食品与发酵工业 >

2023 , Vol. 49 >Issue 19: 22 - 29

DOI: https://doi.org/10.13995/j.cnki.11-1802/ts.034342

研究报告

黑曲霉酸性果胶裂解酶在大肠杆菌中的表达和酶学性质研究

  • 张海云 ,
  • 马佳宁 ,
  • 李阳阳 ,
  • 阳鹏辉 ,
  • 宋伟艳 ,
  • 刘松
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  • 1(江南大学 未来食品科学中心,江苏 无锡,214122)
    2(江南大学 生物工程学院,江苏 无锡,214122)
    3(大连理工大学 化工学院,辽宁 大连,116024)
第一作者:硕士研究生(刘松教授为通信作者,E-mail:liusong@jiangnan.edu.cn)

收稿日期: 2022-11-22

  修回日期: 2022-12-23

  网络出版日期: 2023-11-01

基金资助

国家重点研发计划项目(2019YFA0706900)

收起

Expression of Aspergillus niger pelanase in Escherichia coli and enzymatic property characterization

  • ZHANG Haiyun ,
  • MA Jianing ,
  • LI Yangyang ,
  • YANG Penghui ,
  • SONG Weiyan ,
  • LIU Song
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  • 1(Science Center for Future Foods, Jiangnan University, Wuxi 214122, China)
    2(School of Biotechnology, Jiangnan University, Wuxi 214122, China)
    3(School of Chemical Engineering, Dalian University of Technology, Dalian 116024, China)

Received date: 2022-11-22

  Revised date: 2022-12-23

  Online published: 2023-11-01

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摘要

酸性果胶裂解酶能以β-反式消去作用断裂果胶中的α-1,4糖苷键形成不饱和寡聚半乳糖醛酸,对食品等工业中高酯化果胶降解具有重要意义。该研究将黑曲霉Aspergillus niger AG11来源的酸性果胶裂解酶(pelA)在大肠杆菌Escherichia coli BL21(DE3)中进行表达,并进行了蛋白标签优化和酶学性质研究。最终确定在N端融合碳水化合物结合结构域(carbohydrate-binding structural domain, CBM)时,pelA的胞内酶活力最高,达到3.25 U/mL,比初始条件下提高4.12倍。对CBM切除前后pelA的酶学性质进行测定,结果表明pelA的比酶活力、最适反应温度及最适反应pH分别为15.45 U/mg、40 ℃和5.0,pelA在30~50 ℃孵育2 h保持80%以上活性,这与pelA+CBM相同,在40 ℃、pH为4.0~5.0时孵育2 h,相对酶活力保持在60%以上,此时pelA+CBM具有80%以上的活性。以柑橘果胶为底物,pelA的Km和kcat分别为4.44 mmol/L和31.44 s-1。该研究结果为pelA的分子改造和在工业生产中的应用奠定了基础。

关键词： 黑曲霉; 酸性果胶裂解酶; 胞内表达; 蛋白标签优化; 酶学性质

本文引用格式

张海云 , 马佳宁 , 李阳阳 , 阳鹏辉 , 宋伟艳 , 刘松 . 黑曲霉酸性果胶裂解酶在大肠杆菌中的表达和酶学性质研究[J]. 食品与发酵工业, 2023 , 49(19) : 22 -29 . DOI: 10.13995/j.cnki.11-1802/ts.034342

Abstract

Acid pectin lyase can break the a-1,4 glycosidic bond in pectin by b-trans-elimination to form unsaturated oligogalacturonic acid, which is important for the degradation of highly esterified pectin in food and other industries. In this study, acidic pectin lyase (pelA) from Aspergillus niger AG11 was expressed in Escherichia coli BL21 (DE3), and the protein tag optimization and enzymatic properties were investigated. It was finally determined that the highest intracellular enzyme activity of pelA was achieved at 3.25 U/mL with the N-terminal fusion of carbohydrate-binding structural domain (CBM), 4.12-fold higher than the initial condition. The enzymatic properties of pelA were determined before and after CBM resection. The results showed that the specific enzyme activity, optimum reaction temperature and optimum reaction pH of pelA were 15.45 U/mg, 40°C and 5.0, respectively. PelA maintained more than 80% activity at 30-50°C incubation for 2 h, which was the same as pelA+CBM. The relative enzyme activity was maintained above 60% by incubation at 40 ℃ and pH 4.0-5.0 for 2 h. At this time, pelA+CBM had more than 80% activity. Using citrus pectin as the substrate, the Km and kcat of pelA were 4.44 mmol/L and 31.44 s-1 , respectively. The results of the study laid the foundation for the molecular modification of pelA and its application in industrial production.

Key words： Aspergillus niger; acid pectin lyase; intracellular expression; optimization of melting labels; enzymatic properties

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