生物学杂志

生物学杂志 ›› 2024, Vol. 41 ›› Issue (6): 39-.doi: 10.3969/j.issn.2095-1736.2024.06.039

• 研究报告 • 上一篇    下一篇

重组蔗糖异构酶在枯草芽孢杆菌中的表达及发酵优化

姜　晨， 吴昕曈， 陈慧玲， 刘　乐， 张　谦， 李宪臻， 郭小宇   

  1. 大连工业大学 生物工程学院 微生物资源与催化实验室， 大连 116033
  • 出版日期:2024-12-18 发布日期:2024-12-16
  • 通讯作者: 郭小宇，博士，讲师，研究方向为酶工程与生物催化，E-mail:xiaoguo1987214@126.com
  • 作者简介:姜晨，硕士研究生，研究方向为蔗糖异构酶的表达，E-mail:1004065696@qq.com
  • 基金资助:
    国家自然科学基金项目(31771907)

Expression and fermentation optimization of recombinant sucrose isomerase in Bacillus subtilis

JIANG Chen, WU Xintong, CHEN Huiling, LIU Le, ZHANG Qian, LI Xianzhen, GUO Xiaoyu   

  1. Laboratory of Microbial Resources and Catalysis, School of Biological Engineering, Dalian Polytechnic University,
    Dalian 116033, China
  • Online:2024-12-18 Published:2024-12-16

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摘要： 通过启动子及培养基优化策略构建非诱导型启动子调控的高表达水平蔗糖异构酶，探究不同启动子对重组菌株Bacillus subtilisSCK6表达蔗糖异构酶的影响，通过无限制克隆法分别构建6个非诱导型单启动子PHpal Ⅰ、P43、PgsiB、PsrfA、PamyQ、PaprE和3个双启动子P43-PgsiB、2P43、2PgsiB调控的蔗糖异构酶重组菌株，结果显示，2P43调控下蔗糖异构酶酶活力最高，为 (19.08±0.09 ) U/mL ，是原始启动子Pgrac调控下酶活力的1.37倍。利用培养基优化，进一步提高蔗糖异构酶表达水平。通过单因素法从碳源、氮源和无机盐等3个方面对2P43调控的重组菌株的发酵培养基进行优化，最优发酵培养基配方为25 g/L甘油、30 g/L酵母浸粉、20.75 g/L大豆蛋白胨，Pal Ⅰ酶活力提高至(80.36±9.32) U/mL，是原始培养基 (19.08±0.09) U/mL 的4.2倍。实验成功构建高产物活力的双启动子调控Bacillus subtilis蔗糖异构酶表达系统，为后续研究及生产食品可用的蔗糖异构酶奠定理论基础，并为工业生产和应用异麦芽酮糖提供理论依据。

关键词: 蔗糖异构酶, 启动子, 枯草芽孢杆菌, 培养基优化, 全面实验

Abstract: Through promoter engineering and medium optimization strategies, sucrose isomerase with high expression level regulated by non-inducible promoter was constructed, and the effects of different promoters on sucrose isomerase expression of recombinant strain Bacillus subtilis SCK6 were investigated. Unrestricted cloning method was used to construct 6 non-inducible single promoters （PHpal Ⅰ, P43, PgsiB, PsrfA, PamyQ and PaprE） and 3 double promoters （P43-PgsiB, 2P43 and 2PgsiB） regulated sucrose isomerase recombinant strains, respectively. The results showed that the activity of sucrose isomerase under the control of 2P43 was the highest (19.08±0.09) U/mL, which was 1.37 times of that under the control of the original promoter Pgrac. The expression level of sucrose isomerase was further improved by optimizing the medium. The fermentation medium of recombinant strain regulated by 2P43 was optimized from carbon source, nitrogen source and inorganic salt by single factor method. The optimal fermentation medium formula was 25 g/L glycerol, 30 g/L yeast extract powder and 20.75 g/L soybean peptone. The activity of Pal Ⅰ enzyme was increased to （80.36±9.32） U/mL. It was 4.2 times of the original medium (19.08±0.09) U/mL. In this experiment, the expression system of double-promoter regulated Bacillus subtilis sucrose isomerase with high product activity was successfully constructed, which laid a theoretical foundation for subsequent research and production of sucrose isomerase available for food, and provided a theoretical basis for industrial production and application of Isomaltulose.

Key words: sucrose isomerase, initiator project;Bacillus subtilis, medium optimization, full-scale experiment

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