一个Ⅱ型甲烷氧化菌中甲烷单加氧酶羟基化酶的分离纯化和理化性质

Methane Monooxygenases Hydroxylase from a Type Ⅱ Methanotroph: Purification and Physical-chemical Properties

甲烷氧化细菌能够催化甲烷和一系列小分子烃类化合物的羟基化反应,对控制全球变暖起着重要作用,在工业催化和生物除污中具有非凡的潜能。应用层析方法纯化了Ⅱ型甲烷氧化细菌MethylosinustrichosporiumIMV3011中甲烷单加氧酶的羟基化酶,并对其进行了表征。凝胶过滤法测定了该酶分子量为201.3kD;SDS-PAGE表明羟基化酶含有三个亚基(αβγ),分子量分别为58kD、36kD和23kD,比较两种方法证明该羟基化酶是一个同型二聚体构型(αβγ)2,总分子量为234kD。薄层等电聚焦测定该酶的等电点为5.2。酶的比活力为603.6nmol/(min.mg),活力回收为34.3%。HPLC法测定该酶的纯度在95%以上。原子吸收光谱显示每分子羟基化酶中含有3.02个Fe原子。羟基化酶的稳定性pH值为6.2～7.5,稳定性温度为低于35℃。菌株IMV3011的细胞表观构型呈现了长型、稍微弯曲的杆状形态。

Methanotrophs can catalyze hydroxylate of methane and some hydrocarbon. Which play an important role in mitigating global warming and have also potential significance for industrial applications or bioremediation. A high activity of hydroxylase, a crucial component in sMMO, from Methylosinus trichosporium IMV 3011 has been purified to homologues by using chromatographic techniques. The molecular weight of the hydroxylase determined by gel filtration is 201.3kD, and SDS-PAGE showed that hydroxylase consists of three subunits（αβγ） with molecular weights of 58kD, 36kD and 23kD respectively, drawing a comparison both methods indicated that the hydroxylase is a homodimer with an （αβγ）2 configuration. Purified hydroxylase has a pI at 5.2 judged by thin layer isoelectric focusing. The purified hydroxylase contains 3.02 mol of iron per mol of protein. The stability pH for the hydroxylase in solution is 5.8 - 8.0 and the stability temperature is below 35℃. The cells form show a long, bent, and rod-shaped with even surface observed by scanning electron microscopy.