甲基单胞菌Methylomonas sp.GYJ3中甲烷单加氧酶羟基化酶组分的纯化和性质

Methylomonassp．GYJ3菌株中经DEAE-SepharoseCL-6B阴离子交换层析和SephacrylS300凝胶层析分离纯化出甲烷加氧酶羟基化酶组分．经HPLC分析，纯度大于90％，分子量为240kD，纯化倍数为3．9，比活为225nmol环氧丙烷每分钟毫克蛋白．SDS-PAGE表明，羟基化酶由三个亚基组成，亚基分子量为56、43、27kD．ICPAES测定羟基化酶的Fe含量为2.1molFe每摩尔蛋白．HPLC法用于甲烷单加氧酶羟基化酶组分的纯化，纯化的羟基化酶组分比活为541nmol（环氧丙烷）每分钟毫克蛋白，是两步LC法纯化的羟基化酶的两倍，Fe含量为3．78molFe每摩尔蛋白．催化性质研究表明羟基化酶能够被化学还原剂还原为还原态羟基化酶，还原态的羟基化酶单独存在时表现出MMO活性，说明它是MMO活性中心，天然态的羟基化酶单独存在时无MMO活性，加入粗酶液中MMO活性明显增加，说明GYJ3菌中MMO是一个复合酶系．

Purification and Properties of Hydroxylase Component of Soluble Methane Monooxygenase from Methylomonas sp. GYJ3

The purification of the hydroxylase component of soluble methane monooxygenase from Methylomonas sp. GYJ3 is reported. The hydroxylase was purified by DEAE-Sepharose anion exchange chromatography and Sephacryl S 300 gel filtration chromatography. Analysis of purity of the purified hydroxylase by HPLC indicated that its homogeneity is above 90 %. The purified component was 3. 9 fold increase in specific activity. Specific activity of the component was 225nmol propene oxide per mg protein per min. Fe content on the hydroxylase determined by ICP-AES was 2. 1mol Fe per mol protein. HPLC method was developed to purify the hydroxylase component from the strain GYJ3. The hydroxylase with high specific activity was obtained with 541nmol propene oxide per mg protein per min. Fe content in the component purified by HPLC was 3. 78mol Fe per mol protein. SDS-PAGE and HPLC size exclusion chrornatographic studies indicated that the hydroxylase with molecular mass of 240 kD consist of three subunits with molecular masses of 56, 43, 27 kD,suggesting that the component has an (αβγ)2 subunit structure. The hydroxylase was reduced by chemical reductants. The reduced hydrocylase showed the MMO activity,suggesting that MMO active site was located in this component. No MMO activity was found in native hydroxylase alone. However,when it was added to the crude MMO,sharp increase in MMO activity was observed. It is implied that MMO from the strain GYJ3 was a multiple-component enzyme system.