食品与发酵工业 >

2023 , Vol. 49 >Issue 21: 39 - 44

DOI: https://doi.org/10.13995/j.cnki.11-1802/ts.034544

研究报告

L-赖氨酸脱羧酶的表达、纯化及其酶学性质研究

  • 何世霞 ,
  • 谢文鹏 ,
  • 郭欣欣 ,
  • 吕育财 ,
  • 张文 ,
  • 杨潇 ,
  • 龚大春
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  • 1(湖北省生物酵素工程技术研究中心(三峡大学),湖北 宜昌,443002)
    2(三峡大学 生物与制药学院,湖北 宜昌,443002)
    3(中国轻工业功能酵母重点实验室(三峡大学),湖北 宜昌,443002)
第一作者:硕士研究生(龚大春教授为通信作者,E-mail:185195061@qq.com)

收稿日期: 2022-12-06

  修回日期: 2023-01-31

  网络出版日期: 2023-12-08

基金资助

国家自然科学基金项目(21776162);湖北省技术创新专项项目(2019ABA114)

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Expression, purification, and enzymatic properties of L-lysine decarboxylase

  • HE Shixia ,
  • XIE Wenpeng ,
  • GUO Xinxin ,
  • LYU Yucai ,
  • ZHANG Wen ,
  • YANG Xiao ,
  • GONG Dachun
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  • 1(Hubei Engineering Research Center for Biological Jiaosu, China Three Gorges University, Yichang 443002, China)
    2(College of Biological and Pharmaceutical, China Three Gorges University, Yichang 443002, China)
    3(China Key Laboratory of Light Industry Functional Yeast, China Three Gorges University, Yichang 443002, China)

Received date: 2022-12-06

  Revised date: 2023-01-31

  Online published: 2023-12-08

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摘要

戊二胺可由赖氨酸经过赖氨酸脱羧酶脱羧生成,是生物基聚酰胺PA56的关键单体。该研究以pET-28a(+)质粒为载体,将来源于大肠杆菌K12 MG1655的赖氨酸脱羧酶Ldc基因,经过密码子优化后克隆到大肠杆菌BL21(DE3)中,构建重组菌E.coli BL21(DE3)/pET-28a(+)-Ldc,用Ni-Agarose柱分离纯化出带有His标签的目的蛋白,进行酶学性质研究。重组酶Ldc分子质量在81.2 kDa左右,比酶活力为0.56 U/mg,在pH 5.7～8.0稳定性较好,相对酶活力保持80%上;该酶在20～60 ℃稳定性很好,T50值为72 ℃;金属离子对酶活力有一定的影响,在终浓度为5 mmol/L条件下,Cu2+抑制作用最明显,其次是Ni2+,而Mg2+和Ca2+有微弱的激活作用;对赖氨酸脱羧酶的动力学参数进行了表征,该酶对赖氨酸具有较好的亲和力和催化效率,其Km为0.011 mol/L,Vmax值为0.643 mmol/(L·min),kcat值为0.23 s-1。研究结果为赖氨酸脱羧酶Ldc分子改造和工业化生产应用提供了科学依据。

关键词： 戊二胺; 赖氨酸脱羧酶; 大肠杆菌K12 MG1655; 重组表达; 酶学性质

本文引用格式

何世霞 , 谢文鹏 , 郭欣欣 , 吕育财 , 张文 , 杨潇 , 龚大春 . L-赖氨酸脱羧酶的表达、纯化及其酶学性质研究[J]. 食品与发酵工业, 2023 , 49(21) : 39 -44 . DOI: 10.13995/j.cnki.11-1802/ts.034544

Abstract

Cadaverine is the key monomer of biological polyamide PA56, which is produced by lysine decarboxylase. By constructing recombinant E. coli BL21(DE3)/pET-28a(+)-Ldc, the lysine decarboxylase (Ldc) from Escherichia coli K12 MG1655 was overexpressed. The recombinant Ldc with His-tag was separated and purified by Ni-Agarose affinity column and its enzymatic properties were investigated.The molecular weight of recombinant Ldc was about 81.2 kDa, and its specific activity was 0.56 U/mg. The suitable pH range was 5.7-8.0 with the relative enzyme activity above 80%. The recombinant Ldc was stable in the temperature range of 20-60 ℃, and the T50 value was 72 ℃. Effect of metal ions on the enzyme activity was observed. Cu2+ had a strong inhibition on the recombinant Ldc at 5 mmol/L, followed by Ni2+. On the contrary, Mg2+ and Ca2+ had a certain activating effect. The dynamics parameters of lysine decarboxylase were characterized with Km 0.011 mol/L, Vmax 0.643 mmol/(L·min), and kcat 0.23 s-1. The enzyme showed good affinity and catalytic efficiency to lysine. This study provided a scientific basis for molecular modification and industrial application of lysine decarboxylase Ldc.

Key words： cadaverine; lysine decarboxylase; E. coli K12 MG1655; recombinant expression; enzymatic properties

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