食品科学 ›› 2013, Vol. 34 ›› Issue (23): 200-205.doi: 10.7506/spkx1002-6630-201323042

• 生物工程 • 上一篇    下一篇

三种氨肽酶基因在大肠杆菌中的重组表达与比较

张 洁1,张 梁1,黄武宁2,石贵阳1,*   

  1. 1.粮食发酵工艺与技术国家工程实验室,工业生物技术教育部重点实验室,江南大学,江苏 无锡 214122;
    2.义乌市海之纳生物工程有限公司,浙江 义乌 322006
  • 收稿日期:2012-12-14 修回日期:2013-11-07 出版日期:2013-12-15 发布日期:2014-01-03
  • 通讯作者: 石贵阳 E-mail:zhangl@jiangnan.edu.cn
  • 基金资助:

    国家“863”计划项目(2011AA100905;2012AA021201);教育部“新世纪优秀人才支持计划”项目(NCET-11-0665)

A Comparative Study of Recombinant Expression of Three Aminopeptidases in Escherichia coli

ZHANG Jie1,ZHANG Liang1,HUANG Wu-ning2,SHI Gui-yang1,*   

  1. 1. National Engineering Laboratory for Cereal Fermentation Technology, Key Laboratory of Industrial Biotechnology, Ministry of
    Education, Jiangnan University, Wuxi 214122, China;2. Yiwu Haizhina Bioengineering Co. Ltd., Yiwu 322006, China
  • Received:2012-12-14 Revised:2013-11-07 Online:2013-12-15 Published:2014-01-03
  • Contact: SHI Gui-yang E-mail:zhangl@jiangnan.edu.cn

摘要:

PCR扩增Bacillus licheniformis 14580、Bacillus subtilis 168、Geobacillus stearothermophilus IFO12589氨肽酶基因,分别酶切连接表达质粒pET-28a,构建重组表达载体pET28a-BLAP、pET28a-BSAP、pET28a-GSAP,酶活力检测表明3个氨肽酶基因均在大肠杆菌宿主BL21(DE3)中获得重组表达。进一步对3株重组菌的氨肽酶粗酶液反应条件进行比较研究,结果表明:重组氨肽酶BSAP与GSAP的粗酶活较高,达到1500U/L以上;BLAP、BSAP、GSAP粗酶的最适酶反应温度分别为50、75、60℃,BSAP温度稳定性最好,在30~70℃时比较稳定;3种重组氨肽酶的最适pH值都是9.0,pH值在8.5~9.0时比较稳定;0.lmmol/L Co2+对酶活有较强的激活作用,BSAP的相对酶活力最高达到195.6%,其他二价金属离子对酶活均有不同程度的抑制,其中Zn2+抑制作用最大;重组质粒pET28a-BSAP、pET28a-GSAP在大肠杆菌中较pET28a-BLAP稳定。

关键词: 氨肽酶, 重组表达, 酶反应

Abstract:

The genes encoding aminopeptidase GSAP, BSAP and BLAP were respectively amplified from Geobacillus
stearothermophilus IFO12589, Bacillus subtilis 168 and Bacillus licheniformis 14580. These amplified DNA fragments
were individually inserted into the expression vector E. coli pET-28a to construct plasmids pET28a-BLAP, pET28a-BSAP
and pET28a-GSAP, respectively. The expression of these genes was confirmed by activity analysis, and then the expressed
enzymes were characterized. The activities of both recombinant BSAP and GSAP were 1500 U/L, which were higher than
that of BLAP. The optimum temperatures were 50, 75 ℃ and 60 ℃ for BSAP, GSAP and BLAP, respectively, and the
optimum pH values were all 9.0. They were stable at pH 8.5—9.0. BSAP was obviously activated by 0.l mmol/L Co2+,
reaching a maximum of 195.6% of its original activity, but inhibited to different extents by other divalent metal ions, with
Zn2+ being the strongest inhibitor. In addition, the recombinant plasmids pET28a-BSAP and pET28a-GSAP were more stable
than pET28a-BLAP in E. coli.

Key words: aminopeptidase, recombinant expression, enzymatic reaction

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