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投稿时间：2021-11-10
投稿时间：2021-11-10
中文摘要: 将合成的来源于长孢洛德酵母菌（Lodderomyces elongisporus）属的氨基甲酸乙酯水解酶基因在大肠杆菌中实现异源表达，并进行纯化及其酶学性质研究。摇床培养后酶的比活力为4.52 U/mg，经过镍离子亲和层析柱纯化至显示单一条带后酶的比活力为9.86U/mg。酶学性质研究表明，氨基甲酸乙酯水解酶的最适反应温度为30℃；最适pH值为7.0；金属离子Mg2+对该水解酶的酶活有微弱的促进作用，而Cu2+以及Fe3+对酶的活性有强烈的抑制作用；对低含量的乙醇溶液与NaCl溶液有一定的耐受性；并且该水解酶可以对不同底物的酰胺类化合物进行有效降解，以氨基甲酸乙酯（ethyl carbamate，EC）为底物进行反应时测得水解酶的动力学参数Km值为6.64 mmol/L，最大反应速率Vmax值为8.24 μmol/min。
Abstract:The synthetic urethanase gene derived from Lodderomyces elongisporus was expressed heterologously in Escherichia coli and purified，and its enzymatic properties were investigated.The specific activity of urethanase after culture in a shaker was 4.52 U/mg，and that after purification by nickel ion affinity chromatography to a single band was 9.86 U/mg.As demonstrated by enzymatic properties，the optimum reaction of urethanase occurred at 30 ℃ and pH7.0.Mg2+could slightly potentiate enzyme activity，while Cu2+and Fe3+strongly inhibited the enzyme activity.It showed a certain tolerance to ethanol solution and NaCl solution with low content.Besides，urethanase could effectively degrade amide compounds of different substrates.With ethyl carbamate（EC）serving as the substrate，the measured kinetic parameter Kmof the urethanase was 6.64 mmol/L，and the maximum reaction rate Vmaxwas 8.24 μmol/min.
keywords: ethyl carbamate urethanase heterologous expression enzymatic properties Lodderomyces elongisporus
文章编号：202210026 中图分类号： 文献标志码：
基金项目:国家十三五重点研发计划（2017YFC1601704）；雪莲菌发酵乳中高加索乳杆菌和酿酒酵母的共生机制（C2021204061）
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