甲基单胞菌Methylomonas sp.GYJ3中甲烷单加氧酶还原酶组分的纯化和性质

Ｍｅｔｙｌｏｍｏｎａｓｓｐ．ＧＹＪ３菌的甲烷单加氧酶（ＭＭＯ）粗酶提取液经ＤＥＡＥ－ＳｅｐｈａｒｏｓｅＣＬ－６Ｂ阴离子交换层析、ＳｅｐｈａｄｅｘＧ－１００凝胶过滤层析和ＤＥＡＥ－ＴＳＫｇｅｌＨＰＬＣ分离纯化出ＭＭＯ还原酶组分．经ＨＰＬＣ分析，纯度大于９５％，纯化倍数为４．４，加入至ＭＭＯ羟基化酶和调节蛋白Ｂ的体系中表现比活为２２８ｎｍｏｌ环氧丙烷每分钟毫克蛋白．ＳＤＳ－ＰＡＧＥ电泳表明还原酶由一种亚基组成，分子量４２ｋＤ．ＩＣＰ－ＡＥＳ测定还原酶的Ｆｅ含量为１．８３ｍｏｌＦｅ每ｍｏｌ蛋白．ＵＶ－Ｖｉｓ光谱表明还原酶除２８０ｎｍ蛋白质特征峰外在４６０ｎｍ有最大吸收峰，且Ａ２８０ｎｍ／Ａ４６０ｎｍ为２．５０，与其它黄素一铁硫蛋白相似，推测还原酶可能含一个ＦＡＤ辅基和Ｆｅ２Ｓ２中心．在厌氧条件下，还原酶能够和ＮＡＤＨ作用，ＵＶ－Ｖｉｓ光谱分析表明还原酶４６０ｎｍ处特征吸收峰消失，说明在ＭＭＯ催化过程中还原酶接受ＮＡＤＨ的电子．ＤＥＡＥ－ＳｅｐｈａｒｏｓｅＣＬ－６Ｂ阴离子交换层析分离出调节蛋白Ｂ，部分纯化的调节蛋白Ｂ的分子量大约在２０ｋＤ，它能够提高ＭＭＯ比活性４０倍，ＭＭＯ还原酶和调节蛋白Ｂ单独存在时不具有ＭＭＯ

Purification and Properties of Reductase Component of Soluble Methane Monooxygenase from Mehtylomonas sp GYJ3

The purification of the reductase component was carried out by using DEAE Sepharose anion exchange chromatography,Sephadex G 100 gel filtration chromatography,and DEAE TSK gel anion exchange HPLC The purity of the component is above 95% The purification of the reductase component was by 4 4 fold in specific activity The component exhibited an activity of 228nmol propene oxide per mg protein per min,when it was added to the system of hydroxylase and component B SDS PAGE of the reductase indicated that the component has a single peptide with molecular mass of 42kD The Fe content in the component determined by ICPAES was 1 83 mol Fe per mol protein UV Vis spectrum of the reductase showed maximum absorbance at 280 nm and 460nm ,and A 280nm / A 460nm is 2 50 similar with other flavin Fe 2 S 2 protein It suggests that the reductase might contain a FAD cofactor and Fe 2 S 2 center Under anaerobic condition ,the absorbance of 460nm of the reductase disappeared when the protein was titrated by NADH,suggesting that the reductase should accept the electrons from NADH The component B was resolved by DEAE Sepharose chromatography The molecular mass of the main protein band is below 20 kD It stimulates the methane monooxygenase activity of the system of hydroxylase and reductase by 40 fold ,No methane monooxygenase activity was found in the presence of reductase and component B without the hydroxylase The high specific activity required all three components