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논문명/저자명
Site-directed mutagenesis를 이용한 alginate lyase AlgL의 고활성 mutant 개발 / 박옥란
발행사항
광주 : 조선대학교 대학원, 2011.8
청구기호
TD 615.1 -11-130
형태사항
viii, 82 p. ; 26 cm
자료실
전자자료
제어번호
KDMT1201151958
주기사항
학위논문(박사) -- 조선대학교 대학원, 바이오신약개발학, 2011.8. 지도교수: 조훈, 최철희
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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

Table 1. PCR primer sequences for algl 60

Table 2. Cycling parameters for the PCR method 60

Table 3. The primer sequences of AlgL mutants 61

Table 4. Km, Kcat and Kcat/Km values of mutants K63A, K63E and K63H(이미지참조) 69

Table 5. Km, Kcat and Kcat/Km values of mutants Y64A, Y64F, D68A and D68N(이미지참조) 70

Table 6. Km, Kcat and Kcat/Km values of mutants F60A, F82A, W143A, L145A and S147T(이미지참조) 71

Table 7. Km, Kcat and Kcat/Km values of mutants T86A, T86S, E93A, E93K and E93Q(이미지참조) 72

Table 8. Km, Kcat and Kcat/Km values of mutants K138A, K138E, L148A, L148G and L148I(이미지참조) 73

Table 9. Km, Kcat and Kcat/Km values of mutants Y152A, Y152F, D190A and D190K(이미지참조) 74

Table 10. Km, Kcat and Kcat/Km values of mutants K194A, K194E, W202A, W202D and W202R(이미지참조) 75

Table 11. Km, Kcat and Kcat/Km values of mutants N197A, N197D, N198A, N198D and H199A(이미지참조) 76

Table 12. Km, Kcat and Kcat/Km values of mutants K245A, K245D, K245G, K245L and K245R(이미지참조) 77

Table 13. Km, Kcat and Kcat/Km values of mutants R247A, R247G, and R247L(이미지참조) 78

Table 14. Km, Kcat and Kcat/Km values of mutants Y253A, Y253F, Y256A and Y256F(이미지참조) 79

Table 15. Km, Kcat and Kcat/Km values of mutants Q309A, Q309E, Q309H, Q309K and Q309N(이미지참조) 80

Table 16. Km, Kcat and Kcat/Km values of mutants E312A, E312D, E312K, and E312Q(이미지참조) 81

Table 17. Km, Kcat and Kcat/Km values of mutants K319A, K319E, R350A, R350D, R350G and R350L(이미지참조) 82

Table 18. Km, Kcat and Kcat/Km values of mutants K245A/R247A, K245A/R247G, K245G/R247A and K245G/R247G(이미지참조) 83

Table 19. Km, Kcat and Kcat/Km values of mutants K245D/K194E, K245D/E312D, K245D/K319A, R350L/K245L and R350L/R247L(이미지참조) 84

Table 20. Km, Kcat and Kcat/Km values of mutants K245A/R247A/K194E, K245A/R247A/E312D, K245A/R247A/K319A and K245A/R247A/K319E(이미지참조) 85

Table 21. Km, Kcat and Kcat/Km values of mutants K194E/K245D/E312D, K194E/K245D/K319A and K194E/K245A/R247A/K319A(이미지참조) 86

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

초록보기 더보기

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.

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