Biochemical Analysis of a Fibrinolytic Enzyme Purified from Bacillus subtilis Strain A1

여원식,서민정,김민정,이혜현,강병원,박정욱,최영현,정영기 한국미생물학회 2011 The journal of microbiology Vol.49 No.3

A fibrinolytic enzyme from Bacillus subtilis strain A1 was purified by chromatographic methods, including DEAE Sephadex A-50 column chromatography and Sephadex G-50 column gel filtration. The purified enzyme consisted of a monomeric subunit and was estimated to be approximately 28 kDa in size by SDS-PAGE. The specific activity of the fibrinolytic enzyme was 1632-fold higher than that of the crude enzyme extract. The fibrinolytic activity of the purified enzyme was approximately 0.62 and 1.33 U/ml in plasminogen-free and plasminogen-rich fibrin plates, respectively. Protease inhibitors PMSF, DIFP, chymostatin, and TPCK reduced the fibrinolytic activity of the enzyme to 13.7, 35.7, 15.7, and 23.3%, respectively. This result suggests that the enzyme purified from B. subtilis strain A1 was a chymotrypsin-like serine protease. In addition,the optimum temperature and pH range of the fibrinolytic enzyme were 50°C and 6.0-10.0, respectively. The N-terminal amino acid sequence of the purified enzyme was identified as Q-T-G-G-S-I-I-D-P-I-N-G-Y-N,which was highly distinguished from other known fibrinolytic enzymes. Thus, these results suggest a fibrinolytic enzyme as a novel thrombolytic agent from B. subtilis strain A1.

Bacillus subtilis K-54가 생산하는 Fibrinolytic enzyme의 혈전생성 및 스트레스에 미치는 영향

이홍석,이철수,유천권,서원상,강상모 한국산업미생물학회 2000 한국미생물·생명공학회지 Vol.28 No.1

B. subtilis K-54에서 생산된 fibrinolytic 효소의 혈전증과 스트레스에 대한 효과를 mouse를 이용하여 조사하였다. 부분 정제된 효소의 역가를 4 PCU (Protein Casein Unit)로 조정한 후 매일 1회씩 쥐에게 3일간 경구 투여하였다. 효소가 투여된 쥐에 인공적으로 collagen과 epinephrine을 이용하여 혈전을 유발시킨 후 생존 여부를 관찰한 결과 쥐의 수명이 대조군에 비하여 연장됨을 확인하였다. 또한 효소를 투여 한 group의 5-hydroxyindoleacetatic acid(HIAA) 수치가 감소하지 않아 이 효소는 스트레스 자체에는 영향을 미치지 못하는 것으로 판단된다. 간 및 뇌 조직의 과산화 지질 값 및 혈액내의 antiplasmin, Activated Partial Thromboplastin Time(APTT)과 Protrombin Time(PT) 값의 경우 효소를 투여한 스트레스군의 수치가 대조군에 비하여 낮았다. 그러나 단백질 분해의 경우에는 각 군당 별 차이를 나타내지 않았다. The effect of fibrinolytic 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, an 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 treated with the enzyme was lower than that of control. However, protein degradation (PD) value showed no significant difference between treatment and control groups. In addition, the value of activated partial thromboplastin time (APTT), protrombin time (PT) and antiplasmin in blood were higher in the stress group than that of the enzyme treated group.

Purification and Characterization of a Fibrinolytic Enzyme from Snake Venom of Macrovipera lebetina turanica

Kwon, Ki-Rok,Park, Do-Il,Lee, Seung-Bae,Choi, Suk-Ho KOREAN PHARMACOPUNCTURE INSTITUTE 2011 Journal of pharmacopuncture Vol.14 No.2

Objectives: Fibrinolytic enzyme preparations were isolated from the snake venom of Macrovipera lebetica turanica in this study. Methods: The purity of the preparations was determined using SDS-PAGE and the enzymic characteristics of the purified fibrinolytic enzyme were determined. Results: 1. All of the two preparations with fibrinolytic activity obtained from the snake venom of M. l. turanicat contained the major polypeptide with the molecular weight of 27,500. One of the preparation showed purified fibrinolytic enzyme. 2. The purified fibrinolytic enzyme hydrolyzed ${\alpha}$-chain of fibrinogen faster than ${\beta}$-chain but not ${\gamma}$-chain. 3. The fibrinolytic activity was inhibited completely by EDTA, EGTA, 1,10-phenanthroline, and dithiothreitol. 4. The fibrinolytic activity was inhibited completely by calcium chloride, iron(III) chloride, mercuric chloride, and cobalt (II) chloride. 5. The fibrinolysis zone formed after addition of zinc sulfate was smaller but clearer than the control. Conclusions: These results suggested that the fibrinolytic enzyme purifed from the snake venom of M. l turanica was a metalloprotease containing dithiol group.

Purification and Characterization of a Fibrinolytic Enzyme from Snake Venom of Macrovipera lebetina turanica

권기록,최석호,박도일,이승배 대한약침학회 2011 Journal of pharmacopuncture Vol.14 No.2

Objectives: Fibrinolytic enzyme preparations were isolated from the snake venom of Macrovipera lebetica turanica in this study. Methods: The purity of the preparations was determined using SDS-PAGE and the enzymic characteristics of the purified fibrinolytic enzyme were determined. Results:1. All of the two preparations with fibrinolytic activity obtained from the snake venom of M. l. turanicat contained the major polypeptide with the molecular weight of 27,500. One of the preparation showed purified fibrinolytic enzyme. 2. The purified fibrinolytic enzyme hydrolyzed α-chain of fibrinogen faster than β-chain but not γ-chain. 3. The fibrinolytic activity was inhibited completely by EDTA, EGTA, 1,10-phenanthroline, and dithiothreitol. 4. The fibrinolytic activity was inhibited completely by calcium chloride, iron(III) chloride, mercuric chloride, and cobalt (II) chloride. 5. The fibrinolysis zone formed after addition of zinc sulfate was smaller but clearer than the control. Conclusions: These results suggested that the fibrinolytic enzyme purifed from the snake venom of M. l turanica was a metalloprotease containing dithiol group.

피브린 Zymographic Gel에서 Bacillus amyloliquefaciens G-13으로부터 혈전용해효소의 생성에 영향을 미치는 물리화학적 요인들의 비교

이현호(Hyun-Ho Lee),차민지(Min-Ji Cha),오계헌(Kye-Heon Oh) 한국생물공학회 2020 KSBB Journal Vol.35 No.3

Our previous research demonstrated the characteristics and fibrinolytic activities of NaCl- and capsaicin-resistant bacterium Bacillus amyloliquefaciens G-13, isolated from mustard leaf kimchi [1]. In this study, we extended the work to the comparison of various physicochemical factors (e.g., temperature, pH, NaCl, capsaicin, metal ions, and inhibitors) influencing the production of fibrinolytic enzyme by strain G-13. Initially, a time course of the fibrinolytic activity of strain G-13 was measured by the fibrin plate assay, and after 84 hours of incubation, the maximum fibrinolytic activity was about 3.42 times that of plasmin used as the standard. Through a fibrin zymographic assay, four bands (23, 34, 45, and 67 kDa) of fibrinolytic enzyme from strain G-13 were compared in fibrin zymographic gels. The strain could grow and produce the fibrinolytic enzyme in the presence of NaCl (1-10%) or capsaicin (0-300 ㎍/mL), respectively. The optimum pH and temperature for the enzymatic activity were 8 and 35℃. In the study of the effects of metal ions on fibrinolytic activity, K⁺, Ca<SUP>2+</SUP>, and Mg<SUP>2+</SUP> ions increased the activity and maintained it overall, but Fe<SUP>3+</SUP>, Zn<SUP>2+</SUP>, Ba<SUP>2+</SUP>, and Hg<SUP>2+</SUP> ions rapidly decreased the activity of the fibrinolytic enzyme. The enzyme was completely inhibited by phenylmethanesulfonyl fluroide (PMSF), suggesting that it was a serine protease. These results provide important clues for understanding the characteristics of fibrinolytic bacteria by confirming the effects of B. amyloliquefaciens G-13 on various physicochemical factors influencing the production of fibrinolytic enzymes through fibrin zymography.

Bacillus subtilis BK-17 유래 혈전용해 효소의 특성

백현,임학섭,정경태,최영현,최병태,서민정,김지은,류은주,허만규,주우홍,정영기,Hyun Bek,Lim Hak-Seob,Chung Kyung Kae,Choi Yung Hyun,Choi Byung Tae,Seo Min-Jeong,Kim Ji-Eun,Ryu Eun-Ju,Huh Man Kyu,Joo Woo Hong,Jeong Young Kee 한국생명과학회 2005 생명과학회지 Vol.15 No.6

마른 볏짚으로부터 fibrinolytic enzyme (BK)을 분비하는 균주를 분리하여 동정한 결과, Bacillus subtilis속으로 분류되었다. 분리된 균주(Bacillus subtilis BK-17를 배양하여 그 배양액으로부터 에탄올 침전, ion exchange, gel filtration 등의 과정을 거쳐 fibrinolytic enzyme (BK)를 분리 및 정제하였다. 이 정제된 효소를 SDS-PACE gel 전기영동한 결과 분자량은 약 31 kDa 이었다. BK의 활성 및 안정성에 대한 pH의 영향을 조사해본 결과, pH $6\∼8$범위에서 매우 높았고, 최적 pH는 7과 8이었다. 본 효소의 활성 및 안정성에 대한 최적 온도는 $50^{\circ}C$이었으며, $20^{\circ}C\∼50^{\circ}C$범위에서는 안정성이 그대로 유지되었지만 $60^{\circ}C\∼80^{\circ}C$범위에서는 현저히 감소하여 $50^{\circ}C$에 비해 $20\%\∼40\%$의 활성을 보였다. BK에 대한 활성 역시 안정성과 비슷한 양상을 보였고, 다만 $20^{\circ}C$에서 약 $62\%$의 활성을 보였고, 그 이후 $50^{\circ}C$까지 지속적으로 증가하여 $50^{\circ}C$에서 최대의 활성을 보였다. 실험한 금속이온들 중 1 mM의 $Zn^{2+}$와 $Ca^{2+}$에서 각각 $35\%$와 $23\%$의 저해활성을 보였지만 나머지 이온들에서는 의미 있는 영향이 없었다. 동일농도의 EDTA에 대해서는 $45\%$의 저해활성을 보였기 때문에 BK는 metallo enzyme으로 생각된다. Fibrinogen-rich fibrin plate 와 plasminogen-free fibrin plate에서의 분해활성을 검토해본 결과, 두 plate에서 비슷한 분해활성을 보였다. 따라서 본 효소는 plasminogen activator type 보다는 fibrin에 직접 활성을 가지는 것으로 생각된다. A bacterium, producing a fibrinolytic enzyme, was screened from a decaying rice plant. The bacterium was identified as Bacillus subtilis by morphological, biochemical, and physiological properties and named Bacillus subtilis BK-17. The fibrinolytic enzyme (BK) was purified from supernatant of Bacillus subtilis BK-17 culture broth. The molecular weight was 31 kDa as determined by SDS-PAGE. The effect of temperature, pH, and plasminogen on the activity of the bacillokinase (BK) was analysed and the activity was compared with urokinase. The optimal temperature and pH were $50^{circ}C$ and pH 7, pH 8, respectively. The BK activity was inhibited to $45\%$, $35\%$, and $23\%$ with 1mM EDTA, $Zn^{2+}$, and $Ca^{2+}$, respectively. However, $Mg^{2+}$, $Mn^{2+}$, and $Co^{2+}$ ions did not have any significant effect on the enzyme activity The BK showed the artivity in the both plates, plasminogen-free fibrin plate and plasminogen-rich fibrin plate. The result indicates that the BK can directly act the fibrin. In comparison of fibrinolytic activity with urokinase on the fibrin plate, the BK shows about 20 folds higher activity than that of the urokinase.

Fibrinolytic Enzyme Production by Bacillus subtilis KH-4 Isolated from Deonjang

Kim, J.M.,Suh, H.J.,Ahn, S.W.,Kim, M.S.,Oh, S.H. The Korean Society of Food Science and Nutrition 2002 Preventive Nutrition and Food Science Vol.7 No.4

A strong fibrin-specific fibrinolytic enzyme was produced from Bacillus subtilis KH-4 isolated from Deonjang, a Korean fermented soybean paste similar to Japanese miso. The addition of glucose as a carbon source resulted in the highest levels of caseinolytic and fibrinolytic activities. Likewise, the addition of yeast extract as the nitrogen source resulted in the highest caseinolytic and fibrinolytic activities (3473.2 unit and 47.4 munit, respectively), It was observed that out of all metal ion sources only calcium (chloride) enhanced caseinolytic and fibrinolytic activities, with increases of 4949.3 unit and 58.2 unit/mg, respectively. The optimal temperature for the production of the enzyme was found to be 4$0^{\circ}C$ in the optimal medium (glucose 20 g, yeast extract 5 g, CaCl$_2$l g, and NaCl 2 g). The maximum fibrinolytic activity was observed at the late stationary phase. B. subtilis KH-4 produced a fibrinolytic enzyme at 4$0^{\circ}C$, after 30 h growth, which increased up to 54 h and then remained constant. These results suggest that Deonjang has potential as a source of physiologically active anti-thromotic enzymes.

Purification and Biochemical Characterization of a 17 kDa Fibrinolytic Enzyme from Schizophyllum commune

In Suk Park,Jeong Uck Park,Min Jeong Seo,Min Jeong Kim,이혜현,Sung Ryeal Kim,강병원,최영현,주우홍,Yong Kee Jeong 한국미생물학회 2010 The journal of microbiology Vol.48 No.6

A fibrinolytic enzyme of the mushroom, Schizophyllum commune was purified with chromatographic methods,including a DEAE-Sephadex A-50 ion-exchange column and gel filtrations with Sephadex G-75 and Sephadex G-50 columns. The analysis of fibrin-zymography and SDS-PAGE showed that the enzyme was a monomeric subunit that was estimated to be approximately 17 kDa in size. The fibrinolytic activity of the enzyme in plasminogen-rich and plasminogen-free fibrin plates was 1.25 and 0.44 U/ml, respectively. The N-terminal amino acid sequence of the purified enzyme was identified as HYNIXNSWSSFID, which was highly distinguished from known fibrinolytic enzymes. The relative activity of the purified enzyme with an addition of 5 mM EDTA, Phosphoramidon, and Bestatin was about 76, 64, and 52%, respectively, indicating that it is a metalloprotease. The optimum temperature for the purified enzyme was approximately 45°C, and over 87% of the enzymatic activity was maintained as a stable state in a pH range from 4.0 to 6.0. Therefore, our results suggest that the potential thrombolytic agent from S. commune is a unique type of fibrinolytic enzyme.

청국장으로 부터 분리한 Bacillus subtilis KCK-7에 의한 fibrin분해 효소 생산 배지 최적화

이시경,허석,배동호,최기현 한국산업미생물학회 1998 한국미생물·생명공학회지 Vol.26 No.3

청국장으로부터 분리한 체내혈액의 응고기작에 의해 생성된 단백질인 fibrin을 분해할 수 있는 효소를 분비하는 Bacillus subtilis KCK-7을 이용하여 fibrinolytic enzyme의 생성을 위한 최적 배지조건을 조사하였다. 탄소원으로서 soluble starch 5%와 cellobiose 0.5% 첨가시 가장 높은 효소생성을 보였으며 , 질소원으로는 peptone, 생대두분이 우수하였고 특히, 생대두분 2%첨가시에 가장 높은 효과를 보였다. 최적 배지조건은 0.5% peptone, 0.3% beef extract, 0.5% cellobiose, 5% soluble starch, 2% soybean meal, 0.02% Na_2HPO_4이었다. 본 배지를 효소생산용 배지로 사용할 때 배양 48시간에 효소생성이 가장 높은 것으로 나타났다. The medium optimization was investigated to maximize the production of fibrinolytic enzyme by Bacillus subtilis KCK-7 isolated from Chungkookjang which could hydrolyze the fibrin produced through the blood coagulation mechanism in human body. The simultaneous addition of 5% soluble starch and 0.5% cellobiose to the medium as carbon sources resulted in the highest production of the fibrinolytic enzyme. Likewise, the optimized composition of medium appeared to be 0.5% peptone, 0.3% beef extract, 0.5% cellobiose, 5% soluble starch, 2% raw soybean meal and 0.02% Na_2HPO_4. In addition, the fibrinolytic enzyme production by Bacillus subtilis KCK-7 reached to the maximum level after the cultivation for 48 hr, using the optimized medium.

Isolation of Bacteria with Protease Activity from Cheonggukjang and Purification of Fibrinolytic Enzyme

Yeon Hee Choi(최연희),Jun Seung Lee(이준승),So Young Bae(배소영),Keun Jae Yang(양근재),Kyu Won Yeom(염규원),Dong Hyeok Jo(조동혁),Ock hwa Kang(강옥화),Hyung Suk Baik(백형석) 한국생명과학회 2013 생명과학회지 Vol.23 No.2

혈전용해효소를 생산하는 균주 분리를 위해서 우선 한국, 일본 등지에서 모은 21개의 청국장 시료를 준비하였고 총 268개의 균주를 분리하였다. 이 중에서 1% skim milk가 포함된 nutrient agar 배지에서 protease를 생산하는 bacteria를 분리하였고, 이 결과로 22개의 균주가 분리되었다. 균주들은 apiweb을 통해 생화학적 특성에 근거하여 동정하였다. 또한 세균동정을 위해 16S rRNA 염기서열 분석을 수행하였다. 분리된 대부분의 균주는 Bacillus subtilis와 Bacillus amyloliquefaciens였다. 혈전용해효소의 활성은 fibrin plate 방법에 의해 측정되었고 A2-14, A2-20, C1-05, C1-09, F2-01로 명명된 다섯 균주가 선택되었다. 이중에서 A2-20 균주는 강한 혈전용해 활성을 보였고 동정결과 Bacillus amyloliquefaciens에 가까웠다. A2-20 균주에 의해 생산되는 혈전용해효소는 균 상등액을 이용한 gel filtration과 ion exchange chromatography를 거쳐 부분정제 되었다. 부분 정제된 효소의 최적 pH는 7.0이었고, 최적 온도는 35℃였다. 정제된 단백질의 분석은 SDS-PAGE와 zymography로 이루어졌다. 이와 더불어 혈전용해효소의 유전자적 분석도 수행되었으며 A2-20 균주가 생산하는 혈전용해효소의 부분적인 염기서열과 유전적 상동성을 보이는 서열을 규명하였다. To isolate the fibrinolytic enzyme, 268 strains from 21 samples were morphologically isolated from Cheonggukjang collected from Korea and Japan. Among the 268 strains, protease-producing bacteria were isolated in nutrient agar medium including 1% skimmed milk. As a result of this, 22 strains were isolated. Apiweb site was used to identify these strains based on their biochemical properties. In addition, 16S rRNA sequencing was performed to identify the strain. Most of the identified strains were Bacillus subtilis and B. amyloliquefaciens. Fibrinolytic enzyme activity was measured with the fibrin plate method. Five strains were finally selected: A2-14, A2-20, C1-05, C1-09, and F2-01. Of those five strains, the A2-20 strain, which is close to B. amyloliquefaciens, showed the strongest fibrinolytic activity. The fibrinolytic enzyme produced by the A2-20 strain was partially purified from culture supernatant by gel filtration and ion exchange chromatography. The optimal pH and temperature values of the partially purified enzyme were 7.0 and 35°C, respectively. Purified protein analysis was carried out with SDS-PAGE and zymography. A genetic analysis was also conducted by PCR based on the consensus sequence of fibrinolytic enzyme. Corresponding genes with a partial sequence of the A2-20 strain were identified.