热带海洋学报 ›› 2022, Vol. 41 ›› Issue (2): 170-176.doi: 10.11978/2021065

• 海洋生物学 • 上一篇    下一篇

海洋弧菌HN897 β-琼脂糖酶基因vas1-1339异源表达及活性分析

喻飞(), 金兴坤, 雷天影, 曹海航, 陈锵辉, 阳耀帆, 李佳航, 赵哲()   

  1. 河海大学海洋学院, 海洋生物技术与资源利用研究所, 江苏 南京 210098
  • 收稿日期:2021-05-21 修回日期:2021-06-24 出版日期:2022-03-10 发布日期:2021-06-30
  • 通讯作者: 赵哲
  • 作者简介:喻飞(1990— ), 男, 讲师, 博士, 研究方向为海洋微生物。email: 20190029@hhu.edu.cn
  • 基金资助:
    国家自然科学基金(31872597)

Heterologous expression and enzymatic characterization of marine Vibrio astriarenae-derived β-Agarase gene vas1-1339

YU Fei(), JIN Xingkun, LEI Tianying, CAO Haihang, CHEN Qianghui, YANG Yaofan, LI Jiahang, ZHAO Zhe()   

  1. Department of Marine Biology, College of Oceanography, Hohai University, Nanjing 210098, China
  • Received:2021-05-21 Revised:2021-06-24 Online:2022-03-10 Published:2021-06-30
  • Contact: ZHAO Zhe
  • Supported by:
    National Natural Science Foundation of China(31872597);Jiangsu Agriculture Science and Technology Innovation Fund(CX(19)2033)

摘要:

海洋弧菌HN897株是一株高产琼脂糖酶的Vibrio astriarenae菌, 前期功能缺失实验证明其β-琼脂糖酶基因vas1-1339的缺失可显著降低弧菌HN897株的琼脂水解活性。文章在大肠杆菌中异源表达Vas1-1339基因编码蛋白, 分析该基因的表达及蛋白功能活性。首先, 构建含vas1-1339基因开放阅读框全长的表达载体pET28a-1339, 利用原核表达技术在大肠杆菌中成功地异源表达了Vas1-1339琼脂糖酶; 然后, 利用卢戈氏碘液染色分析发现异源表达Vas1-1339琼脂糖酶的大肠杆菌能够高效水解琼脂, 且异丙基硫代半乳糖苷(IPTG)最优诱导浓度为10μmol·L-1; 免疫印迹分析发现胞内基因表达产物羧基端可能存在切割现象, 推测Vas1-1339琼脂糖酶存在复杂的成熟过程。对纯化的琼脂糖酶活性分析发现海洋弧菌HN897株β-琼脂糖酶Vas1-1339能够独立发挥琼脂糖降解功能。

关键词: β-琼脂糖酶, 海洋弧菌, 琼脂糖酶解, 异源蛋白表达

Abstract:

The marine Vibrio HN897 strain is an agarose-producing Vibrio astriarenae. Studies indicated that the absence of the gene Vas1-1339, encoding a β-agarase, significantly reduced the hydrolysis effect of Vibrio HN897 strain on agarose. Herein, we further analyzed the expression and enzymatic characterization of the β-agarase gene in heterologous bacteria, Escherichia coli (E. coli). Vas1-1339 agarase was successfully expressed in E. coli, and the optimal concentration of inducer, IPTG, was at 10 μmol·L-1. Immunoblotting analysis showed that the C-terminal of the gene expression product might be cleavage in E. coli, suggesting complex maturation process of this protein. Lugol’s iodine staining analysis revealed that E. coli, expressing Vas1-1339 agarase, was highly effective in degrading agarose, and purified protein also had this similar function, which suggests that the β-agarase Vas1-1339 of the HN897 strain can independently play the role on agarolytic degradation. These results lay a preliminary foundation for the functional study and related technology application of β-agarase derived from marine vibrion.

Key words: β-agarase, marine Vibrio, agarose hydrolysis, protein expression

中图分类号: 

  • P735.51