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)