Liu Dongqi, Zhu Tingting, Fan Li, Quan Junming, Guo Hongchun, Ni Jinren
College of Environmental Sciences, Peking University, Beijing, 100871, P.R. China.
Biotechnol Lett. 2007 Oct;29(10):1529-35. doi: 10.1007/s10529-007-9421-7. Epub 2007 Aug 8.
A 1,125-bp long ORF encoding a novel gentisate 1,2-dioxygenase with two-domain bicupins was cloned from Silicibacter pomeroyi DSS-3 and expressed in Escherichia coli. The resulting product was purified to homogeneity and partially characterized. Non-reductive SDS-PAGE and gel filtration showed that the active recombinant gentisate 1,2-dioxygenase had an estimated molecular mass of 132 kDa, and reductive SDS-PAGE indicated an approximate size of 45 kDa. The enzyme thus appears to be a homotrimeric protein. This is in contrast to the homotetrameric or dimeric protein of the gentisate 1,2-dioxygenases that have been characterized thus far. The K (m) and K (cat)/K (m) for gentisate were 12 muM and 653 x 10(4) M(-1 )s(-1); the pI was 4.6-4.8. It was optimally active at 40 degrees C and pH 8.0.
从波氏硅杆菌DSS-3中克隆出一个1125bp长的开放阅读框，其编码一种具有双结构域双杯状结构的新型龙胆酸1,2-双加氧酶，并在大肠杆菌中表达。对所得产物进行了纯化，直至达到均一性，并对其进行了部分特性鉴定。非还原十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）和凝胶过滤显示，活性重组龙胆酸1,2-双加氧酶的估计分子量为132 kDa，还原SDS-PAGE表明其大小约为45 kDa。因此，该酶似乎是一种同三聚体蛋白。这与迄今为止已鉴定的龙胆酸1,2-双加氧酶的同四聚体或二聚体蛋白形成对比。龙胆酸的米氏常数（K(m)）和催化常数与米氏常数的比值（K(cat)/K(m)）分别为12 μM和653×10⁴ M⁻¹ s⁻¹；其等电点为4.6 - 4.8。该酶在40℃和pH 8.0时活性最佳。
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