|Budget Amount *help
¥2,300,000 (Direct Cost : ¥2,300,000)
Fiscal Year 1997 : ¥900,000 (Direct Cost : ¥900,000)
Fiscal Year 1996 : ¥1,400,000 (Direct Cost : ¥1,400,000)
In order to clarify that reductive TCA cycle is operative in a thermophilic hydrogenxidizing bacterium, Hydrogenobater thermophilus, the followings were demonstrated.
1.Purification and characterization of ferredoxin
Purification of ferredoxin was performed by applying cell-free extract successively. to Q-Sepharose, DEAE-5PW,and Superdex 75. Characterization of the prorein was also perfprmed.
2.Purification and characterization of pyruvate synthase
Purification of pyruvate synthase was performed by applying cell-free extract successively to DEAE-Sepharose CL-6B,PA-QA,Hydroxyapatite, and Superdex 200. Characterization of the protein was also performed.
3.Reactivity of ferredoxin toward 2-oxoglutarate synthase or pyruvate synthase
In the first step, reaction was performed toward CO_2 evolution direction ; both enzymes reacted with ferredoxin. In the next step, reduced ferredoxin was used as a reductant to show the carboxylation reaction. However, only a little carboxylation reaction occurred probably due to the instability of CoA-compounds. Finally, CoA-compounds were produced through enzymatic reactions ( ATP : citrate lyase and succinyl-CoA synthetase ) . By this system carboxylation reaction was detected and also the expected products were identified.