulfate precipitation, Sephadex G-200 and G-75 gel filtration column chromatography. The molecular weight of the purified enzyme was 28,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A gene encoding the fibrinolytic enzyme was cloned into a plasmid vector pBluescript, transforming E.coli XL-1 Blue. The clone was able to degrade fibrin, This indicated that the gene could encode a fibrinolytic enzyme. The nucleotide sequence of the 2.7 kb insert was determined in both direction. One open reading frame composed of 1023 nucleotides was found to be a potential protein coding region. There was the putative Shine-Dalgano sequence and TATA box upstream of the open reading frame. The homology search data in the genome database showed that both the 2.7 kb insert and 1 kb open reading frame carried no significance in the nucleotide sequence of known fibrinolytic enzyme from Bacillus serovars. The recombinant cell harboring the novel gene involved in fibrinolysis was subjected to protein purification. The molecular mass of the purified fibrinolytic enzyme was determined to be 31864 Dalton, which was highly in accordance with the molecular mass(33 kDa) of the fibrinolytic gene deduced from the insert. The fibrinolytic enzyme was Purified 50.5 folds to homogeneity in overall yield of 10.7% by DEAE Sephadex A-50 ion exchange, 85% ammonium sulfate precipitation, Sephadex G-50, Superdex 75 HR FPLC gel filtration. In conclusion, a novel fibrinolytic gene from Bacillus subtilis was identified and characterized by cloning a genomic library of Bacillus subtilis into pBleuscript. For the soybean fermented by this strain, it is found that there increased assistant protein about 20% compared to the soybean not fermented and increased about 30% according to amino acid analysis and, in particular, essential amino acid increased about 40%. When keeping this fermented soybean powder at room temperature for about 70days, it showed very high stability maintaining almost perfect activity and, therefore, it gave us great suggestion its possibility of development as a new functional food.

• Preventive Nutrition and Food Science
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• 제11권2호
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• pp.127-132
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• 2006

Bacillus sp. strain K-l, which produces a strong fibrinolytic enzyme, was isolated from chongkukjang, a traditional Korean fermented soybean paste. The fibrinolytic enzyme was purified from chongkukjang base by using ammonium sulfate fractionation and chromatographic techniques. Purified enzyme, CK K-1 was demonstrated to be homogeneous by SDS-PAGE and isoelectric focusing electrophoresis, and has molecular mass of a 12.4 kDa and a pI of 8.0. The optimal reaction pH value and temperature were 8.0 and $40^{\circ}C$, respectively. Phenyl-methyl-sulfonyl-fluoride (PMSF; serine protease inhibitor), ethylene-diamine-tetra-acetic acid (EDTA; metallo protease inhibitor), copper ion, ferric ion and lead ion inhibited the enzyme activity. These results indicated that the fibrinolytic enzyme is a metallo-serine protease and different from nattokinase and chongkukjangkinase.

• 한국미생물·생명공학회지
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• 제28권5호
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• pp.258-263
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• 2000

A fibrinolytic enzyme was purified to homogeneity from Bacillus sp. S19 using DEAE and CM column chromatograhies, and gel filtration with a recovery yield of 13%. Its molecular mass was estimated to be 42 kDa by SDS-PAGE. The pH and temperature optima were 8.0 and $40^{\circ}C$, respectively. The enzyme was stable up to $45^{\circ}C$ and over a pH range of 6-9. The N-terminal amino acid sequence of the enzyme was determined as Alsa-Gln-Asp-Ala-Thr-Val-Asn-Ile-Ser-Ala-Glu-Arg-Gln-Val-Ile. The fibrinolytic activity was increased by $Cu^{2+}$ while it was strongly inhibited by metal ions such as $Cd^{2+}$ and $Ba^{2+}$ . In addition, the enzyme was inhibited by EDTA, but not by PMSF, suggesting that it is a metallorprotease.

• 생명과학회지
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• 제12권1호
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• pp.43-48
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• 2002

토양으로 부터 혈전용해효소를 생산하는 방선균을 분리하여, 본 방선균이 생산하는 혈전용해효소의 최적 생산 조건을 검토하였다. 생산균주는 회색의 기균사를 생성하며 약 0.6~0.8$\times$0.4~0.8$\mu\textrm{m}$ 크기의 원통형 포자를 가지며 포자의 표면은 매끈하며, 약 10~40개의 포자가 고리를 이루고 있는 형태를 나타내었다. 이상의 결과로 본 균주는 Sfreptomyces 속으로 추정되어 Streptomyces sp. JK-20로 명명하였다. 본 균주의 생리학적 특성으로 20~32$^{\circ}C$의 온도에서 생육 가능하며 최적 생육 온도 및 pH는 24$^{\circ}C$ 및 pH 6이었다. 본 균주의 혈전용해효소의 생산을 위한 최적 생산 조건을 검토한 결과 탄소원으로 1% xylose였고 질소원은 0.5% yeast extract, 0.5% polypeptone, 금속염은 0.1% MgSO$_4$.7$H_2O$였으며 인산염은 0.1% NaH$_2$Po$_4$.2$H_2O$이었다. 본 생산 균주를 최적 배지 조건에서 배양하였을 때 배양 4일째에 혈전용해효소의 생산력이 가장 높았다.

• 한국미생물·생명공학회지
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• 제28권1호
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• pp.52-58
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• 2000

The effect of fivrinolytic enzyme produced from Bacillus subtilis K-54 on the thrombosis and stress in vivo was investigated. Each partially purified fibrinolytic enzyme of 4 protein casein unit was administered orally for 3 days before intravenously injection with collagen and epinephrine. In the mice group administered with the enzyme and increased life span of mice was observed in comparison with that of control. The result suggest that the enzyme may prevent the formation of thrombos in vivo. Administration of the enzyme did not influence to stress itself because 5-hydroxyindoleacetatic acid concentration of brain in the mice group with stress did not decreased after the administration of the enzyme. The value of lipid peroxide (LPO) of the liver and brain cells in the group treted with the enzyme was lower than that of control. However, protein degradation (PDP value showed no significant difference between treatment and control groups. In addition, the value of activated partial thromboplastin time (APTT), protrombin time (PT0 and antiplasmin in blood were higher in the stress group than that of the enzyme treated group.

• 생명과학회지
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• 제8권6호
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• pp.737-740
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• 1998

멸치액젓으로부터 분리한 혈전용해효소를 rat에 경구투여하여 혈액내에서 효소활성을 확인 하였다. 경구투여 후 0, 1, 2, 3, 4시간 후에 혈액을 채취하여 fibrin분해활성을 본 결과 1시간부터 활성이 나타나기시작하여 3시간째에 가장 높았다. Euglobulin Iysis time(ELT)도 경구투여 2, 3시간후에 활성을 보였다. 또한, euglobulin에 의한 amidolysis acti-vity도 1시간부터 활성이 보이기 시작하여 3시간후에 최고활성에 달했다. 이상의 결과로 동물실험을 통한 혈전용해효소의 경구투여에 의한 활성을 혈액내에서 확인할 수 있었다.

• 대한의생명과학회지
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• 제8권4호
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• pp.245-250
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• 2002

Fibrinolytic enzyme has been purified from the edible mushroom, Tricholoma sejunctum using DEAE-cellulose chromatography, Phenyl-Sepharose chromatography and Mono-S column chromatography. The apparent molecular mass of purified enzyme was estimated to be 17100 Da by SDS-polyacrylamide gel electrophoresis and 19000 Da by gel filtration, Indicating that it was a monomer. The N-terminal amino acid sequence of the enzyme was Ala-Thr-Tyr-Lys-Ile-X-Ser-Ala-Thr-His-Gln-X-X-Leu-Val. It has a pH optimum at pH 9.5, suggested that purified enzyme was a alkaline protease. The activity of purified enzyme was inhibited by EDTA and 1,10-phenanthroline, indicating that purified enzyme is a metalloprotease. The activity of purified enzyme was increased by Zn$^{2+}$ and Co$^{2+}$, however, the enzyme activity was totally inhibited by Hg$^{2+}$.

• Journal of Microbiology and Biotechnology
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• 제25권12호
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• pp.2090-2099
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• 2015

Recently, the cardiovascular disease has been widely problematic in humans probably due to fibrin formation via the unbalanced Western style diet. Although direct (human plasmin) and indirect methods (plasminogen activators) have been available, bacterial enzyme methods have been studied because of their cheap and mass production. To detect a novel bacterial fibrinolytic enzyme, 111 bacterial strains with fibrinolytic activity were selected from kimchi. Among them, 14 strains were selected because of their stronger activity than 0.02 U of plasmin. Their 16S rRNA sequence analysis revealed that they belong to Bacillus, Leuconostoc, Propionibacterium, Weissella, Staphylococcus, and Bifidobacterium. The strain B. subtilis ZA400, with the highest fibrinolytic activity, was selected and the gene encoding fibrinolytic enzyme (bsfA) was cloned and expressed in the E. coli overexpression system. The purified enzyme was analyzed with SDS-PAGE, western blot, and MALDI-TOF analyses, showing to be 28.4 kDa. Subsequently, the BsfA was characterized to be stable under various stress conditions such as temperature (4-40oC), metal ions (Mn²⁺, Ca²⁺, K²⁺, and Mg²⁺), and inhibitors (EDTA and SDS), suggesting that BsfA could be a good candidate for development of a novel fibrinolytic enzyme for thrombosis treatment and may even be useful as a new bacterial starter for manufacturing functional fermented foods.

• 한국미생물·생명공학회지
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• 제31권3호
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• pp.284-291
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• 2003

본 연구는 우리나라의 전통발효식품인 재래간장에서도 혈전용해효소 생산 미생물이 존재할 가능성이 있다고 판단하여, 지역의 재래간장으로부터 혈전용해효소 생산능이 우수한 균주 K42를 최종 선발하였다. 선발된 균주는 B. amyloliquefaciens로 동정되었으며, l2% NaCl 함유된 배지에서도 균생육이 양호하였으며 10% NaCl 농도까지 효소생산이 유지되는 내염성 세균임을 알 수 있었다. 혈전용해효소의 정제는 40% ammonium sulfate로 분별 염석 후 DEAE-Sephadex A-50, Sephadex G-100, Sephadex G-75를 이용하여 단일단백의 효소로 정제하였다. 효소의 활성도는 배양여액에 비해 17.1배 증가하였고, 회수율은 1.1%로 나타났다. Disc-electrophoresis와 SDS-PAGE를 실시하여 분자량 45,000 Dalton의 단일 단백질로 확인하였다. 혈전용해효소의 특성조사에 있어서는 최적 pH는 8.0의 약알칼리성을 나타내었고 안정성에 있어서는 pH 4.0에서 pH 10.0 범위에서 80% 이상의 잔존활성을 나타내었으며, 최적반응 온도는 $50^{\circ}C$로 조사되었고 열 안정성에 있어서는 3$0^{\circ}C$의 온도에서 50분간 열처리시 80%이상 잔존 활성을 보였다. 금속이온of B. amyloliquefaciens K42가 생산하는 fibrinolytic enzyme의 활성에 미치는 영향을 검토한 결과 $Mg^{2+}$ $Cu^{ 2+}$에 대해서는 효소 활성이 증가하는 양상을 보였다. 또한 B. amyioliquefaciens K42가 생산하는 fibrinolytic enzyme은 metallo enzyme의 활성저해제인 EDTA등에 대해서는 15%이하로 효소 활성이 감소되었는데 이러한 효소 활성 저해로 보아 metallo enzyme이거나 활성에 금속이온이 필요한 것으로 추정된다. 그리고 기질인 fibrin과의 친화성을 알아보기 위해 Km값을 측정하였는데 Km값은 2.03 mg/ml 나타났다.

• 한국식품영양과학회지
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• 제33권2호
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• pp.439-442
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• 2004

재래식 된장으로부터 혈전용해능과 $\beta$-glucosidase효소를 생산하는 미생물을 분리하였으며, 분리한 균의 형태학적, 생화학적인 분류를 수행하여 Bacillus속으로 동정하였으며, Bacillus sp. KHI-15로 명명하였다 그리고 분리한 균주의 효소생산을 위한 배지조건과 배양조건을 측정하였다. 선발균주는 2% glucose, 0.5% yeast extract, 0.1% calcium chloride의 최적 배지에서 최대의 효소생산을보였다. 초기 pH7∼8과 4$0^{\circ}C$에서는 혈전용해능이 최대를 보였으며, 초기 pH6∼8과 30∼4$0^{\circ}C$에서는 $\beta$-glucosidase 효소활성의 생산이 가장 높았다.