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地衣芽孢杆菌几丁质酶在枯草芽孢杆菌中的重组表达及其制备氨基寡糖的研究

袁源 宿玲恰 张康 朱昫飏 夏伟 吴敬

袁源, 宿玲恰, 张康, 朱昫飏, 夏伟, 吴敬. 地衣芽孢杆菌几丁质酶在枯草芽孢杆菌中的重组表达及其制备氨基寡糖的研究[J]. 南方水产科学, 2022, 18(2): 39-47. doi: 10.12131/20210297
引用本文: 袁源, 宿玲恰, 张康, 朱昫飏, 夏伟, 吴敬. 地衣芽孢杆菌几丁质酶在枯草芽孢杆菌中的重组表达及其制备氨基寡糖的研究[J]. 南方水产科学, 2022, 18(2): 39-47. doi: 10.12131/20210297
YUAN Yuan, SU Lingqia, ZHANG Kang, ZHU Xuyang, XIA Wei, WU Jing. Recombinant expression of Bacillus licheniformis chitinase in B. subtilis and preparation of amino-oligosaccharides[J]. South China Fisheries Science, 2022, 18(2): 39-47. doi: 10.12131/20210297
Citation: YUAN Yuan, SU Lingqia, ZHANG Kang, ZHU Xuyang, XIA Wei, WU Jing. Recombinant expression of Bacillus licheniformis chitinase in B. subtilis and preparation of amino-oligosaccharides[J]. South China Fisheries Science, 2022, 18(2): 39-47. doi: 10.12131/20210297

地衣芽孢杆菌几丁质酶在枯草芽孢杆菌中的重组表达及其制备氨基寡糖的研究

doi: 10.12131/20210297
基金项目: 国家重点研发计划项目 (2019YFD0901901)
详细信息
    作者简介:

    袁源:袁 源 (1996—),女,硕士研究生,研究方向为酶工程与技术。E-mail: 6200208066@stu.jiangnan.edu.cn

    通讯作者:

    吴 敬 (1969—),女,教授,博士,从事酶工程与技术研究。E-mail: jingwu@jiangnan.edu.cn

  • 中图分类号: Q 556

Recombinant expression of Bacillus licheniformis chitinase in B. subtilis and preparation of amino-oligosaccharides

  • 摘要: 将地衣芽孢杆菌 (Bacillus licheniformis) 来源的几丁质酶基因blchiA在枯草芽孢杆菌 (B. subtilis) 中进行重组表达,构建了枯草芽孢杆菌工程菌株 (B.subtilis WS9/pHY300PLK-blchiA),发酵上清液的酶活为0.73 U·mL−1。对重组酶BLCHIA的酶学性质进行表征,其在pH 6.0和60 ℃时表现出最佳活性,比活为3.68 U·mg−1。10 mmol·L−1的铜离子 (Cu2+) 和亚铁离子 (Fe2+) 对其活性有一定促进作用,在pH 5.0~8.0和50~60 ℃下稳定性较好。此外,研究表明重组酶对脱乙酰度>95%的壳聚糖的催化能力明显优于胶体几丁质,并且水解胶体几丁质和壳聚糖的产物类型具有明显差异,以胶体几丁质为底物主要生成几丁二糖,以壳聚糖为底物可生成聚合度为2~7的壳寡糖。研究获得的重组酶BLCHIA在壳聚糖降黏和制备不同聚合度的低聚糖方面具有良好的应用前景。
  • 图  1  pHY300PLK-blchiA表达载体的鉴定

    Figure  1.  Identification of pHY300PLK-blchiA expression vector

    图  2  重组几丁质酶BLCHIA摇瓶发酵过程的酶活

    Figure  2.  Enzyme activity of recombinant chitinase BLCHIA in shake flask fermentation

    图  3  重组几丁质酶BLCHIA的分离纯化

    Figure  3.  Separation and purification of recombinant BLCHIA

    图  4  重组几丁质酶BLCHIA的最适pH (a) 和pH稳定性 (b)

    Figure  4.  Optimal pH (a) and pH stability (b) of recombinant chitinase BLCHIA

    图  5  重组几丁质酶BLCHIA的最适温度 (a) 和热稳定性 (b)

    Figure  5.  Optimal temperature (a) and thermal stability (b) of recombinant chitinase BLCHIA

    图  6  金属离子对重组几丁质酶BLCHIA酶活的影响

    Figure  6.  Effect of metal ions on activity of recombinant chitinase BLCHIA

    图  7  BLCHIA水解胶体几丁质的薄层色谱分析

    Figure  7.  TLC analysis of BLCHIA hydrolyzed colloidal chitin

    图  8  BLCHIA水解壳聚糖的薄层色谱分析

    Figure  8.  TLC analysis of BLCHIA hydrolyzed chitosan

    表  1  BLCHIA水解壳聚糖的产物峰面积占比

    Table  1.   Proportion of peak area of BLCHIA           hydrolyzed chitosan         %

    产物类型
    Product type
    反应时间 Reaction time
    30 min1 h3 h5 h
    氨基葡萄糖 Aminoglucose 0.00 0.42 0.80 0.79
    壳二糖 Chitobiose 12.87 17.35 22.65 26.54
    壳三糖 Chitotriose 24.37 30.63 36.70 38.44
    壳四糖 Chitotetraose 27.73 27.49 26.26 24.94
    壳五糖 Chitopentaose 21.27 17.26 7.98 4.21
    壳六糖 Chitosan 8.85 4.56 2.99 2.67
    壳七糖 Chitoheptaose 3.37 2.28 2.61 2.40
    下载: 导出CSV
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出版历程
  • 收稿日期:  2021-10-12
  • 修回日期:  2022-01-06
  • 录用日期:  2022-01-17
  • 网络出版日期:  2022-02-07
  • 刊出日期:  2022-04-01

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