食品科学 ›› 2019, Vol. 40 ›› Issue (6): 113-120.doi: 10.7506/spkx1002-6630-20180305-031

• 生物工程 • 上一篇    下一篇

海洋氧化节杆菌KQ11右旋糖苷酶催化位点关键氨基酸

刘 乐1,丁 一1,王紫玄1,房耀维1,王淑军1,2,吕明生1,*   

  1. 1.淮海工学院海洋生命与水产学院,江苏 连云港 222005;2.江苏省海洋资源开发研究院,江苏 连云港 222005
  • 出版日期:2019-03-25 发布日期:2019-04-02
  • 基金资助:
    国家自然科学基金面上项目(31471719);江苏省科技厅社会发展项目(BE2016702)

Dextranase from Arthrobacter oxydans KQ11: Identification of Key Residues in Catalysis

LIU Le1, DING Yi1, WANG Zixuan1, FANG Yaowei1, WANG Shujun1,2, Lü Mingsheng1,*   

  1. 1. College of Marine Life and Fisheries, Huaihai Institute of Technology, Lianyungang 222005, China; 2. Marine Resources Development Institute of Jiangsu, Lianyungang 222005, China
  • Online:2019-03-25 Published:2019-04-02

摘要: 通过同源比对,对来自海洋氧化节杆菌(Arthrobacter oxydans KQ11)的右旋糖苷酶(记作AoDex)催化域及关键氨基酸进行预测,运用定点突变将AoDex催化域418-QTDGIELYKGSTMKNTFFNANDD-440中的5 个氨基酸突变为甘氨酸,获得5 种突变型右旋糖苷酶原核表达载体:pColdIII-KQN-Q418G、pColdIII-KQN-D420G、pColdIII-KQN-E423G、pColdIII-KQN-D439G、pColdIII-KQN-D440G,表达产物分别记为Q418GDex、D420GDex、E423GDex、D439GDex、D440GDex。经过发酵表达,Q418GDex、D420GDex、E423GDex、D439GDex几乎没有酶活力。D440GDex酶活力与AoDex一致;所不同的是,D440GDex在25~40 ℃时的酶活力提高了2~3 倍,最适pH值也从AoDex的5.5变为6.5。数据表明,Q418、D420、E423、D439四个氨基酸残基是AoDex催化域中的关键氨基酸。D440突变为甘氨酸对该酶的性质有较大影响,也表明其不是催化域中的广义碱。本研究表明AoDex的催化机制与糖苷酶49家族是一致的,为AoDex的功能改造提供了理论支持。

关键词: 右旋糖苷酶, 定点突变, 催化域, 关键氨基酸, 结构预测

Abstract: The catalytic domain and key amino acid residues of dextranase from Arthrobacter oxydans KQ11 (AoDex) were predicted by homologous sequence alignment. The mutants Gln418→Gly, Asp420→Gly, Glu423→Gly, Asp439→Gly, and Asp440→Gly were obtained from the catalytic domain 418-QTDGIELYKGSTMKNTFFNANDD-440 by site-directed mutagenesis. The mutant dextranases Q418GDex, D420GDex, E423GDex, and D439GDex had almost no enzymatic activity, while D440GDex had equal enzymatic activity to AoDex. However, at temperatures between 25 and 40 ℃, D440GDex activity increased by 2–3 folds compared to AoDex activity. The optimum pH for D440GDex was 6.5 while that for AoDex was 5.5. Q418, D420, E423, D439 were the key amino acid residues in the catalytic domain of AoDex. Mutation of D440 had a great impact on the enzymatic properties, suggesting it to be not the general base of the AoDex catalytic domain. These findings suggest that AoDex and the GH family 49 share a similar catalytic mechanism, which will provide theoretical support for improving the enzymatic properties of AoDex.

Key words: dextranase, site-directed mutagenesis, catalytic domain, key amino acid residues, structure prediction

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