Abstract：
Urethane hydrolase can degrade the carcinogen ethyl carbamate (EC) in fermented food, but its stability and activity limit its application. In this study, a mutant G246A and a double mutant N194V/G246A with improved cpUH activity and stability of Candida parapsilosis were obtained by site-directed mutagenesis. The catalytic efficiency (Kcat/Km) of mutant G246A and double mutant N194V/G246A are 1.95 times and 1.88 times higher than that of WT, respectively. In addition, compared with WT, the thermal stability and pH stability of mutant G246A and double mutant N194V/G246A were enhanced. The ability of mutant G246A and double mutant N194V/G246A to degrade EC in rice wine was also stronger than that of WT. The mutation increased the stability of the enzyme, as evidenced by decreased root mean square deviation (RMSD) and increased hydrogen bonds between the enzyme and substrate by molecular dynamics simulation and molecular docking analysis. The molecule modification of new cpUH promotes the industrial process of EC degradation.
摘要：
氨基甲酸酯水解酶能降解发酵食品中的致癌物质氨基甲酸乙酯(EC),但其稳定性和活性限制了其应用。在这项研究中,通过定点诱变获得了突变株G246A和双突变株N194V/G246A，它们的cpUH活性和稳定性得到改善。突变体G246A和双突变体N194V/G246A的催化效率(Kcat/Km)分别是WT的1.95倍和1.88倍,分别。此外,与WT相比，突变体G246A和双突变体N194V/G246A的热稳定性和pH稳定性增强。突变体G246A和双突变体N194V/G246A降解米酒中EC的能力也强于WT。突变增加了酶的稳定性，通过分子动力学模拟和分子对接分析，证明了均方根偏差（RMSD）的降低和酶与底物之间氢键的增加。新型cpUH的分子修饰促进了EC降解的工业过程。
