异养硝化好氧反硝化菌株Agrobacterium tumefaciens LAD9羟胺氧化酶的分离纯化

Separation and Purification of Hydroxylamine Oxidase from Agrobacterium tumefaciens LAD9

羟氨氧化酶(Hydroxylamine oxidase,HAO)的作用方式直接决定了异养硝化好氧反硝化细菌的代谢途径,分离得到纯度较高的HAO也就成为研究这类细菌脱氮机制的重要环节。以异养硝化好氧反硝化细菌Agrobacterium tumefaciens LAD9为代表,建立了该菌株HAO的分离纯化方法:首先采用渗透压休克法提取细胞周质液,然后采用DEAE Sepharose CL-6B离子交换层析和Sephacryl S-100凝胶过滤层析对细胞周质液进行分离纯化。结果表明,经过离子交换层析可得到分子量分别为55.3、35.7和19.2kD的杂蛋白,进一步经过凝胶过滤层析即可得到电泳纯的HAO,纯化倍数为5.79,产率为39.71%。对其酶学性质的初步研究表明,该菌株HAO的分子量为18.8 kD,能够将羟胺氧化为亚硝酸盐氮,且Fe2+的加入可显著增强其酶活。

The metabolic pathway of heterotrophic nitrification-aerobic denitrification bacteria was directly determined by the actions of their hydroxylamine oxidase（HAO）. Isolating high-purity HAO from this kind of bacteria has become particularly important to explain the mechanism of nitrogen removal. In this study, the separation and purification technic of HAO from a novel heterotrophic nitrification-aerobic denitrification strain Agrobacterium tumefaciens LAD9 was established. Electrophoretic purity of HAO could be sucessfully purified through DEAE Sepharose CL-6B ion-exchange chromatography and Sephacryl S-100 gel filtration from its periplasm. The final purification fold was 5.79 and the yield was 39.71%. SDS-PAGE eclectrophoresis results revealed that the molecular weight of HAO in the strain LAD9 was 18.8 kD. Studies on enzymatic properties showed that the purified enzyme could oxidize hadroxylamine to nitrite and its activity could be enhanced by the addition of Fe2＋.