耐盐氨基甲酸乙酯水解酶的分离纯化及酶学性质

Purification and characterization of a halophilic urethanase from Klebsiella pneumoniae

摘要:

氨基甲酸乙酯是发酵食品中存在的一种致癌物质，酶法去除发酵食品中的氨基甲酸乙酯是消除氨基甲酸乙酯危害的一种重要方法。从小鼠的胃部获得了一株产氨基甲酸乙酯水解酶的肺炎克雷伯氏菌，为了解该氨基甲酸乙酯水解酶的酶学性质，从肺炎克雷伯氏菌中提取获得氨基甲酸乙酯水解酶粗酶液，经硫酸铵沉淀、离子交换层析和凝胶过滤层析分离得到氨基甲酸乙酯水解酶纯酶。通过十二烷基硫酸钠聚丙烯酰胺电泳(SDS-PAGE) 分析，估计该酶的分子量约为55 kDa。其水解氨基甲酸乙酯的Km值为74 mmol/L。酶反应的最适温度为55 ℃，最适pH为7.0。乙二胺四乙酸 (EDTA) 和二硫苏糖醇 (DTT) 对该酶有较强的激活作用，而Cu2+和Zn2+则有较强的抑制作用。该酶可耐受高浓度NaCl，对低浓度乙醇也有一定的耐受性，对于酱油中氨基甲酸乙酯的消除有一定的参考意义。

Abstract:

Ethyl carbamate (EC) is a carcinogenic substance in many fermented foods. Enzymatic removal of ethyl carbamate from fermented foods is an important way to eliminate its potential health damage to consumers. To study the enzymatic properties of an ethyl carbamate hydrolase (urethanase) from Klebsiella pneumoniae, a strain isolated from murine somach, we purified the enzyme using ammonium sulfate precipitation, ion exchange chromatography and gel filtration chromatography. The molecular mass of this enzyme was estimated to be 55 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Its Km was 74 mmol/L when EC was used as the substrate. Moreover, its optimal reaction temperature was 55 ℃, and the optimum pH was 7.0. The activity was enhanced by ethylene diamine tetraacetic acid (EDTA) and dithiothreitol (DTT), but strongly inhibited by Cu2+ and Zn2+. The enzyme was halophilic and tolerant to low concentration of ethanol. Therefore, it has the potential to remove EC from fermented foods.