Citrobacter amalonaticus Y19로부터 일산화탄소 탈수소화효소의 클로닝과 이형 발현 및 특성 조사 = Cloning, expression and characterization of carbon monoxide dehydrogenase from citrobacter amalonaticus Y19

다국어 초록 (Multilingual Abstract)

Hydrogen is pure and clean energy for next generation phase. It can be produced by using microorganisms. One of them, water gas shift reaction is simple and high efficiency hydrogen production from carbon monoxide. previously, Citrobacter amalonaticus Y19 was reported the high growth rate (0.7 h-1) and hydrogen productivity (27.1 mmol g cell-1 h-1). To get a deeper insight of the water gas shift reaction, genome of Y19 was explored. From the contiguous gene analysis it was found that, apart from the metabolic genes for catabolic pathway of hexose sugars, unique genes for CO-dependent hydrogen production are present. Carbon monoxide dehydrogenase (codh) gene cluster was found on contig 00120. The hydrogenase gene cluster was structurally found to be in the reverse direction with respect to codh cluster on the same contig. Functional analyses of genes suggest that the CODH and hydrogenase proteins in Y19 are closely related to other type IV CODH and Ni-Fe hydrogenases. The other maturation genes also have very similar amino acid sequence. CODH cluster consist of many genes out of which, CooFSCTJ and HypB were identified as very essential protein, the genes responsible for there essential accessory protein along with CODH was over expressed in E. coli BL 21(DE3). CODH specific activity in crude cell was 4.53 μmol CO min-1 mg protein-1. It was 7.5 times higher than CODH from C. amalonaticus Y19. Also soluble fraction and pariculate fraction were detected 1.27 and 9.06 μmol CO min-1 mg protein-1, respectively. In this study, we report the first genetic evidence of CO induced H2 production in Citrobacter species.

목차 (Table of Contents)

• 1. 서론 1
• 1.1 배경 1
• 1.2 일산화탄소와 일산화탄소 전환반응 2
• 1.3. 일산화탄소 전환반응을 위한 미생물 3
• 1.4. 일산화탄소 탈수소화효소와 수소화효소 5
• 1.4.1. 일산화탄소 탈수소화효소 6
• 1.4.2. 수소화효소 9
• 1.5. Citrobacter amalonaticus Y19의 특성 10
• 1.6. 이 연구의 목적 11
• 2. 재료 및 방법 12
• 2.1. 균주 및 성장 조건 12
• 2.2. 유전자 sequencing과 Annotation 12
• 2.3. Operon 판별 13
• 2.4. 계통수 분석 13
• 2.5. 플라스미드 구축 13
• 2.6. 세포 준비 14
• 2.7. SDS-PAGE 15
• 2.8. 활성 측정 15
• 3. 결과 및 논의 17
• 3.1. 유전자 분석 17
• 3.1.1. 일산화탄소 탈수소화효소 유전자와 단백질 21
• 3.1.2. 수소화효소 유전자와 단백질 26
• 3.1.3. 일산화탄소 탈수소화효소 수소화효소의 계통수 29
• 3.2. 일산화탄소 탈수소화효소 유전자의 클로닝 및 발현 31
• 3.2.1 일산화탄소 탈수소화효소의 maturation 시스템 가설 31
• 3.2.2. 플라스미드의 구축 33
• 3.2.3. 일산화탄소 탈수소화효소 발현의 온도 영향 35
• 3.2.4. 일산화탄소 탈수소화효소 발현의 IPTG 영향 36
• 3.2.5. 활성 측정 37
• 4. 결 론 39
• 참고문헌 41
• Abstract 50