Carbon Monoxide Dehydrogenase Activity in Bradyrhizobium japonicum
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Transcript of Carbon Monoxide Dehydrogenase Activity in Bradyrhizobium japonicum
Carbon Monoxide Dehydrogenase Activity in Bradyrhizobium japonicum
이정은
Introduction Carbon monoxide (CO) dehydrogenases (CODHs) - a key enzyme in the CO metabolism - carboxydotrophic bacteria - catalyzes the oxidation of CO to CO2
CO + H2O -> CO2 +2e- +2H+
- contains 1) molybdopterin 2) [2Fe – 2S]
Bradyrhizobium japonicum species - gram-negative soil bacteria - N2- fixing symbiosis - utilize H2 and CO2 as a sole sources of energy and carbon - use CO as a a sole sources of energy and carbon
Results
CO- dependent growth
B. japonicum110spc4 -can utilize CO as a sole sourceof carbon and energy (aerobic) -growth under an atmosphere of 50%CO- 50% air for 22 days -the number of cells increased from 1.26 x 104 to 1.58 x 108
Assay of CODH activity -CO-dependent reduction of NBT with PMS -utilization methylene blue, thionine, NAD,NADP, FAD, FMN as potential electron acceptors (negative result)
Purification and properties of CODH
Anion-exchange chromatography - 특정 pH 에서 단백질의 전하와 밀도의 차이에 의해서 분리 하는 방법
Hydrophobic interaction chromatography - liquid chromatography technique 중 하나로써 bimolecule 들의 hydrophobicity
에 의해 분리 하는 방법
Gel filtration chromatography - 분자의 size 의 차이를 이용해서 분리 하는 방법
Purification and properties of CODH
Anion-exchange chromatography
-특정 pH 에서 단백질의 전하와 밀도의 차이에 의해서 분리 하는 방법 - CODH 의 pI 값 : 5.8
Gel filtration chromatography
- 분자의 size 의 차이를 이용해서 분리 하는 방법
Hydrophobic interaction chromatography
liquid chromatography technique 중 하나로써 biomolecule 들의 hydrophobicity 에 의해 분리 하는 방법
Purification and properties of CODH
•Single protein band of about 230 kDa / Single band CODH activity ( native PAGE , Lane 1-2)• three protein bands (SDS-PAGE) - 75 , 28.5, 17.2 kDa• immunostaining with IgG antibodies•Densitometric analysis of CODH polypeptide - 63.4, 20.2, 16.4 %
Purification and properties of CODH
• 338 nm – molybdenum 440 nm – FAD 550 nm – Fe-S
• A280 /A450 : 4.6 A450 /A550 : 3.03
Metal contents and analysis of flavins and carboxamidomethylated proteins
• Iodoacetamide treated protein 에서 pterin –like compound 가 나옴
• 17.28 min 에 homogeneous peak 가 elute 됨
Discussion
B. japonicum 110spc4 - grew under CO-autotrophic condition. - hydrogen bacteria - utilize H2 and CO2 as a sole sources of energy and carbon
- use CO as a a sole sources of energy and carbon - contain a CODH enzyme CODH in 110 spc4 - NBT/ PMS - value of CO-oxidizing activity - nitrate reductase activity - heterohexameric subunit (LMS)2
- 75-kDa polypeptide /Cox L subunit of the O.carboxydovorans (immunological relation)