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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 CO 2 CO + H 2 O -> CO 2 +2e - +2H +

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Carbon Monoxide Dehydrogenase Activity in Bradyrhizobium japonicum

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  1. Carbon Monoxide Dehydrogenase Activity in Bradyrhizobium japonicum 이정은

  2. 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

  3. Results

  4. CO- dependent growth B. japonicum110spc4 -can utilize CO as a sole source of 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)

  5. Purification and properties of CODH

  6. Purification and properties of CODH • Anion-exchange chromatography - 특정 pH에서 단백질의 전하와 밀도의 차이에 의해서 분리 하는 방법 • Hydrophobic interaction chromatography - liquid chromatography technique 중 하나로써 bimolecule들의 hydrophobicity 에 의해 분리 하는 방법 • Gel filtration chromatography - 분자의 size의 차이를 이용해서 분리 하는 방법

  7. Anion-exchange chromatography • 특정 pH에서 단백질의 전하와 • 밀도의 차이에 의해서 분리 하는 방법 • - CODH의 pI 값: 5.8

  8. Gel filtration chromatography -분자의 size의 차이를 이용해서 분리 하는 방법

  9. Hydrophobic interaction chromatography • liquid chromatography technique 중 하나로써 biomolecule들의 hydrophobicity 에 의해 분리 하는 방법

  10. 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 %

  11. Purification and properties of CODH • 338 nm – molybdenum • 440 nm – FAD • 550 nm – Fe-S • A280 /A450 : 4.6 • A450 /A550 : 3.03

  12. Metal contents and analysis of flavins and carboxamidomethylated proteins • Iodoacetamide treated protein에서 • pterin –like compound가 나옴 • 17.28 min에 homogeneous peak가 • elute 됨

  13. 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)

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