食品科学 ›› 0, Vol. ›› Issue (): 0-0.

• 生物工程 •    下一篇

小麦蛋白质二硫键异构酶基因的克隆、表达及重组酶性质研究

刘光1,胡松青2,张婷婷1,王敬敬3,李琳4,侯轶1   

  1. 1. 华南理工大学
    2. 华南理工大学食品科学与工程学院
    3. 上海海洋大学
    4. 华南理工大学轻工与食品学院
  • 收稿日期:2016-03-04 修回日期:2016-10-26 出版日期:2017-01-25 发布日期:2017-01-16
  • 通讯作者: 胡松青 E-mail:fesqhu@scut.edu.cn
  • 基金资助:
    草鱼肉气味形成机理研究;高等学校博士学科点专项科研基金;广东省自然科学基金

Gene cloning, expression and characterization of protein disulfide isomerase from wheat (Triticum aestivum L.)

2, Lin LI 2   

  • Received:2016-03-04 Revised:2016-10-26 Online:2017-01-25 Published:2017-01-16

摘要: 目的:克隆小麦蛋白质二硫键异构酶(wheat protein disulfide isomerase,wPDI)基因,实现其在大肠杆菌中的表达并探究其酶学性质。方法:以小麦种子总RNA为模板,逆转录并扩增得到wpdi,并以pET-30b为表达载体、大肠杆菌BL21(DE3)为宿主菌进行原核表达,表达产物经金属螯合层析纯化后进行了酶学性质研究。结果:克隆的基因全长1 548 bp,与“Wyuna”品质小麦PDI基因相似性达99%。构建了pET-30b-wpdi表达载体,获得wPDI的最佳表达条件为:诱导温度22 ℃,诱导时间6 h,诱导剂浓度0.5 mmol/L。该酶含4 个硫氧还蛋白结构域,分子量约为66.2 kDa,具有二硫键的还原酶和异构酶活性以及分子伴侣活性。结论:对重组wPDI的表达和酶学性质研究,为wPDI在面制品加工及其它方面的应用奠定了基础

关键词: 小麦蛋白质二硫键异构酶, 克隆, 表达, 酶学性质

Abstract: Purpose: The wheat protein disulfide isomerase (wPDI) gene was cloned and expressed in Escherichia Coli, and its enzymatic properties were investigated. Methods: wpdi gene was obtained by reverse transcription polymerase chain reaction (RT-PCR) amplification. The constructed recombinant plasmid pET-30b-wpdi was expressed in BL21(DE3). After metal chelating chromatography, the enzymatic properties of purified wPDI were determined. Results: A 1 548 bp gene fragment was amplified and sequenced as the wpdi gene that had 99% identity with that of wheat (Triticum aestivum L.), named Wyuna. The optimized conditions for wPDI expression were as follows: induction at 22 °C using IPTG concentration of 0.5 mM, the induction time was 6 h. The recombinant wPDI consisted of four thioredoxin-like domains and had the molecular weight of ~66.2 kDa. The recombinant wPDI exhibited enzymatic activities (including reductase activity and isomerase activity of disulfide bonds) and chaperone activity. Conclusions: The expression of wPDI and its enzymatic properties provided the fundamental to the application in flour-processing industry, etc.

Key words: wheat protein disulfide isomerase, cloning, expression, enzymatic activities

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