食品与发酵工业 >

2023 , Vol. 49 >Issue 2: 47 - 53

DOI: https://doi.org/10.13995/j.cnki.11-1802/ts.031405

研究报告

嗜盐白蚁菌褐藻胶裂解酶的重组表达与改造优化

  • 吴雯 ,
  • 朱风帅 ,
  • 俞嘉乐 ,
  • 李恒 ,
  • 龚劲松 ,
  • 许正宏 ,
  • 史劲松
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  • 1(糖化学与生物技术教育部重点实验室,江南大学 生命科学与健康工程学院,江苏 无锡,214122)
    2(粮食发酵与食品生物制造国家工程研究中心,江南大学,江苏 无锡,214122)
    3(江南大学 生物工程学院,江苏 无锡,214122)
第一作者：硕士研究生(李恒副教授为通信作者,E-mail:liheng@jiangnan.edu.cn)

收稿日期: 2022-03-04

  修回日期: 2022-04-05

  网络出版日期: 2023-02-15

基金资助

国家重点研发计划专项子课题(2021YFC2102000);宁夏回族自治区重点研发计划(2020BFH02011)

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Recombined expression and optimized modification of alginate lyase from Isoptericola halotolerans

  • WU Wen ,
  • ZHU Fengshuai ,
  • YU Jiale ,
  • LI Heng ,
  • GONG Jinsong ,
  • XU Zhenghong ,
  • SHI Jinsong
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  • 1(Key Laboratory of the Ministry of Education of Sugar Chemistry and Biotechnology, College of Life Sciences and Health Engineering, Jiangnan University, Wuxi 214122, China)
    2(National Engineering Research Center for Food Fermentation and Food Biomanufacturing, Jiangnan University, Wuxi 214122, China)
    3(College of Bioengineering, Jiangnan University, Wuxi 214122, China)

Received date: 2022-03-04

  Revised date: 2022-04-05

  Online published: 2023-02-15

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摘要

为进一步拓展褐藻胶裂解酶在枯草芽孢杆菌体系中的高效表达,该研究以来源于海洋嗜盐白蚁菌WX (Isoptericola halotolerans WX)的一段褐藻胶裂解酶基因aly-ih为目的片段,成功构建Bacillus subtilis-pMA5-aly-ih工程菌,并利用启动子改造与培养基优化的方法提高酶催化活性。采用同源重组方法,将内源性启动子Hpa Ⅱ替换为mpr,Aly-IH酶活力较初始酶活力提高至4倍;进一步通过启动子的串联多拷贝提高酶活力,当拷贝数为3时,重组酶酶活力较初始酶活力提高至5.1倍。在此基础上,优化发酵培养基配方,在16 g/L甘油、16 g/L大豆蛋白胨、25 mmol/L Mn2+的培养条件下,Aly-IH酶活力提高到320 U/mL,是初始酶活力的10.7倍。该酶可高效降解褐藻胶得到平均聚合度为2.3的寡糖产物。研究结果表明,经枯草芽孢杆菌表达并改造优化的褐藻胶裂解酶Aly-IH具有良好的催化性能和应用潜力。

关键词： 褐藻胶裂解酶; 枯草芽孢杆菌; 启动子改造; 培养基优化

本文引用格式

吴雯 , 朱风帅 , 俞嘉乐 , 李恒 , 龚劲松 , 许正宏 , 史劲松 . 嗜盐白蚁菌褐藻胶裂解酶的重组表达与改造优化[J]. 食品与发酵工业, 2023 , 49(2) : 47 -53 . DOI: 10.13995/j.cnki.11-1802/ts.031405

Abstract

For further expanding the high-efficiency expression of alginate lyase in Bacillus subtilis, a segment of the alginate lyase gene named aly-ih from Isoptericola halotolerans WX was employed to construct an engineered strain of B. subtilis-pMA5-aly-ih. The catalytic activity of the enzyme was successively improved by the strategies of promoter modification and medium optimization. Herein, the enzymatic activity was increased by 4-fold compared with the initial enzyme through replacing the endogenous promoter HpaⅡ by mpr with homologous recombination method. The activity of the recombinase was further increased by 5.1-fold when the copy number of promoter mpr reached 3. The optimized fermentation medium was as following: 16 g/L glycerol, 16 g/L soy peptone, 25 mmol/L Mn2+. With the above conditions, the enzymatic activity of Aly-IH reached 320 U/mL which was 10.7 times as much as the initial enzyme. Aly-IH could efficiently degrade alginate to oligosaccharides with an average degree of polymerization of 2.3. Conclusively, the modified engineered Aly-IH in B. subtilis exerted prominent catalytic performance and potential application.

Key words： alginate lyase; Bacillus subtilis; promoter modification; media optimization

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