氨基甲酸乙酯水解酶的分离纯化及酶学性质

Purification and Characterization of an Urethanase

摘要:

氨基甲酸乙酯是发酵食品生产过程中的副产物, 具有致癌性和遗传毒性, 影响食品安全。酶法降解氨基甲酸乙酯是一种高效安全的去毒方法。从小鼠肠道筛选得到一株具有降解氨基甲酸乙酯活性的赖氨酸芽孢杆菌。通过对其细胞破碎上清液进行硫酸铵沉淀、离子交换层析、疏水层析和凝胶层析分离纯化, 得到了氨基甲酸乙酯水解酶纯酶。SDS-PAGE电泳图显示, 该酶亚基分子量约为50 kDa。该酶最适反应温度为35 ℃, 最适pH值为7.0, 在10~45 ℃时, 具有良好的热稳定性。以氨基甲酸乙酯为底物反应, 其Km和Kcat分别为37.2 mmol/L和1 176.4 s-1。金属离子显著抑制酶活力, 而EDTA对酶活力无影响, 表明此酶为非金属酶。此外, 该酶对低浓度的NaCl和乙醇有一定的耐受性, 具有在酱油和黄酒中去除氨基甲酸乙酯的应用潜力。

Abstract:

Ethyl carbamate(EC, or urethane), a by-product of fermented food, has been shown to be possible carcinogenic and genotoxic to human. Enzymatic removal of EC from fermented foods and beverages is an efficient and safe method. Lysinibacillus fusiformis, a strain isolated from the gastrointestinal tracts of mice, showed the activity of hydrolyzing urethane. The protein with urethanase activity was purified to electrophoretic homogeneity by ammonium sulfate precipitation, ion exchange chromatography, hydrophobic interaction chromatography and gelfiltration chromatography. The monomeric molecular weight of this urethanase was detected to be approximate 50 kDa by SDS-PAGE. Enzymatic properties analysis demonstrated that the purified enzyme was stable at temperature range 10~45 ℃, the optimal reaction conditions of it was determined to be at 35 ℃ and at pH 7.0. Using urethane as the substrate, the kinetic constants of Km and Kcat were detected to be 37.2 mmol/L and 1 176.4 s-1, respectively. The activity was inhibited by metal ions while EDTA had no effect on enzyme activity. The results showed that the urethanase was not an ion-associated enzyme. In addition, the enzyme showed tolerance to low concentration of ethanol and NaCl, indicating its potential applications in removing urethane from fermented products(rice wine and soy sauce).