분리균주 Variovoriax sp. DSH의 Medium-Chain-Length Poly(hydroxyalkanoate) (MCL-PHA) depolymerase 유전자 클로닝, 대장균에서의 발현 및 효소 특성연구

다국어 초록 (Multilingual Abstract)

A betaproteobacteria strain, Variovorax sp. DSH, capable of degrading MCL-PHA was isolated from a soil sample and the gene of an extracellular MCL-PHA depolymerase from the isolate was cloned and sequenced. The gene (phaZDSH) of extracellular MCL-PHA depolymerase of Variovorax sp. DSH consisted of and 837 bp open reading frame encoding a protein of 278 amino acids with a deduced molecular mass of 30.19 kDa. The amino acid sequence of the phaZDSH had a 100% (278/278) sequence homology with that of the MCL-PHA depolymerase from Pseudomonas alcaligenes LB19. The results are expected on the HGT (Horizontal Gene Transfer).
The N-terminal regions of the phaZDSH contained many hydrophobic amino acid, suggesting that substrate binding domain is located in this region. In contrast, a catalytic triad of the phaZDSH (172Ser, 228Asp, 260His) existed in the C-terminal region, indicating the presence of a catalytic domain in the C-terminal region. The phaZDSH was inserted into pBluescript II KS (-) expression vector and expressed in Escherichia coli DH5α. The expressed protein in E. coli was purified from the active form in crude lysate by hydrophobic interaction chromatography using Octyl-Sepharose CL-4B and subsequently by anion exchange chromatography using Hiprep 16/10 DEAE FF column. The enzyme consisted of monomeric subunit having a molecular mass of 28 kDa, as determined by SDS-PAGE. The maximum activity was observed at pH 8.5 and 45℃. The enzyme was significantly inactivated by EDTA and PMSF but insensitive to DTT. It was also markedly inhibited by 0.2 mM Tween 80 and Triton X-100. When the depolymerase was treated with 1 mM of divalent cations such as Mn2+, Zn2+, and Ag2+, its hydrolysis activity of PHO at pH 8.5 was significantly inhbited, while it was practically insensitive to Ca2+ and Mg2+. The results of characterization is very similar to PhaZLB19
This is the first describing characterization of a MCL-PHA depolymerase gene from Betaproteobacteria and its gene product.

목차 (Table of Contents)

• 서론 1
• 재료 및 방법 8
• 1. 균주 분리 및 배양조건 8
• 1-1. 균주 분리 및 배지조성 8
• 1-2. 시료의 준비 8
• 1-2-1. MCL-PHA 시료 제조 8
• 1-2-2. MCL-PHA 현탁액 제조 8
• 1-3. 분리 균주의 동정 9
• 1-4. 균주 및 배양조건 9
• 1-4-1. 분리균주 및 플라스미드 9
• 1-4-2. 형질 전환체의 배양조건 10
• 2. DNA 분리 조작 및 형질전환 10
• 3. 유전자 (phaZ_(DSH))의 클로닝 10
• 3-1. Genomic DNA 준비 10
• 3-2. Polymerase Chain Reaction (PCR) 10
• 3-3. DNA 염기서열의 결정 11
• 3-4. 발현 vector 의 제조 11
• 3-4-1. 관련 효소의 활성측정 및 단백질 정량 12
• 3-4-2. 전기영동 및 분자량측정 12
• 4. 효소의 정제 12
• 4-1. 조효소액의 제조 12
• 4-2. Hydrophobic interaction chromatography (HIC) 13
• 4-3. Desalting 13
• 4-4. Anion exchange chromatography 13
• 5. 관련효소의 특성분석 14
• 5-1. 효소활성에 대한 온도 및 pH의 영향 14
• 5-2. 효소활성에 대한 저해제 (inhibitors)의 영향 14
• 5-3. 효소활성에 대한 이온 (metal ion)의 영향 14
• 결과 및 고찰 15
• 1. 분리균주의 동정 15
• 2. phaZ_(DSH) 유전자의 클로닝 20
• 3. phaZ_(DSH) 유전자의 분석 22
• 4. 재조합 E. coli 에서 발현 벡터 제조 26
• 5. 재조합 균주에서의 MCL-PHA depolymerase 의 정제 28
• 5-1. MCL-PHA depolymerase 제조 28
• 5-2. MCL-PHA depolymerase의 정제 28
• 5-3. 전기영동 및 분자량 측정 29
• 6. 관련효소의 특성분석 34
• 6-1. 효소활성에 대한 pH 및 온도의 영향 34
• 6-2. 효소활성에 대한 저해제 (inhibitors) 및 이온의 영향 39
• 결론 42
• 참고문헌 45