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  • ISSN 1006-3080
  • CN 31-1691/TQ

Kex2蛋白酶K291突变体性质和动力学研究

杨帆 刘晓 王之可 李素霞

杨帆, 刘晓, 王之可, 李素霞. Kex2蛋白酶K291突变体性质和动力学研究[J]. 华东理工大学学报(自然科学版). doi: 10.14135/j.cnki.1006-3080.20201009001
引用本文: 杨帆, 刘晓, 王之可, 李素霞. Kex2蛋白酶K291突变体性质和动力学研究[J]. 华东理工大学学报(自然科学版). doi: 10.14135/j.cnki.1006-3080.20201009001
YANG Fan, LIU Xiao, WANG Zhike, LI Suxia. Properties and Kinetics of Kex2 K291 Mutants[J]. Journal of East China University of Science and Technology. doi: 10.14135/j.cnki.1006-3080.20201009001
Citation: YANG Fan, LIU Xiao, WANG Zhike, LI Suxia. Properties and Kinetics of Kex2 K291 Mutants[J]. Journal of East China University of Science and Technology. doi: 10.14135/j.cnki.1006-3080.20201009001

Kex2蛋白酶K291突变体性质和动力学研究

doi: 10.14135/j.cnki.1006-3080.20201009001
详细信息
    作者简介:

    杨帆:杨 帆(1995-),女,硕士生,主要研究方向为重组蛋白酶。E-mail:2258361193@qq.com

    通讯作者:

    李素霞,E-mail:lisuxia@ecust.edu.cn

  • 中图分类号: Q78

Properties and Kinetics of Kex2 K291 Mutants

  • 摘要: 利用毕赤酵母GS115表达系统,成功表达并纯化获得了两种Kex2蛋白酶突变体Kex2-K291L和Kex2-K291H。对比研究了天然Kex2和这两种突变体的酶学性质和稳定性。实验结果表明,与天然Kex2相比,Kex2-K291H和Kex2-K291L两种突变体的稳定性得到了明显的改善。两种突变体的最适pH和最适温度与天然Kex2保持一致,均为pH 9.0和37 ℃,两种突变体的温度稳定性也与天然Kex2基本一致。与天然Kex2蛋白酶相比,突变体Kex2-K291H的pH值稳定性范围,从5.0~6.0扩大至5.0~7.0。酶促反应动力学研究表明,突变体Kex2-K291H和Kex2-K291L的Kcat/Km值分别为天然Kex2的1.8倍和2.0倍。突变体Kex2-K291H和Kex2-K291L在改善天然Kex2自降解问题的同时,提高了pH稳定性和催化效率。

     

  • 图  1  Kex2蛋白酶结构示意图

    Figure  1.  Three-dimensional structure of Kex2 protease

    Blue−calcium ion; Red−the catalytic triad Ser385-His213-Asp175; Orange−K291 residue; Yellow−K290 residue; Green-Whole protein structure

    图  2  天然Kex2蛋白酶和突变体的诱导表达结果(a)和Q-FF纯化后的天然Kex2蛋白酶和突变体(b)

    Figure  2.  The expression of the wild type Kex2 protease and its mutants (a) and Purification of the wild type Kex2 protease and its mutants with Q-FF (b)

    图  3  天然Kex2蛋白酶和突变体的最适温度

    Figure  3.  Optimal temperature of wild-type Kex2 protease and mutants   

    图  4  天然Kex2蛋白酶与突变体的温度稳定性比较

    Figure  4.  Comparison of temperature stability between wild-type Kex2 protease and mutants

    图  5  天然Kex2蛋白酶与突变体的最适pH值

    Figure  5.  Optimal pH value of wild-type Kex2 protease and its mutants   

    The substrates were dissolved in buffers of different pH value and kept at 25 ℃. Then add protease and react.

    图  6  天然Kex2蛋白酶与突变体pH稳定性

    Figure  6.  pH stability of wild-type Kex2 protease and its mutants

    图  7  Lineweaver-Burk双倒数图分析蛋白酶的动力学参数

    Figure  7.  Lineweaver-Burk plots analysis of the kinetics

    表  1  突变引物

    Table  1.   Primers of mutants

    PrimersSequences for primers (5’ to 3’)
    FCCGGAATTCTTGGTCTCCTCTCAACAAATC
    RATAGTTTAGCGGCCGCTTATGGGGTATTTGGATC
    F291HCAGATTTGGTGAAGCATGCTTTGGTTAAGG
    R291HCCTTAACCAAAGCATGCTTCACCAAATCTG
    F291LCAGATTTGGTGAAGTTGGCTTTGGTTAAGG
    R291LCCTTAACCAAAGCCAACTTCACCAAATCTG
    下载: 导出CSV

    表  2  Kex2蛋白酶动力学参数

    Table  2.   Kinetic parameters of Kex2 proteases

    ProteasesKm/(mmol·L−1)Kcat/s−1Kcat/Km/(mol−1·L·s−1)
    Wild Kex20.0371514.081$ \times {10}^{7} $
    Kex2-K291H0.0181367.556$ \times {10}^{7} $
    Kex2-K291L0.0191598.368$ \times {10}^{7} $
    下载: 导出CSV
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出版历程
  • 收稿日期:  2020-10-09
  • 网络出版日期:  2021-01-07

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