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ABSTRACT 11
I. 서론 13
1. 해조류 알긴산(seaweed alginate) 13
1.1. 해조류 알긴산 종류 및 용도 13
1.2. 알긴산 분해 효소의 종류 14
2. 세균 생물막 및 생물막(bacterial biofilm, BBF) 감염에 의해 생성된 박테리아 알긴산 15
2.1. 세균 생물막의 역사 15
2.2. 세균 생물막의 정의 및 구조특성 15
2.3. 세균 생물막의 형성 16
2.4. 세균 생물막에 의한 감염 16
2.5. 박테리아 알긴산 연구에 의한 생물막 병의 예방과 치료 17
3. 연구목적 18
4. AlgL과 mutant 18
II. 재료 및 방법 20
1. AlgL을 encoding 하는 유전자의 subcloning 20
1.1. A. vinelandii로 부터 AlgL을 encoding 하는 유전자 분리 20
1.2. 분리한 algl gene을 pET28a(+) vector로 도입 20
2. 대장균에 의한 단백질 발현과 정제 21
2.1. 단백질 발현 과정 21
2.2. 단백질 정제 22
2.3. 단백질 정량 23
2.4. Agarose gel 전기영동 및 DNA elution 23
2.5. AlgL의 SDS-PAGE 23
3. algl로부터 mutant 제작 24
4. 알긴산 분해효소의 활성 측정 실험 25
4.1. Thiobarbituric acid (TBA) 발색 원리 25
4.2. TBA 활성 실험 25
5. Michaelis-Menten 및 Lineweaver-Burk kinetics 26
III. 결과 및 고찰 27
1. TBA를 이용한 seaweed alginate와 bacterial alginate에서 AlgL의 mutant 활성 측정 결과 27
1.1. Mutant K63A, K63E, K63H 및 wild type의 활성비교 27
1.2. Mutant Y64A, Y64F, D68A, D68N 및 wild type의 활성비교 27
1.3. Mutant F60A, F82A, W143A, L145A, S147T 및 wild type의 활성비교 28
1.4. Mutant T86A, T86S, E93A, E93K, E93Q 및 wild type의 활성비교 29
1.5. Mutant K138A, K138E, L148A, L148G, L148I 및 wild type의 활성비교 30
1.6. Mutant Y152A, Y152F, D190A, D190K 및 wild type의 활성비교 31
1.7. Mutant K194A, K194E, W202A, W202D, W202R 및 wild type의 활성비교 31
1.8. Mutant N197A, N197D, N198A, N198D, H199A 및 wild type의 활성비교 32
1.9. Mutant K245A, K245D, K245G, K245L, K245R 및 wild type의 활성비교 33
1.10. Mutant R247A, R247G, R247L 및 wild type의 활성비교 34
1.11. Mutant Y253A, Y253F, Y256A, Y256F 및 wild type의 활성비교 35
1.12. Mutant Q309A, Q309E, Q309H, Q309K, Q309N 및 wild type의 활성비교 36
1.13. Mutant E312A, E312D, E312K, E312Q 및 wild type의 활성비교 36
1.14. Mutant K319A, K319E, R350A, R350D, R350G, R305L 및 wild type의 활성비교 37
1.15. Mutant K245A/R247A, K245A/R247G, K245G/R247A, K245G/R247G 및 wild type의 활성비교 38
1.16. Mutant K245D/K194E, K245D/E312D, K245D/K319A, R350L/K245L, R350L/R247L 및 wild type의 활성비교 39
1.17. Mutant K245A/R247A/K194E, K245A/R247A/E312D, K245A/R247A/K319A, K245A/R247A/K319E 및 wild type의 활성비교 40
1.18. Mutant K194E/K245D/K312D, K194E/K245D/K319A, K194E/K245A/R247A/K319A 및 wild type의 활성비교 41
IV. 요약 및 결론 43
참고문헌 87
Figure 1. Types of alginate: poly β-D-mannuronate, poly α-L-guluronate, poly β-D-mannuronate and poly α-L-guluronate. 46
Figure 2. Block sites of alginate polymer and alginate lyse reactions. 47
Figure 3. Mature biofilm structure contaning layers, including bulk of biofilm, linking film, conditioning film, and the substratum to which the film is attached. 48
Figure 4. Diagram showing the development of a biofilm as a five-stage process. 49
Figure 5. The nucleotide sequence of algl gene. (pubmed locus number AF037600) 50
Figure 6. Ribbon like structure of AlgL. 51
Figure 7. Cloning of algl gene into pET28a(+). 52
Figure 8. Function of isopropyl β-D-1-thiogalactopyranoside to transcription of mRNA from DNA. 53
Figure 9. SDS-PAGE of the recombinant AlgL alginate lyase in Escherichia coli BL21(DE₃). 54
Figure 10. SDS-PAGE of purified AlgL and its mutants. 54
Figure 11. Interaction between neighboring residues in the 6xHis tag and Ni-NTA matrix. 55
Figure 12. Protein purification with the Ni-NTA protein purification system. 55
Figure 13. The principle of TBA assay. 56
Figure 14. Structure of acetylate poly-β-D-mannuronate. 56
Figure 15. Michaelis-Menten plot and Lineweaver-Burk plot. 57
Figure 16. Schematic view of the interactions of AlgL. 57
Figure 17. Schematic representation of alginate degradation mechanism 58
Figure 18. Schematic view of the increased activity site of AlgL. 59
Figure 19. Schematic view of the decreased activity site of AlgL. 59
Alginate is a copolymer of beta-D-mannuronic acid and alpha-L-guluronic acid(GulA), linked together by 1-4 linkages. The polymer is a well-established industrial product obtained commercially by harvesting brown seaweeds. Some bacteria, mostly derived from Azotobacter vinelandii, A. chroococcum and several species of Pseudomonas, are also capable of producing copious amounts of this polymer. This uniform polymer is then further modified by acetylation at positions O-2 and/or O-3 but not in seaweed alginates.
A biofilm is a structured consortium of bacteria embedded in a self-produced polymer matrix consisting of polysaccharide, protein and DNA. Bacteria biofilms cause chronic infections because they show increased tolerance to antibiotics system. Cystic fibrosis patients with chronic lung infection is caused by biofilm-growing mucoid Pseudomonas aeruginosa strains. Therefore, alginate lyase mutants with high enzyme activity are needed to decompose the biofilm efficiently.
Alginate lyase(AlgL) were cloned from the Azotobacter vinelandii using plasmid pET28a(+) for mutagenesis and were prepared 79 mutants. Mutants were expressed as His tag fusion enzymes in E. coli, and purified using Ni-NTA agarose bead. The alginate lyase activity was quantitatively measured by thiobarbituric acid assay using sodium alginate(seaweed alginate) and acetylated algiante(bacterial alginate).
K194E/K245D/K319A was the mutants the highest activity in acetylated alginate and K194E/K245A/R247A/K319A was the mutants the highest activity in sodium alginate. The activity was increased more than 6-fold. However, of various mutants, K63A, K63E, N198A, N198D, H199A, Y253A and Y253F were found to be inactive. Asn-197, His-199 and Tyr-253 of wild type AlgL contributed to its high catalytic activity by interacting alginate.
These improved mutants of AlgL would be valuable for the eradication of biofilm-forming bacteria when combined with antibiotics.*표시는 필수 입력사항입니다.
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