食品与发酵工业 >

2022 , Vol. 48 >Issue 3: 56 - 62

DOI: https://doi.org/10.13995/j.cnki.11-1802/ts.028038

研究报告

假丝酵母氨基甲酸乙酯水解酶的分子克隆及酶学性质

  • 张献 ,
  • 冯治平 ,
  • 张耀 ,
  • 张雪怡 ,
  • 杨丽娟
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  • 1(四川轻化工大学 生物工程学院,四川 宜宾,644000)
    2(酿酒生物技术及应用四川省重点实验室(四川轻化工大学),四川 宜宾,644000)
硕士研究生(杨丽娟副教授为通信作者,E-mail:yanglijuan@suse.edu.cn)

收稿日期: 2021-05-21

  修回日期: 2021-07-22

  网络出版日期: 2022-03-04

基金资助

四川轻化工大学人才引进项目(2018RCL23);四川轻化工大学专业学位研究生课程案例库建设项目(KA202008);大学生创新创业训练计划项目(B50104314)

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Molecular cloning and biochemical characterization of urethanase from Candida parapsilosis

  • ZHANG Xian ,
  • FENG Zhiping ,
  • ZHANG Yao ,
  • ZHANG Xueyi ,
  • YANG Lijuan
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  • 1(School of Biotechnology,Sichuan University of Science & Engineering,Yibin 644000,China)
    2(Liquor Making Bio-Technology & Application of Key Laboratory of Sichuan Province,Sichuan University of Science & Engineering,Yibin 644000,China)

Received date: 2021-05-21

  Revised date: 2021-07-22

  Online published: 2022-03-04

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摘要

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1 656 bp,编码552个氨基酸;重组蛋白大小约为60 kDa,纯化后比酶活力为3.10 U/mg;cpUH的最适反应温度和pH分别为40 ℃和7.5,其在25~30 ℃和pH 6.0~9.0均有较好的稳定性;金属离子Cu2+、Zn2+明显抑制酶活力,其余Fe3+、Mn2+、Ca2+、Co2+、Mg2+都对酶活力有抑制作用,而EDTA对酶活力无影响;此外,以氨基甲酸乙酯为底物时,其Km和Vmax值分别为6.01 mmol/L和3.34 μmol/min,且cpUH对乙醇具有一定的耐受性,有望应用到酒饮料生产中。

关键词： 假丝酵母; 氨基甲酸乙酯; 氨基甲酸乙酯水解酶; 异源表达; 酶学性质

本文引用格式

张献 , 冯治平 , 张耀 , 张雪怡 , 杨丽娟 . 假丝酵母氨基甲酸乙酯水解酶的分子克隆及酶学性质[J]. 食品与发酵工业, 2022 , 48(3) : 56 -62 . DOI: 10.13995/j.cnki.11-1802/ts.028038

Abstract

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 1 656 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 Km and Vmax were 6.01 mmol/L and 3.34 μmol/min, respectively. At the same time, cpUH had certain tolerance to ethanol, which was expected to be applied in the production of alcoholic beverages.

Key words： Candida parapsilosis; ethyl carbamate; urethanase; heterologous expression; enzymatic properties

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