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食品与发酵工业  2020, Vol. 46 Issue (8): 1-6    DOI: 10.13995/j.cnki.11-1802/ts.023374
  研究报告 本期目录 | 过刊浏览 | 高级检索 |
食品级高产亮氨酸氨肽酶重组Bacillus subtilis的构建和发酵优化
张大伟, 刘德华, 黄钦钦, 田亚平*
(江南大学,工业生物技术教育部重点实验室,江苏 无锡, 214122)
Construction and fermentation optimization of food-grade recombinant Bacillus subtilis for the production of leucine aminopeptidase
ZHANG Dawei, LIU Dehua, HUANG Qinqin, TIAN Yaping*
(Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, Wuxi 214122, China)
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摘要 为解决枯草芽孢杆菌(Bacillus subtilis)在发酵产亮氨酸氨肽酶(leucine aminopeptidase,LAP)需额外添加抗生素的问题,该研究构建了1株食品级产LAP重组B. subtilis。首先通过Cre/lox系统敲除B. subtilis 168基因组中D-丙氨酸消旋酶基因(dal)得到缺陷型菌株BS168 (dal)-。其次使用高斯组装的方法构建以dal作为标记基因的质粒pMA5-lap-dal (AmpR, Ori)-并转化至BS168 (dal)-中得到食品级重组菌BS168 (dal)-/pMA5-lap-dal (AmpR, Ori)-。该工程菌发酵产LAP无需添加抗生素且不含抗性基因。对该菌株进行5 L发酵罐条件优化,在转速300 r/min、温度33 ℃、pH 7.0、分阶段补料的条件下,酶活最高达到302 U/mL。结果表明,构建的食品级重组B. subtilis产LAP具有潜在的工业应用前景。
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张大伟
刘德华
黄钦钦
田亚平
关键词:  亮氨酸氨肽酶  D-丙氨酸消旋酶  枯草芽孢杆菌  食品级表达  发酵优化    
Abstract: To solve the problem that antibiotics has to be added during the production of leucine aminopeptidase (LAP) using Bacillus subtilis fermentation, a food-grade recombinant B. subtilis strain was constructed. The D-alanine racemase gene (dal) in the B. subtilis 168 genome was knocked out with the Cre/lox recombination system to obtain a defective strain BS168 (dal)-. By employing the Gibson assembly method, the plasmid pMA5-lap-dal (AmpR, Ori)- with the dal as a selection marker was introduced into the auxotrophic strain BS168 (dal)- to obtain food-grade recombinant strain BS168 (dal)-/pMA5-lap-dal (AmpR, Ori)-. This strain contained no resistance gene and did not need adding antibiotics during LAP fermentation. The condition of 5 L bioreactor was optimized for this strain. The LAP activity reached 302 U/mL under the following conditions: agitation speed 300 r/min; temperature 33°C; pH 7.0 and fed-batch. The results show that the food-grade LAP recombinant B. subtilis constructed in this study has potential industrial application.
Key words:  leucine aminopeptidase    Bacillus subtilis    D-alanine racemase    food-grade expression    fermentation optimization
收稿日期:  2020-01-14                出版日期:  2020-04-25      发布日期:  2020-05-20      期的出版日期:  2020-04-25
基金资助: 企业横向协作项目(180625);国家自然科学基金(31601558)
作者简介:  硕士研究生(田亚平教授为通讯作者,E-mail:biochem@jiangnan.edu.cn)
引用本文:    
张大伟,刘德华,黄钦钦,等. 食品级高产亮氨酸氨肽酶重组Bacillus subtilis的构建和发酵优化[J]. 食品与发酵工业, 2020, 46(8): 1-6.
ZHANG Dawei,LIU Dehua,HUANG Qinqin,et al. Construction and fermentation optimization of food-grade recombinant Bacillus subtilis for the production of leucine aminopeptidase[J]. Food and Fermentation Industries, 2020, 46(8): 1-6.
链接本文:  
http://sf1970.cnif.cn/CN/10.13995/j.cnki.11-1802/ts.023374  或          http://sf1970.cnif.cn/CN/Y2020/V46/I8/1
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