Studies on Function and Activity of Fibrinolytic Enzymes from Bacillus spp. and Staphylococcus sp. Strains = 바실러스 균주와 스타피로코커스 균주로부터 분리한 혈전용해효소의 기능 및 효소활성에 대한 연구

국문 초록 (Abstract)

Bacillus 균주와 Staphylococcus 균주들로부터 5 종류의 세포외 혈전용해효소 (2종류의 스브틸리신, Vpr, peptidase T, and AJ)를 분리하고 이들효소의 기능을 특성화하였다.
첫째, Bacillus sp. DJ-4로부터 우수한 혈전용해능을 가진 스브틸리신 DJ-4를 분리하였다. 스브틸리신 DJ-4의 유전자를 증폭한 후, 이 유전자의 nucleotide와 아미노산 서열을 결정한 결과, B. amyloliquefacens에 의해 분비되는 스브틸리신 BPN’과 79%의 유사성을 보였다. 대장균을 통해 전체- 스브틸리신 DJ-4와 성숙-스브틸리신 DJ-4를 발현시킨 후, 이들의 혈전용해능 및 플라스미노겐 활성인자 효과를 연구하였다. 재조합 전체-스브틸리신 DJ-4의 경우, Bacillus sp. DJ-4에 의해 자연적으로 분비되는 스브틸리신 DJ-4와 비교했을 때, 열 (60oC)과 산성조건 (pH 3.0-4.0)에서 보다 높은 안정성을 보였다. 재조합 전체-스브틸리신 DJ-4의 플라스미노겐 활성인자 효과는 플라스민 보다 2.75배의 높은 활성을 나타내었다. 그리고, 기질 특이성 (카제인 분해능에 대한 혈전분해능)을 스브틸리신 BPN’와 스브틸리신 Carlsberg등과 비교한 결과, 각각 2.67배와 3.97배의 높은 기질 특이성을 보였다. 재조합 성숙- 스브틸리신 DJ-4은 사람 섬유소 단백질의 구성 성분인, Aα-, Bβ-, 그리고 γ-사슬,을 5분내에 모두 분해하였다. 그러나, 아주 낮은 농도 (50ng)에서는 스브틸리신 BPN’와는 달리 γ-사슬은 분해하지 않았다. 반면에, 재조합 성숙-스브틸리신 DJ-4의 경우는 효소활성이 없었다.
재조합 스브틸리신 (스브틸리신 BPN’와 Carlsberg)의 효소활성을 확인하기위해, 서로 다른 zymography 방법, SDS-fibrin zymography (SDS-FZ), reverse fibrin zymography (RFZ), 그리고 isoelectric focusing-fibrin zymography, 을 사용하였다. 재조합 스브틸리신 BPN’와 Carlsberg의 경우, Laemmili 완충용액 조건에서는 분해 젤 상으로 이동하지 않고 젤 상단 부분에 걸려 있는 “binding mode”를 형성하는 것을 SDS-FZ와 RFZ 방법을 통해 알았다. 이런 문제를 3에서 10의 pH 범위를 가진 IEF-FZ 방법을 이용함으로써 해결할 수 있었다.
둘째, Bacillus subtilis KCTC 3014의 배양액으로부터 세 종류의 세포외 효소 (Vpr, PepT, 그리고 스브틸리신)을 확인하였다. 세 종류 모두 DEAE-셀룰로즈 음이온 교환수지를 이용하여 성숙된 단백질 형태로 부분적으로 분리 되었다. Zymography와 밀도측정계를 이용하여 그들의 효소활성을 측정하였다. Vpr과 PepT 단백질의 상대적인 분자량은 SDS 젤과 zympgraphy를 이용하여 각각 68과 48 kDa으로 결정되었다. 하지만, 스브틸리신의 경우, 분해 젤의 상단 부위에 “binding mode”를 형성하였다. 100oC에서 5 분간 변성한 후, 스브틸리신의 분자량을 29 kDa로 결정할 수 있었다. 반면에, 효소활성은 띄지 않았다. Vpr, PepT, 그리고 스브틸리신의 효소활성의 최적 pH는 각각 9.0, 6.0-7.0, 그리고 7.0-8.0 이었다. 그리고, 최적 온도는 각각 40, 50, 그리고 40oC 였다. 효소 저해제 실험결과, Vpr과 스브틸리신은 serine계 효소, 그리고 PepT는 metal계 효소로 분류되었다. 세 종류의 유전자, vpr, pepT, 그리고 apr (스브틸리신을 인지하는 유전자)를 증폭하여 그들의 nucleotide와 아미노산 서열을 결정하였다.
마지막으로, 한국의 젓갈로부터 분리한 Staphylococcus 균주로부터 새로운 혈전용해효소 (AJ)를 분리하였다. AJ 단백질의 상대적인 분자량은 SDS 젤과 zympgraphy를 이용하여 26 kDa으로 결정되었다. AJ의 효소활성의 최적 pH와 온도는 각각 2.5-3.0과 85oC 였다. AJ를 100oC에서 20 분간 가열한 후 효소 활성을 측정한 결과, 초기 활성의 85%를 유지하였다. AJ는 사람 섬유소 단백질의 구성 성분인, Aα-, Bβ-, 그리고 γ-사슬 중, Aα-사슬만 선택적으로 분해하였다. 이 결과는 AJ의 경우 α-fibrinogenase 계열에 속하는 단백질을 의미한다. 효소 저해제 실험결과, AJ는 serine계 효소 저해제인 diisoproryl fluorophosphate에 의해 효소 활성이 저해되는 즉, AJ는 serine계 효소로 분류되었다. 흥미로운 것은, AJ는 강산 조건 및 100oC에서도 아주 안정성을 보였다. 반면에 중성과 알칼리 조건에서는 쉽게 분해되는 특징을 보였다. 특히, pH 7.0에서 8.0 범위에서는 효소활성을 가진 dimer 형태를 이루었다. 현재까지 AJ와 같은 강산과 열에 대한 안정성을 가진 혈전용해효소에 대한 내용은 보고되어 있지않다.

다국어 초록 (Multilingual Abstract)

Five extracellular fibrinolytic enzymes (two subtilisins, Vpr, peptidase T, and AJ) from Bacillus spp. Strains and Staphylococcus sp. strain were isolated and their enzymatic properties were characterized.
First, we purified a strong fibrinolytic enzyme (subtilisin DJ-4) from Bacillus sp. DJ-4 and characterized its enzymatic activity. We cloned the gene subtilisin DJ-4, and determines its nucleotide sequence, which showed 97% identity with subtilisin BPN’ from B. amyloliquefacens. Recombinant full-subtilisin DJ-4 (rf-subDJ-4) and mature-subtilisin DJ-4 (rm-subDJ-4) were expressed using a pET29 vector system, and their fibrin(ogen)olytic and plasminogen activator activities were studied. Rf-subDJ-4 was found to have a higher stability to heat (60oC) and to acidic conditions (pH 3.0-4.0) than the native subtilisin DJ-4 of Bacillus sp. DJ-4. The plasminogen activator activity of rf-subDJ-4 was 2.75 times greater than that of plasmin on a molar basis. And its specific activity (F/C, the ratio of fibrinolytic activity to caseinolytic activity) was 2.67 and 3.97 times higher than those of subtilisin BPN’ and subtilisin Carlsberg, respectively. Rf-subDJ-4 rapidly hydrolyzed the Aα-, Bβ-, and γ-chains of fibrinogen within 5 min. But, unlike subtilisin BPN’ at a very low concentration (50 ng), the γ-chain was not cleaved. On the other hand, rm-subDJ-4 did not show enzyme activity. To identify the activity of recombinant subtilisins (subtilisin BPN’ and subtilisin Carlsberg), three different zymography methods, SDS-fibrin zymography (SDS-FZ), reverse fibrin zymography (RFZ), and isoelectric focusing-fibrin zymography (IEF-FZ), were used. The recombinant subtilisins BPN’ and Carlsberg did not migrate into the electrophoretic field based on a Laemmili buffer system, instead forming a “binding mode” at the top part of the separating gels with the SDS-FZ and RFZ techniques. This problem was resolved when using IEF-FZ with a pH range from 3 to 10.
Secondly, three extracellular proteases (Vpr, peptidase T, and subtilisin) from the culture supernatant of Bacillus subtilis KCTC 3014 were identified. All proteins were partially purified by using a DEAE-cellulose ion exchange column chromatography as a mature form. Their activities were determined by using zymography and densitometer. The relative molecular masses of Vpr and peptidase T (PepT) were determined to be 68 and 48 kDa by SDS-PAGE and zymography, respectively. However, subtilisin formed a “binding mode” at the top part of the separating gel. After denaturation by boiling at 100oC for 5 min, its molecular mass was determined to be 29 kDa, whereas its activity was disappeared. The optimal pH of Vpr, PepT, and subtilisin were at pH 9.0, 6.0-7.0, and 7.0-8.0, respectively. And, the optimal temperature of Vpr, PepT, and subtilisin were exhibited at 40, 50, and 40oC, respectively. With inhibitor test, Vpr and subtilisin were classified as a serine protease, and PepT was a metalloprotease. Three genes, vpr, pepT, and apr (encoding subtilisin protein), were cloned and their nucleotide and deduced amino acid sequences were determined.
Lastly, a new fibrinolytic enzyme, designated as AJ, was purified from Staphylococcus sp. strain AJ screened from Korean salt-fermented Anchovy-jeot. Relative molecular weight of AJ was determined as 26 kDa by using SDS-PAGE and fibrin zymography method. AJ exhibited optimum pH and temperature at 2.5-3.0 and 85oC, respectively. AJ kept 85% of the initial activity after heating at 100oC for 20 min on the zymogram gel. It cleaved the Aα-chain of fibrinogen but did not affect the Bβ-and γ-chains, indicating that AJ is a α-fibrinogenase. The fibrinolytic activity was inhibited by diisopropyl fluorophosphate, indicating AJ is a serine protease. Interestingly, AJ was very stable at acidic condition and heat (100oC), whereas it was easily degraded at neutral and alkaline conditions. In particular, in the pH range from 7.0 to 8.0, AJ formed a dimer, showing an activity on SDS gel and fibrin zymogram gel. AJ showed an exceptional activity at extremely low pH and was thermoacid-stable. To our knowledge, a similar combination of acid and heat stability has not yet been reported for other fibrinolytic enzymes.

목차 (Table of Contents)

• CONTENTS
• ABSTRACT = ⅰ
• CONTENTS = ⅵ
• LIST OF FIGURES = ⅹ
• LIST OF TABLES = ⅹⅲ
• ABBREVIATIONS = ⅹⅳ
• CHAPTER Ⅰ. Introduction = 1
• CHAPTER Ⅱ. Fibrinolytic Enzymes from Bacillus spp. Strains = 13
• 1. Introduction = 14
• 2. Materials and methods = 20
• 2.1. Reagents = 20
• 2.2. Bacterial strain and culture condition = 20
• 2.3. Cloning and expression of subtilisin DJ-4 = 21
• 2.4. Fibrinolytic activity assay = 22
• 2.5. Caseinolytic activity assay = 23
• 2.6. Amidolytic activity assay = 23
• 2.7. Fibrinogenolytic activity = 24
• 2.8. SDS-PAGE and IEF gels = 24
• 2.9. Fibrin zymography = 27
• 2.10. Isoelectric focusing-fibrin zymography (IEF-FZ) = 27
• 2.11. Partial purification of extracellular proteases from B. subtilis KCTC 3014 = 28
• 2.12. Enzymatic digestion of protein in-gel and MALDI-TOF mass spectrometric analysis and atabase search = 29
• 2.13. 2-dimensional gel electrophoresis = 30
• 2.14. N-terminal amino acid sequencing = 31
• 2.15. Cloning of vpr, pepT, and apr genes = 31
• 3. Results = 34
• 3.1. Cloning, expression, and fibrin(ogen)olytic properties of a subtilisin DJ-4 gene from Bacillus sp. DJ-4 = 34
• 3.1-1. Cloning of subtilisin DJ-4 = 34
• 3.1-2. Expression of subtilisin DJ-4 = 38
• 3.1-3. Fibrinolytic assays = 40
• 3.1-4. Effect of pH and temperature on rf-subDJ-4 = 44
• 3.1-5. Fibrinogenolytic activity of rf-subDJ-4 = 47
• 3.2. Identification of recombinant subtilisins = 50
• 3.2-1. Identification of recombinant subtilisins (BPN’ and Carlsberg) by SDS-PAGE and SDS-firin zymography analyses = 50
• 3.2-2. Identification of recombinant subtilisins (BPN’ and Carlsberg) by IEF gel and IEF-FZ analyses = 54
• 3.2-3. Identification of recombinant pro- and mature subtilisin DJ-4 by SDS-fibrin and IEF-FZ analyses = 56
• 3.2-4. Fibrin plate assay of recombinant pro- and mature subtilisin DJ-4 = 59
• 3.3. Identification of three extracellular proteases from B. subtilis KCTC 3014 = 61
• 3.3-1. Identification of extracellular proteases from B. subtilis KCTC 3014 = 61
• 3.3-2. Partial purification of extracellular proteases = 61
• 3.3-3. Identification of Vpr and PepT = 65
• 3.3-4. Identification of subtilisin = 68
• 3.3-5. Effects of pH and temperature on the activity of Vpr, PepT, and subtilisin = 70
• 3.3-6. Cloning of vpr, pepT, and apr = 74
• CHAPTER Ⅲ. Purification and Characterization of a Thermoacid-stable Fibrinolytic Enzyme from Staphylococcus sp. Strain AJ Isolated from Korean Salt-fermented Anchovy-joet = 80
• 1. Introduction = 81
• 2. Materials and methods = 83
• 2.1. Reagents = 83
• 2.2. Bacterial strain and culture condition = 83
• 2.3. Purification of the fibrinolytic enzyme = 83
• 2.4. Zymography = 84
• 2.5. Fibrinolytic and caseinolytic activity assay = 85
• 2.6. Amidolytic activity assay = 86
• 2.7. Fibrinogenolytic activity = 86
• 2.8. N-terminal amino acid sequence of AJ = 87
• 2.9. PCR cloning of AJ gene = 87
• 2.10. Thrombolytic activity of the purified enzyme = 88
• 3. Results = 89
• 3.1. Isolation of bacterial strain = 89
• 3.2. Purification of the fibrinolytic enzyme (AJ) from Staphylococcus sp. strain AJ = 91
• 3.3. Enzymatic characterization of AJ = 94
• 3.4. Stability of primary structure of AJ on the zymogram and SDS gels = 97
• 3.5. Comparison of AJ with other proteases for fibrinolytic activity = 99
• 3.6. Fibrinogenolytic activity of AJ = 99
• 3.7. N-terminal amino acid sequence of the purified enzyme = 102
• 3.8. Cloning of AJ gene = 102
• 3.9. Thrombolytic activity of the purified enzyme = 104
• 4. Discussion = 106
• References = 112
• Summary (in Korean) = 131
• Acknowledgement = 136