Methylosinus trichosporium OB3b의 methanol dehydrogenase 활성조절에 의한 메탄올 생합성

Abstract

A methanotrophic bacterium, Methylosinus trichosporium OB3b, was used to synthesize methanol from methane. Cell cultivation was performed by applying mixed gas (methane : air = 1:1, v/v) which was prepared in the tightly sealed gas reservoir to the nitrate minimal salt medium (NMS) medium using gas pump. The optimum pH and temperature for growth were pH 7.0 and 30℃, respectively, and the initial copper concentration was 5 μM. Cells were harvested by centrifugation and washed three times using 10 mM phosphate buffer (pH 7.0). To biosynthesize methanol, methanol dehydrogenase (MDH) activity should be inhibited. The 200 mM of sodium chloride (Nacl) and EDTA 1 mM was used as a MDH inhibitor for the maximum production of methanol. The reaction temperature was 30℃, and the treated concentrations of cell and sodium formate in the reaction mixture were 0.6 mg dry cell wt/ml and 20 mM, respectively. During 36 hr reaction, 7.7 mmole/g dry cell methanol has been accumulated maximally in the reaction mixture. Also to prevent further oxidation of methanol, NaCl and EDTA, inhibitors for methanol dehydrogenase, were treated to the cell suspension. However, the batch type methanol synthesis by M. trichosporium OB3b was terminated at ca. 8 mmole/g dry cell of methanol, because increasing methanol concentration inhibits methane monooxgense (MMO) activity. For the prolonged methanol accumulation, a semicontinuous process was carried out. In this process, the reaction was repeated three times and the produced methanol was 18 mmole/g dry cell compared to 8 mmole/g dry cell in batch reaction under the same condition. During 1hr reaction, 1.1 mmole/g dry cell methanol was accumulated in the reaction mixture. In this reaction Km and Vmax values were found to be 532.6 mM and 1.749 mmol/hr, respectively, and the conversion rate was approximately 37%.