假丝酵母氨基甲酸乙酯水解酶的分子克隆及酶学性质

氨基甲酸乙酯是存在于发酵食品中的一种潜在致癌物.生物酶法可有效降解氨基甲酸乙酯.该研究从假丝酵母CCTCC AY 2017001基因组中克隆了氨基甲酸乙酯水解酶基因cpUH,并实现了其在Escherichia coli中的高效表达.实验结果表明,cpUH基因大小为1656 bp,编码552个氨基酸;重组蛋白大小约为60...

Molecular cloning and biochemical characterization of urethanase from Candida parapsilosis

Ethyl carbamate is a potential carcinogen in fermented food,and it can be effectively degraded by bioenzyme.The carbamate hydrolase gene cpUH was cloned from a strain of Candida parapsilosis and expressed in Escherichia coli.The results showed that the amplified gene encoded 552 amino acids,with a total length of 1656 bp and recombinant protein with 60 kDa.The cpUH obtained by Ni²⁺ affinity chromatography was electrophoretically pure,and its specific activity was 3.10 U/mg.The optimal temperature and pH for the purified recombinant cpUH were 40℃and 7.5,respectively,while good stability in 25-30℃and pH 6.0-9.0 were revealed.The enzyme was significantly inhibited by Cu²⁺and Zn²⁺,and generally inhibited by Fe³⁺,Mn²⁺,Ca²⁺,Co²⁺and Mg²⁺,while EDTA did not.When cpUH catalyzed ethyl carbamate,its K m and V max were 6.01 mmol/L and 3.34μmol/min,respectively.At the same time,cpUH had certain tolerance to ethanol,which was expected to be applied in the production of alcoholic beverages.