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  • ISSN 1006-3080
  • CN 31-1691/TQ

基于长链底物亲和的脂肪酶MAS1理性设计改造

唐秀云 王嘉伟 张建国 缪雨露 赵玥 高蓓 张鲁嘉

唐秀云, 王嘉伟, 张建国, 缪雨露, 赵玥, 高蓓, 张鲁嘉. 基于长链底物亲和的脂肪酶MAS1理性设计改造[J]. 华东理工大学学报(自然科学版). doi: 10.14135/j.cnki.1006-3080.20220328001
引用本文: 唐秀云, 王嘉伟, 张建国, 缪雨露, 赵玥, 高蓓, 张鲁嘉. 基于长链底物亲和的脂肪酶MAS1理性设计改造[J]. 华东理工大学学报(自然科学版). doi: 10.14135/j.cnki.1006-3080.20220328001
TANG Xiuyun, WANG Jiawei, ZHANG Jianguo, MIAO Yulu, ZHAO Yue, GAO Bei, ZHANG Lujia. Rational Design of Lipase MAS1 for the Long-chain Substrate Affinity[J]. Journal of East China University of Science and Technology. doi: 10.14135/j.cnki.1006-3080.20220328001
Citation: TANG Xiuyun, WANG Jiawei, ZHANG Jianguo, MIAO Yulu, ZHAO Yue, GAO Bei, ZHANG Lujia. Rational Design of Lipase MAS1 for the Long-chain Substrate Affinity[J]. Journal of East China University of Science and Technology. doi: 10.14135/j.cnki.1006-3080.20220328001

基于长链底物亲和的脂肪酶MAS1理性设计改造

doi: 10.14135/j.cnki.1006-3080.20220328001
基金项目: 国家重点研发计划(2020YFA0908400);国家自然科学基金(31772007, U1805235);上海市自然科学基金(21ZR1416500);纽约大学全球种子基金;纽约大学-华东师范大学计算化学中心(ECNU创新001公众平台)
详细信息
    作者简介:

    唐秀云(1994—),女,安徽省人,硕士,主要研究方向:脂肪酶理性设计改造。E-mail:txy13865167321@163.com

    通讯作者:

    高 蓓,E-mail:gaobei@ecust.edu.cn

    张鲁嘉,E-mail:ljzhang@chem.ecnu.edu.cn;ljzhang@nyu.edu

  • 中图分类号: Q55

Rational Design of Lipase MAS1 for the Long-chain Substrate Affinity

  • 摘要: 探讨了脂肪酶MAS1对长链底物4-硝基苯酚豆蔻肉酸酯(pNP-C14)催化的的最适温度、最适pH、pH稳定性,并通过分子动力学模拟计算(MD),根据结合自由能初步筛选出了G145W及T141L两个单点突变。实验表明,与野生型MAS1相比,结合自由能降低显著的突变体G145W的Km值减小了11%,kcat/Km值为野生型MAS1的1.29倍,对长链底物的亲和能力和催化效率均有提高;而结合自由能降低较小的突变体T141L的Km值增大了22%,kcat/Km值为野生型MAS1的0.88倍,说明结合能差的绝对零值并非准确的突变筛选标准。通过分子力学/广义波恩表面积(MM/GBSA)残基拆解分析得出:残基T38、F39、L149、F153、V202、V233对脂肪酶活性口袋与长链底物结合的稳定性贡献较其他残基大;残基T38、G40、N41、N45和T237在脂肪酶对长链底物的亲和能力的提高中具有重要贡献,预测为热点残基,其结合自由能的降低可以作为突变筛选的参考标准。

     

  • 图  1  最适温度

    Figure  1.  Optimum temperature curve

    图  2  (a)最适反应 pH(b)pH稳定性

    Figure  2.  (a) Optimal pH curve (b) stability of pH curve

    图  3  分子对接分析

    Figure  3.  Results of molecular docking

    Orange—the catalytic triad Ser109-His232-Asp200; Gray—substrate (pNP-C14)

    图  4  MAS1, G145W, T141L动力学测定双倒数图

    Figure  4.  Lineweaver-Burk plots for the kinetic investigations of the wild-type MAS1, mutants G145W and T141L.

    表  1  引物序列

    Table  1.   Primers

    PrimersSequence(5´→3´)Restriction sites
    MAS1-FATAGAATTCGATGGCGACCGCG
    ACCGCGGCC
    EcoRI
    MAS1-RATACTCGAGTCAATGGTGATGGT
    GATGGTGATGATGG
    XhoI
    T7-FTAATACGACTCACTATA/
    T7-RGCTAGTTATTGCTCAGCG/
    下载: 导出CSV

    表  2  引物序列

    Table  2.   Primers

    PrimersSequence(5´→3´)
    T141L-1RGAGGCCGAGCAGCGTGAGGCCGTGGTTGTCCGG
    T141L-2FCCGGACAACCACGGCCTCACGCTGCTCGGCCTC
    G145W-1RCGGCAGCAGCTTGGTGAGCCAG
    AGCAGCGTGGTGCCGTG
    G145W-2FCACGGCACCACGCTGCTCTGGC
    TCACCAAGCTGCTGCCG
    MAS1-FATAGAATTCGATGGCGACCGCGACCGCGGCC
    MAS1-RATACTCGAGTCAATGGTGATGGTGATGGTGATGATGG
    下载: 导出CSV

    表  3  结合自由能计算结果

    Table  3.   Calculation of total free energy

    LipasesΔG/(kJ·mol−1)ΔΔG/(kJ·mol−1)
    WT−159.896N/A
    G145W−180.611−20.715
    T141L−165.833−5.937
    A135W−157.4062.490
    F47K−159.6070.289
    G76H−119.53340.363
    V35W−151.0728.824
    S230K−151.8628.034
    下载: 导出CSV

    表  4  酶动力学参数测定

    Table  4.   Kinetic parameters of lipases

    LipasesKm (mM)kcat (s−1)kcat/Km (mM−1·s−1)
    WT0.7028.236×1051.173×106
    G145W0.6259.431×1051.510×106
    T141L0.8608.852×1051.030×106
    下载: 导出CSV

    表  5  MM/GBSA残基分解结果

    Table  5.   The results of MM/GBSA residue decomposition

    Amino acid residuesΔG/(kJ·mol−1
    WTT141LG145W
    G37−4.904−0.469−4.820
    T38−9.573−4.485−15.573
    F39−12.619−5.665−12.142
    G40−3.4230.134−5.699
    N41−0.5190.042−1.314
    N45−1.1300.159−3.506
    H75−0.895−0.218−0.243
    H108−3.582−0.527−5.657
    S109−5.565−1.573−4.577
    T141−4.084−2.678−3.197
    L146−6.996−14.577−6.058
    L149−11.255−8.770−17.782
    L150−3.774−11.757−7.841
    F153−15.104−6.561−12.460
    P154−5.4730.126−2.176
    L167−3.372−18.769−5.623
    Q170−4.510−1.925−2.962
    V202−8.535−1.448−8.393
    H232−1.4900.042−5.724
    V233−15.138−1.423−14.544
    A234−4.619−0.033−0.527
    T237−2.7530.017−5.079
    下载: 导出CSV
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  • 网络出版日期:  2022-05-27

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