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过程工程学报 ›› 2020, Vol. 20 ›› Issue (10): 1210-1217.DOI: 10.12034/j.issn.1009-606X.219370

• 生化工程 • 上一篇    下一篇

多铜氧化酶在大肠杆菌中的分泌表达

杨 涛1， 陈 坚1,2*， 方 芳1,2   

  1. 1. 江南大学工业生物技术教育部重点实验室，江苏 无锡 214122 2. 江南大学食品科学与技术国家重点实验室，江苏 无锡 214122
  • 收稿日期:2019-12-16 修回日期:2020-01-19 出版日期:2020-10-22 发布日期:2020-10-16
  • 通讯作者: 陈坚 jchen@jiangnan.edu.cn
  • 基金资助:
    国家重点研发计划项目;Magnéli相亚氧化钛的可控制备、微结构与性能基础研究;国家轻工技术与工程一流学科自主课题

Secretory of a multicopper oxidase in Escherichia coli

Tao YANG1, Jian CHEN1,2*, Fang FANG1,2   

  1. 1. Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, Wuxi, Jiangsu 214122, China 2. State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi, Jiangsu 214122, China
  • Received:2019-12-16 Revised:2020-01-19 Online:2020-10-22 Published:2020-10-16
  • Supported by:
    ;National First-class Discipline Program of Light Industry Technology and Engineering

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摘要： 生物胺是存在于发酵食品中的一类有机物，过量摄入会危害人体健康。多铜氧化酶中的某些酶具有降解多种生物胺的活性，在减控发酵食品中的氨(胺)类危害物方面具有良好的应用前景。研究多铜氧化酶的分泌表达，对酶的特性改造和工业化生产与应用具有重要意义。本研究通过在解淀粉芽孢杆菌来源的多铜氧化酶N端融合信号肽PhoA实现了多铜氧化酶在大肠杆菌中的分泌表达，胞外酶活为69.8 U/L。通过优化诱导条件和酶的分泌确定了多铜氧化酶最优发酵条件为诱导温度25℃、IPTG浓度0.05 mmol/L、诱导时菌体OD600=1.0、诱导6 h后添加150 mmol/L甘氨酸；发酵40 h时胞外多铜氧化酶酶活达到238.1 U/L，是优化前的3.4倍。

关键词: 多铜氧化酶, 生物胺, 分泌表达, 信号肽, 发酵优化

Abstract: Biogenic amines (BAs) are organic compounds that present in fermented foods. The excessive intake of BAs is harmful to human health. Some enzymes belonging to the multicopper oxidase (MCO) family exhibit the activity of degrading a variety of BAs. Thus, they may have good application prospects in reducing ammonia (amine) hazards levels in fermented foods. It is of great significance to accomplish the secretion of multicopper oxidase for the purpose of modification of enzyme catalytic properties for its industrial production and applications. In this work, the secretion of multicopper oxidase in Escherichia coli was achieved by fusing the signal peptide PhoA to the N-terminal of MCOB from Bacillus amyloliquefaciens with an extracellular activity of 69.8 U/L. Secretory of MCOB was improved by optimizing the induction and secretion conditions. The optimal fermentation conditions for MCOB were determined to be: the induction temperature was 25℃, the IPTG concentration was 0.05 mmol/L, induced when the cell density (OD600) reached 1.0, and 150 mmol/L glycine was added after 6 h of induction. After 40 h of fermentation, the extracellular activity of MCOB reached 238.1 U/L, which was 3.4 times of that before optimization.

Key words: multicopper oxidase, biogenic amines, secretory, signal peptides, fermentation optimization