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  • ISSN 1006-3080
  • CN 31-1691/TQ

亚胺还原酶的改造及其催化合成1-苯基-1,2,3,4-四氢异喹啉的性能

曹文斌 李昊 陈飞飞 潘江 郑高伟 许建和

曹文斌, 李昊, 陈飞飞, 潘江, 郑高伟, 许建和. 亚胺还原酶的改造及其催化合成1-苯基-1,2,3,4-四氢异喹啉的性能[J]. 华东理工大学学报(自然科学版), 2022, 48(4): 511-518. doi: 10.14135/j.cnki.1006-3080.20210505001
引用本文: 曹文斌, 李昊, 陈飞飞, 潘江, 郑高伟, 许建和. 亚胺还原酶的改造及其催化合成1-苯基-1,2,3,4-四氢异喹啉的性能[J]. 华东理工大学学报(自然科学版), 2022, 48(4): 511-518. doi: 10.14135/j.cnki.1006-3080.20210505001
CAO Wenbin, LI Hao, CHEN Feifei, PAN Jiang, ZHENG Gaowei, XU Jianhe. Modification and Characterization of Imine Reductases for Synthesis of 1-Phenyl-1,2,3,4-Tetrahydro-Isoquinoline[J]. Journal of East China University of Science and Technology, 2022, 48(4): 511-518. doi: 10.14135/j.cnki.1006-3080.20210505001
Citation: CAO Wenbin, LI Hao, CHEN Feifei, PAN Jiang, ZHENG Gaowei, XU Jianhe. Modification and Characterization of Imine Reductases for Synthesis of 1-Phenyl-1,2,3,4-Tetrahydro-Isoquinoline[J]. Journal of East China University of Science and Technology, 2022, 48(4): 511-518. doi: 10.14135/j.cnki.1006-3080.20210505001

亚胺还原酶的改造及其催化合成1-苯基-1,2,3,4-四氢异喹啉的性能

doi: 10.14135/j.cnki.1006-3080.20210505001
基金项目: 国家自然科学基金(21878085,21871085)
详细信息
    作者简介:

    曹文斌(1993-),男,硕士生,主要研究方向为生物催化。E-mail:494186758@qq.com

    通讯作者:

    许建和,E-mail:jianhexu@ecust.edu.cn

  • 中图分类号: Q81

Modification and Characterization of Imine Reductases for Synthesis of 1-Phenyl-1,2,3,4-Tetrahydro-Isoquinoline

  • 摘要: 对亚胺还原酶酶库的筛选获得了性能优良的候选酶AdIR1,基于蛋白结构分析对AdIR1进行分子改造,获得了催化效率更高的突变体酶F172Y,并对其进行动力学与热稳定性表征;最后利用所开发的突变酶实现了(S)-1-苯基-1,2,3,4-四氢异喹啉((S)-1-Ph-THIQ)克级制备,证明了该酶法合成路线的实用性。

     

  • 图  1  亚胺还原酶催化的不对称还原反应

    Figure  1.  Asymmetric reduction reaction catalyzed by IRED

    图  2  SnIR与AdIR1的结构比对结果

    Figure  2.  Results for structural alignment of AdIR1 with SnIR

    图  3  pH对母本(a)与F172Y(b)酶活的影响

    Figure  3.  Effect of pH on the enzyme activity of WT(a) and F172Y(b)

    图  4  母本(a)与F172Y(b)的热稳定性

    Figure  4.  Thermostability of WT (a) and F172Y (b)

    图  5  母本与F172Y的动力学特征曲线

    Figure  5.  Kinetic characteristic curves of WT and F172Y

    图  6  母本(黄色)与突变体F172Y(绿色)的反应进程曲线

    Figure  6.  Reaction process curves of WT (yellow) and F172Y (green) of AdIR1

    图  7  不同助溶剂浓度下的反应曲线

    Figure  7.  Reaction curves with different cosolvent volume fractions

    图  8  克级制备反应的反应进程曲线

    Figure  8.  Reaction process curves of gram-scale preparative reaction

    表  1  目的基因克隆所用的引物

    Table  1.   Primers used in target gene cloning

    EntryEnzymePrimers (5’ to 3’)Restriction site
    1AdIR1GGAATTCCATATGATCACACTGATCGGGCTCGGT
    CCGGAATTCCTACTTGCGGTCCGCCTTGATG
    Nde I
    EcoR I
    2SeIR2GGGAATTCCATATGAGCACGCCGCTGACGCTGAT
    CCGGAATTCCTACTTCGCGGGCTTGATC
    Nde I
    EcoR I
    下载: 导出CSV

    表  2  目的基因定点突变所用PCR引物

    Table  2.   PCR primers used for site-directed mutations of target gene

    EntryNamePrimers (5’ to 3’)Length
    1118_FPGGCGTGATGNDTCCTGCCGAGTTGGTGGGCAAGGAG36
    2118_RPCTCGGCAGGAHNCATCACGCCGCCCGCGACGAACCG36
    3172_FPCTGGACATCNDTCTGACCTCGCTTTCGGTGTTCATG36
    4172_RPCGAGGTCAGAHNGATGTCCAGCTGCGCCTGGTAGAA36
    5216_FPGCCGAGCAGNDTGAAAAGGGCGAGCACCCCGGCGAC36
    6216_RPGCCCTTTTCAHNCTGCTCGGCCGCGGCGTCCAGGTA36
    下载: 导出CSV

    表  3  亚胺还原酶不对称还原1-苯基-3,4-二氢异喹啉的反应

    Table  3.   Asymmetric reduction of 1-phenyl-3,4-dihydroisoquinoline by IRED

    EntryEnzymeTime/hC/%ee*/%
    1AdIR1249898
    2SeIR2245494
    * S configuration
    下载: 导出CSV

    表  4  亚胺还原酶的关键位点比对结果

    Table  4.   Alignment results of key residues of the imine reductases

    EntryEnzyme1st site2nd site3rd site
    1SnIRT123F178G228
    2AdIR1V118F172G220
    下载: 导出CSV

    表  5  AdIR1突变体的测活结果

    Table  5.   Activity assay results of AdIR1 mutants

    EntryEnzymeMutation siteSpecific activity/(mU·mg−1)Fold
    1WT7.901.0
    2M1V1188.811.1
    3M2V1185.920.7
    4M3V1188.881.2
    5M4F17215.31.9
    WT—Wild type
    下载: 导出CSV

    表  6  F172Y与母本的半衰期

    Table  6.   Half-lives of WT and F172Y at different temperatures

    Enzymet1/2/h
    30 ℃40 ℃50 ℃
    WT70.050.41.0
    F172Y324.0314.03.9
    下载: 导出CSV

    表  7  母本与F172Y的催化动力学参数

    Table  7.   Kinetic parameters of WT and F172Y

    EnzymeT/ ℃Km/
    (mmol·L−1)
    kcat/ (s−1)kcat/Km/
    (L·s−1·mmol−1)
    WT300.05 ± 0.010.0050.10
    F172Y300.05 ± 0.010.0170.34
    F172Y400.07 ± 0.010.0270.38
    下载: 导出CSV

    表  8  F172Y在30 ℃与40 ℃下的反应转化结果

    Table  8.   Reaction conversion results of F172Y at 30 ℃ and 40 ℃

    T/℃Time/hConversion/ %ee/%
    3066999
    128499
    4068499
    129199
    下载: 导出CSV
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出版历程
  • 收稿日期:  2021-05-05
  • 网络出版日期:  2021-07-13
  • 刊出日期:  2022-08-26

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