两个不同启动子及其组合对碱性蛋白酶AprE异源表达的影响 <sup>*</sup>

doi:10.13523/j.cb.20191003

中国生物工程杂志

2019, Vol. 39

Issue (10): 17-23 DOI: 10.13523/j.cb.20191003

研究报告

两个不同启动子及其组合对碱性蛋白酶AprE异源表达的影响 ^*

史超硕,李登科,曹雪,袁航,张钰文,于江悦,路福平,李玉

天津科技大学生物工程学院工业发酵微生物教育部重点实验室天津 300457

The Effect on Heterologous Expression of Alkaline Protease AprE by Two Different Promoter and Combinatorial

SHI Chao-shuo,LI Deng-ke,CAO Xue,YUAN Hang,ZHANG Yu-wen,YU Jiang-yue,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：

背景碱性蛋白酶(alkaline protease)是一种具有广泛用途的工业酶制剂,其发酵活力目前仍不能满足工业生产需要。目的旨在通过优化启动子及其组合来提高Bacillus subtilis WB600 中碱性蛋白酶AprE的产量。方法以Bacillus subtilis WB600 为出发菌株,成功构建了含有4种不同类型启动子(P1、P2、P-1-2、P-2-1)的碱性蛋白酶AprE表达菌株。结果含不同启动子的4株重组菌均可成功表达碱性蛋白酶,发酵48h,含单一启动子P2的重组菌株表达碱性蛋白酶的活力为4 041U/ml,是P1的1.23倍。双启动子重组菌B.subtilis WB600/P-2-1-aprE表达的酶活性最高,是双启动子P-1-2的1.35倍,达到了6 125U/ml。结论为工业化高产碱性蛋白酶提供了一种有效策略。

关键词： 启动子; 碱性蛋白酶; 异源表达; 酶活力

Abstract:

Background: Alkaline protease is an important industrial protease with many applications, and its fermentative activity can’t meet the needs of industrial production.Objective: The aim is to improve the alkaline protease AprE production by screening promoters.Methods: Bacillus subtilis WB600, constructed in previous research, was served as the host strain for AprE production and four promoters (P1,P2,P-1-2,P-2-1) were screened to improve AprE production.Results: The results showed that four recombinant strains containing different promoters could successfully express alkaline protease AprE. After 48h of fermentation, the activity of AprE of P2 was 4 041U/ml, which was 1.23 fold of promoter P1.The highest enzyme activity of the double promoter recombinant B. subtilis WB600/P-2-1-aprE reached 6 125U/ml, which was 1.35 fold of the double promoter P-1-2.Conclusion: Collectively,an effective strategy for enhanced production of alkaline protease was provided.

Key words: Promoter Alkaline protease Heterologous expression Enzyme activity

收稿日期: 2019-03-09 出版日期: 2019-11-12

ZTFLH:

Q814

基金资助: * 国家重点研发计划(2017YFB0308401)

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	史超硕
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	于江悦
	路福平
	李玉

引用本文:

史超硕,李登科,曹雪,袁航,张钰文,于江悦,路福平,李玉. 两个不同启动子及其组合对碱性蛋白酶AprE异源表达的影响 ^*[J]. 中国生物工程杂志, 2019, 39(10): 17-23.

SHI Chao-shuo,LI Deng-ke,CAO Xue,YUAN Hang,ZHANG Yu-wen,YU Jiang-yue,LU Fu-ping LI Yu. The Effect on Heterologous Expression of Alkaline Protease AprE by Two Different Promoter and Combinatorial. China Biotechnology, 2019, 39(10): 17-23.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20191003 或 https://manu60.magtech.com.cn/biotech/CN/Y2019/V39/I10/17

表1 实时荧光定量PCR引物

图1 重组载体中不同启动子的结构示意图

图2 aprE的PCR扩增产物、不同启动子及载体 pWB980 酶切片段电泳图

图3 aprE基因在B. subtilis WB600 中的表达

图4 4株重组菌的酪蛋白水解圈与菌落直径比值(** P<0.01)

图5 发酵上清液的SDS-PAGE分析

图6 4株重组菌株酶活比较(** P<0.01)

图7 4株重组菌株提取 RNA 凝胶电泳检测

图8 4株重组菌株的real time PCR 结果(* P<0.05)

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