Purification and Characterization of Methanol Dehydrogenase from Methylobacterium sp. Strain SY1

국문 초록 (Abstract)

메탄올을 이용하며 성장한 Methylobacterium sp. strain SY1으로부터 다섯 단계의 과정을 거쳐 14배 정제된 methanol dehydrogenase를 얻었다. 95% 이상의 순도를 지닌 정제된 효소의 크기는 128,000이고, 63,000의 분자량을 지닌 두 개의 동일한 소단위로 구성되어 있으며, pH 9.5와 $25^{\circ}C$에서 가장 높은 활성을 나타내었다. 이 효소는 효소활성을 나타내기 위해 암모니아 이온을 필요로 하며, phenazine methosulfate와 2,6-dichlorophenolindophenol을 이용한 효소활성 측정법으로 활성을 측정할 수 있었다. 이 효소는 여러 가지 종류의 1차 알코올, 2차 알코올, 알데하이드, 메틸아민 등을 기질로 이용할 수 있었으며, 메탄올에 대한 $K_m$ 값은 $850{\mu}M$ 이었다. 정제된 효소는 345 nm에서의 absorption peak를 나타내었으나, apoenzyme은 345 nm에서의 peak는 물론 효소활성도 나타내지 않았다. 이 효소는 KCN, EDTA, $NaN_3$에 의해서는 활성이 저해되지 않았으나 $Mn^{2+}$, $Cu^{2+}$, $Mg^{2+}$, $Co^{2+}$ 등 몇 가지 2가 양이온에 의해 강한 저해를 받았다. Glycine과 lysine을 많이 가지고 있는 이 효소의 pI는 8.5로 밝혀졌고, N-terminal 아미노산은 aspartate로 확인되었다. 정제된 효소는 Methylobacterium sp. strain AMI methanol dehydrogenase의 항원과 동일한 항원을 가지고 있는 것으로 밝혀졌다.

다국어 초록 (Multilingual Abstract)

A methanol dehydrogenase from methanol-grown cells of Methylobacterium sp. strain SY1 was purified 14-fold in five steps to better than 95% homogeneity. The molecular weight of the native enzyme was determined to be 128,000. Sodium dodecyl sulfate-gel electrophoresis revealed the enzyme to be a dimer with two identical subunits of molecular weight 63,000. The optimal pH and temperature for enzyme activity were found to be 9.5 and $25^{\circ}C$, respectively. Enzyme activity could be coupled to phenazine methosulfate and required the presence of ammonium ions in the assay mixture. The $K_m$ for methanol was found to be $850{\mu}M$. The enzyme possessed broad substrate specificity for primary and secondary alcohols, aldehydes, and methylamine. The purified enzyme had an absorption peak at 345 nm. Apoenzyme lacked the 345 nm peak and was catalytically inactive. The enzyme was insensitive to KCN, EDTA, and $NaN_3$, but was inhibited strongly by several divalent cations such as $Mn^{2+}$, $Cu^{2+}$, $Mg^{2+}$, and $Co^{2+}$. The isoelectric point of the native enzyme was found to be 8.5. The N-terminal amino acid of the native enzyme was found to be aspartate. The enzyme contained large amounts of glycine and lysine. The methanol dehydrogenase of Methylobacterium sp. strain SY1 was found to have antigenic sites in common with those of Methylobacterium sp. strain AM1 enzyme.