大肠杆菌JM105中硝酸盐还原酶的制备及其性质(英文)

Preparation and properties of nitrate reductase from E. coli JM105

近年来,硝酸盐和亚硝酸盐的污染已引起普遍关注。目前,蔬菜和粮食的NO3-含量过高主要由于农药和化肥的使用、工业废水或生活污水灌溉等,食品类主要来自腌制食品和食品添加剂或防腐剂等。另外,向乳制品或食品中参碱、食盐、化肥、脏水、碱性水等也是硝酸盐污染的来源之一。因此,迫切需要快速、简便、可用于现场的硝酸盐检测方法。这就需仰赖生物酶方法,而生物酶方法的关键是酶制剂的制备和检测方法的建立。本研究通过筛选、厌氧和硝酸盐诱导培养、超声波细胞破碎和差速离心提取等方法,从大肠杆菌(Escherichia coli)JM105细胞膜中制备了硝酸盐还原酶并对其性质进行了研究。结果表明:从大肠杆菌JM105中制得的酶制剂活力很高,且在酶过量的情况下可将NO3-完全转化为NO2-,在用磷酸缓冲液清洗并冷冻保藏过夜后不含有亚硝酸盐还原酶。在加入黄素单核苷酸辅酶(FMN)后,该酶的活力可提高64%,比活力达0.42U·mg-1蛋白。该酶十分稳定,在40℃下24h活力无影响,在浓度为1mmol·L-1的Cu2+、Fe3+、Ca2+、Zn2+、Mg2+和Mo6+存在下,其活力亦不改变。因此,该酶可用于测定食品、蔬菜和环境中硝酸盐的含量。

Nitrate reductase can be used to detect nitrate content in food, vegetables and environment. Membrane-bound enzyme nitrate reductase （NR） was prepared from Escherichia coil JM 105 and properties of NR were analyzed. E. coil strain was anaerobically cultured in the media containing nitrate to induce NR in cells. The cells were crashed by ultrasonic and cell membrane fragments with NR were collected as NR preparation by centrifuging the suspension at different speeds. To remove nitrite reductase activity both cells and NR preparation were washed with the phosphate buffer and then frozen and thawed. NR activity was measured with an assay system to which FMN was introduced to improve the NR activity. Influence of temperature and metals on the NR preparation activity was determined to test stability of the preparation. The results indicated that NR could be largely induced from E. coil JM105; the NR preparation possessed extremely high NR activity and could almost completely convert NO3^- to NO2^-, and did not possess nitrite reductase activity after being washed with the phosphate buffer and frozen and thawed; activity of the NR preparation could be increased by 64% by addition of FMN and consequently specific activity of the NR preparation reached to 0.42 U·mg^-1 protein; NR activity did not change under 40 ℃ for 24 h and with 1 mmol·L^-1 of Cu^2＋, Fe^3＋, Ca^2＋, Zn^2＋, Mg^2＋ or Mo^6＋. Therefore, NR from E. coil JM105 can be used for quick detect of nitrate.