重组1,3-丙二醇氧化还原酶酶学性质和稳定性的研究

1,3-丙二醇氧化还原酶(PDOR)是以甘油为底物生产1,3-PD途径中的关键酶之一。将dhaT在E.coliBL21(DE3)pLysS进行了表达,对含有6×His标记的PDOR进行了纯化,同时考察了重组PDOR的酶学性质和稳定性。重组PDOR反应的最适pH和温度分别是10.0和55°C;在pH7.0～8.0,酶保持了较高的稳定性,酶在30°C保温表现出较高的稳定性;Ca2+,Mg2+和Cu2+对酶活性有抑制作用,而Fe2+,Na+,NH4+和Mn2+对酶活有促进作用;冷冻干燥处理后,PDOR酶活有一定的损失;添加适当浓度的保护剂——海藻糖、葡萄糖、蔗糖、聚乙二醇,对酶在冷冻干燥时有保护作用;添加5%蔗糖的固体酶制剂在保存过程中表现出较好的稳定性。

Study on the Characteristics and the Stability of Recombinant 1,3-propanediol Oxidoreductase

1,3-Propanediol oxidoreductase(PDOR) is the key enzyme in converting glycerol to 1,3-propanediol.In this paper,a recombinant PDOR had been over-expressed in E.coli BL21(DE3)pLysS.The 6×His-tagged PDOR could be purified by Histrap HP and gel-filtration chromatography.Then the characteristics and stability of recombinant PDOR were investigated.The results showed that the optimal pH and temperature for purified enzyme were 10.0 and 55 °C,respectively.PDOR was stable between pH 7.0~8.0,and stable at 30°C.The enzyme could be inhibited by metal ions including Ca2+,Mg2+ and Cu2+,while increased by Fe2+,Na+,NH4+ and Mn2+.The activity of PDOR was lost partly after the freeze dried.During the freeze dried,trehalose,glucose,sucrose or polyethylene increased the stability of PDOR.The enzyme preparation which contained 5% sucrose showed good stability during preservation.