基于共轭微孔聚合物的高性能多酶级联催化系统

doi:10.1016/S1872-2067(24)60088-4

催化学报 ›› 2024, Vol. 63: 213-223.DOI: 10.1016/S1872-2067(24)60088-4

基于共轭微孔聚合物的高性能多酶级联催化系统

吴振华^a, 石家福^a^,^c^,^d^,^*(), 张博禹^a, 焦彧帅^b, 孟祥萱^e, 储子仪^a, 陈裕^a, 程怡然^a, 姜忠义^b^,^c^,^f^,^*()

^a天津大学环境科学与工程学院, 天津 300072
^b天津大学化工学院, 教育部绿色化工重点实验室, 天津 300072
^c物质绿色创造与制造海河实验室, 天津 300192
^d中国科学院过程工程研究所, 生物化学工程国家重点实验室, 北京 100190
^e福州大学化工学院, 福建福州 350108
^f天津大学-新加坡国立大学福州联合学院, 福建福州 350207

收稿日期:2024-04-27 接受日期:2024-06-21 出版日期:2024-08-18 发布日期:2024-08-19
通讯作者: *电子信箱: shijiafu@tju.edu.cn (石家福),zhyjiang@tju.edu.cn (姜忠义).
基金资助:
国家重点研发计划(2021YFC2102300);国家重点研发计划(2022YFC2105900);国家自然科学基金(22122809);生化工程国家重点实验室开放基金项目(2020KF-06);物质绿色创造与制造海河实验室.

Conjugated microporous polymers-scaffolded enzyme cascade systems with enhanced catalytic activity

Zhenhua Wu^a, Jiafu Shi^a^,^c^,^d^,^*(), Boyu Zhang^a, Yushuai Jiao^b, Xiangxuan Meng^e, Ziyi Chu^a, Yu Chen^a, Yiran Cheng^a, Zhongyi Jiang^b^,^c^,^f^,^*()

^aSchool of Environmental Science and Engineering, Tianjin University, Tianjin 300072, China
^bKey Laboratory for Green Chemical Technology of Ministry of Education, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, China
^cHaihe Laboratory of Sustainable Chemical Transformations, Tianjin 300192, China
^dState Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, China
^eCollege of Chemical Engineering, Fuzhou University, Fuzhou 350108, Fujian, China
^fJoint School of National University of Singapore and Tianjin University, International Campus of Tianjin University, Fuzhou 350207, Fujian, China

Received:2024-04-27 Accepted:2024-06-21 Online:2024-08-18 Published:2024-08-19
Contact: *E-mail: shijiafu@tju.edu.cn (J. Shi), zhyjiang@tju.edu.cn (Z. Jiang).
Supported by:
National Key Research and Development Program of China(2021YFC2102300);National Key Research and Development Program of China(2022YFC2105900);National Natural Science Funds of China(22122809);Open Funding Project of the State Key Laboratory of Biochemical Engineering, China(2020KF-06);Haihe Laboratory of Sustainable Chemical Transformations.

摘要/Abstract

摘要：

底物通道效应是维持体内酶级联系统高效代谢的一个重要现象, 在生物体的代谢过程中广泛存在. 自然界通过构筑多酶复合体, 并利用多酶复合体内的连接通道, 促进了中间产物的快速传递, 从而强化底物通道效应, 实现高效物质转化和生命代谢. 受此启发, 本文将多酶分子原位包埋于共轭微孔聚合物(CMPs)中. CMPs作为载体平台, 具有结构规整、化学性质可调以及制备条件温和等优点. 通过这一策略, 成功地在体外建立了多酶催化的底物通道效应, 并深入揭示了其作用机制, 最终实现了多酶催化性能的显著提升.

本文详细研究了以CMPs为载体的集成型酶级联系统(I-ECSs)中的底物通道效应创建与强化机制. 通过机械化学法成功实现了酶级联系统(葡萄糖氧化酶-辣根过氧化物酶, 即GOx-HRP)的原位包埋, 其中过氧化氢(H₂O₂)是GOx-HRP级联反应的中间产物. 通过瞬态时间(级联反应的中间产物达到平衡浓度的时间)、初始反应速率和抑制竞争反应能力等3个参数共同证明了在I-ECSs中底物通道效应的成功创建. 底物通道效应可促进中间产物的快速传递, 因而I-ECSs的催化活性相较于分隔型酶级联系统(S-ECSs)和游离型酶级联系统(F-ECSs)分别提升了4倍和1.4倍. 通过密度泛函理论计算进一步从分子尺度上探究了底物通道效应的形成机制, 结果表明, 孔壁基团通过物理吸附作用富集H₂O₂, 这种物理吸附的相互作用能为‒9.21 kcal mol^‒1, 并通过CMPs对H₂O₂的吸附和脱附实验证明了该相互作用可富集H₂O₂产生局域高浓度, 同时H₂O₂会克服氢键相互作用, 实现在CMPs内部的快速传递. 此外, CMPs具有1.5‒1.6 nm的较规整微孔结构, 为H₂O₂ (分子直径为0.25‒0.28 nm)的快速传递提供了低阻力传质路径. 因此, CMPs可提供适宜的孔壁-H₂O₂相互作用和低阻力传质路径, 实现了底物通道效应的成功创建. 通过调控GOx与HRP间的质量比例, 可在不同程度强化底物通道效应, 从而得到具有不同催化活性的I-ECSs. 此外, I-ECSs还表现出较好的操作稳定性. 经10次循环实验后, I-ECSs仍保留初始活性的76%以上, 且循环过程中的酶基本无泄漏.

综上, 本文以CMPs为载体构建了具有不同活性的I-ECSs, 揭示了底物通道效应的构建和强化机制, 为构建具有高活性和强稳定性的级联催化系统提供借鉴.

关键词: 生物催化, 固定化, 多酶级联催化系统, 底物通道效应, 共轭微孔聚合物

Abstract:

Enhancing catalytic activity of multi-enzyme in vitro through substrate channeling effect is promising yet challenging. Herein, conjugated microporous polymers (CMPs)-scaffolded integrated enzyme cascade systems (I-ECSs) are constructed through co-entrapping glucose oxidase (GOx) and horseradish peroxidase (HRP), in which hydrogen peroxide (H₂O₂) is the intermediate product. The interplay of low-resistance mass transfer pathway and appropriate pore wall-H₂O₂ interactions facilitates the directed transfer of H₂O₂, resulting in 2.4-fold and 5.0-fold elevation in catalytic activity compared to free ECSs and separated ECSs, respectively. The substrate channeling effect could be regulated by altering the mass ratio of GOx to HRP. Besides, I-ECSs demonstrate excellent stabilities in harsh environments and multiple recycling.

Key words: Biocatalysis, Immobilization, Enzyme cascade system, Substrate channeling effect, Conjugated microporous polymers

吴振华, 石家福, 张博禹, 焦彧帅, 孟祥萱, 储子仪, 陈裕, 程怡然, 姜忠义. 基于共轭微孔聚合物的高性能多酶级联催化系统[J]. 催化学报, 2024, 63: 213-223.

Zhenhua Wu, Jiafu Shi, Boyu Zhang, Yushuai Jiao, Xiangxuan Meng, Ziyi Chu, Yu Chen, Yiran Cheng, Zhongyi Jiang. Conjugated microporous polymers-scaffolded enzyme cascade systems with enhanced catalytic activity[J]. Chinese Journal of Catalysis, 2024, 63: 213-223.

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链接本文: https://www.cjcatal.com/CN/10.1016/S1872-2067(24)60088-4

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图/表 5

Fig. 1. Schematic illustration of the preparation of CMPs-scaffolded I-ECSs and the substrate channeling effect.

Fig. 2. Structural feature and characterization of I-ECSs. (a) SEM image of I-ECSs. TEM (b) and EDS elemental mapping images (c-f) of I-ECSs. CLSM images of FITC-labeled GOx (green) (g) and coumarin-labeled HRP (blue) (h). FTIR spectra (i) and pore size distribution (j) of CMPs and I-ECSs.

Fig. 3. Creation of substrate channeling effect in I-ECSs. Catalytic activity curves and τ of different biocatalyst concentrations biocatalysts: 2c (a), c (b), and 0.5c (c). (d) Reaction rate ratios of different concentrations biocatalysts. Catalytic activity curves (e) and reaction rate ratios (f) of I-ECSs and S-ECSs treatment with varying concentrations of free catalase (CAT). Reaction rate ratios calculated from vI-ECSs/vS-ECSs (vI-ECSs and vS-ECSs refer to the reaction rate of I-ECSs and S-ECSs, respectively). (g) The binding energy (ΔE) of pore wall-H2O2 via DFT, and the insert showed the hydrogen-bonded bridges between pore wall and H2O2. (h) Schematic illustration of I-ECSs (above) and the modelling of mass transfer pathway of H2O2 from producing sites to consuming sites (below). (i) Schematic illustration of S-ECSs (above) and the modelling of mass transfer pathway of H2O2 from producing sites to consuming sites (below).

Fig. 4. Fortification of substrate channeling effect in I-ECSs. (a) Catalytic activity curves and τ of I-ECSs-x with different dosages of HRP (I-ECSs-x represented the dosages of HRP in I-ECSs and τx represented τ of I-ECSs-x, x = 1, 3, and 5 mg). (b) Relative activity of I-ECSs-1, I-ECSs-3 and I-ECSs-5 treatment with 25 μg mL-1 free CAT. Reaction rate ratios of I-ECSs-3 or I-ECSs-5 and I-ECSs-1 treatment with varying concentrations of free CAT 0 (c) and 25 (d) μg mL-1, which was calculated from v3/v1 and v5/v1 (v1, v2, and v3 represented the reaction rate of I-ECSs-1, I-ECSs-3 and I-ECSs-5, respectively). (e) Catalytic activity curves of I-ECSs and free GOx-HRP cascade systems (F-ECSs). Notably, the catalytic activity of GOx or HRP in F-ECSs was equivalent to that of GOx or HRP in I-ECSs, obtained by regulating the mass of GOx or HRP in F-ECSs. (f) Reaction rate ratios of I-ECSs and F-ECSs.

Fig. 5. Tolerance of I-ECSs to inhospitable conditions. Stability of I-ECSs and F-ECSs to inhospitable conditions pH resistance (a), heating resistance (b), and proteolytic agent resistance (urea, acetone and N,N-dimethylformamide) (c). (d) Recyclability of I-ECSs.

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基于共轭微孔聚合物的高性能多酶级联催化系统

Conjugated microporous polymers-scaffolded enzyme cascade systems with enhanced catalytic activity

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