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中国生物工程杂志

CHINA BIOTECHNOLOGY
中国生物工程杂志
中国生物工程杂志  2023, Vol. 43 Issue (5): 37-44    DOI: 10.13523/j.cb.2210024
研究报告     
不同信号肽及其组合对果聚糖蔗糖酶异源表达的影响*
王茂军,色依德·斯马依,蔡逸安,李庆刚,路福平,李玉()
工业发酵微生物教育部重点实验室 天津科技大学生物工程学院 天津 300457
Effects of Different Signal Peptides and Their Combinations on Heterologous Expression of Levansucrase
WANG Mao-jun,SIMAYI Seyide,CAI Yi-an,LI Qing-gang,LU Fu-ping,LI Yu()
Key Laboratory of Industrial Fermentation Microbiology,Ministry of Education,College of Biotechnology, Tianjin University of Science&Technology,Tianjin 300457,China
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摘要：

为了实现果聚糖蔗糖酶在解淀粉芽孢杆菌Bacillus amyloliquefaciens 018(简称G3)中的高效表达,选择来源于不同芽孢杆菌的4个果聚糖蔗糖酶基因lsLich、lsAmy、lsSub和lsMega进行异源表达,并将课题组前期确定的对碱性蛋白酶酶活提升较高的5种信号肽进行筛选和组合。其中来源于地衣芽孢杆菌Bacillus licheniformis RN-01的lsLich在重组菌株G3/pLY-2-lsLich中的酶活力最高,酶活力为62.73 U/mL。以LS-Lich作为目的蛋白筛选单信号肽和双信号肽,其中信号肽SPDacB和SPAmyE组合的重组菌株G3/pLY-2-SDA-ls酶活最高,胞外酶活达到125.76 U/mL,较重组菌G3/pLY-2-SD-ls和G3/pLY-2-SA-ls分别提高了31.3%和39.2%,较原始菌株提高了100.49%。该结果表明双信号肽较单信号肽有助于提高LS-Lich的分泌量,同时信号肽组合顺序不同也产生一定的差异。
关键词： 信号肽;  果聚糖蔗糖酶;  解淀粉芽孢杆菌;  异源表达    
Abstract:

To express levansucrase in Bacillus amyloliquefaciens 018 (G3) efficiently. Four levansucrase genes lsLich, lsAmy, lsSub and lsMega from different Bacillus species were heterologously expressed, and five signal peptides with relatively high levels of alkaline protease identified by the research group were screened and combined. lsLich derived from Bacillus licheniformis RN-01 had the highest enzyme activity in the recombinant strain G3/pLY-2-lsLich, with an enzyme activity of 62.73 U/mL. LS-Lich was used as the target protein to screen single signal peptide and double signal peptide. The recombinant strain G3/pLY-2-SDA-ls combined with SPDacB and SPAmyE had the highest enzyme activity, and the extracellular enzyme activity reached 125.76 U/mL. Compared with the recombinant strain G3/pLY-2-SD-ls and G3/pLY-2-SA-ls, they increased by 31.3% and 39.2%, respectively, and increased by 100.49% compared with the original strain. The results indicated that the double signal peptide was helpful in increasing the secretion of LS-Lich compared with the single signal peptide, and the combination order of signal peptides also produced some differences.
Key words: Signal peptide    Levansucrase    Bacillus amyloliquefaciens    Heterologous expression
收稿日期: 2022-10-17 出版日期: 2023-06-01
ZTFLH:  Q819  
基金资助: *国家重点研发计划(2021YFC2100402)
通讯作者: **电子信箱: liyu@tust.edu.cn   
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王茂军
色依德·斯马依
蔡逸安
李庆刚
路福平
李玉

引用本文:

王茂军, 色依德·斯马依, 蔡逸安, 李庆刚, 路福平, 李玉. 不同信号肽及其组合对果聚糖蔗糖酶异源表达的影响*[J]. 中国生物工程杂志, 2023, 43(5): 37-44.

WANG Mao-jun, SIMAYI Seyide, CAI Yi-an, LI Qing-gang, LU Fu-ping, LI Yu. Effects of Different Signal Peptides and Their Combinations on Heterologous Expression of Levansucrase. China Biotechnology, 2023, 43(5): 37-44.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.2210024    或    https://manu60.magtech.com.cn/biotech/CN/Y2023/V43/I5/37

图1  果聚糖蔗糖酶的水解、转糖基和聚合作用
名称 氨基酸序列 来源
SacB MNIKKFAKQATVLTFTTALLAGGATQAFA B.subtilis 168
DacB MRIFKKAVFVIMISFLIATVNVNTAHA B.subtilis 168
YoaW MKKMLMLAFTFLLALTIHVGEASAV B.subtilis 168
NprE MGLGKKLSVAVAASFMSLSISLPGVQA B.subtilis 168
AmyE MFAKRFKTSLLPLFAGFLLLFHLVLAGPAAASA B.subtilis 168
表1  5种信号肽氨基酸序列及其来源
图2  重组载体中不同信号肽的结构示意图
图3  不同表达载体的PCR验证图
图4  重组菌G3/pLY-2-ls不同发酵时间的酶活力测定
图5  不同信号肽重组菌株的酶活力测定
图6  不同信号肽组合重组菌的酶活力比较
图7  信号肽串联重组菌株的SDS-PAGE验证图
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