多元介孔MOFs：通过调节表面亲水/疏水性实现酶固定化的多功能平台

doi:10.1016/S1872-2067(24)60020-3

催化学报 ›› 2024, Vol. 60: 386-398.DOI: 10.1016/S1872-2067(24)60020-3

• 论文 • 上一篇

多元介孔MOFs：通过调节表面亲水/疏水性实现酶固定化的多功能平台

冯玉晓^a, 马庆庆^a, 王子辰^a, 张群力^a, 赵利雪^a, 崔建东^a^,^d^,^*(), 杜英杰^a^,^b^,^c^,^*(), 贾士儒^a

^a天津科技大学生物工程学院, 食品营养与安全国家重点实验室, 工业发酵微生物教育部重点实验室, 天津 300457
^b湖北大学生命科学学院, 生物催化与酶工程国家重点实验室, 湖北武汉 430062
^c天津益倍生物科技集团有限公司, 天津 300457
^d浙江大学生命科学学院, 生物质化工教育部重点实验室, 浙江杭州 310050

收稿日期:2024-01-17 接受日期:2024-03-17 出版日期:2024-05-18 发布日期:2024-05-20
通讯作者: *电子信箱: cjd007cn@163.com (崔建东), yingjiedu@tust.edu.cn (杜英杰).
作者简介:第一联系人：¹共同第一作者.
基金资助:
国家自然科学基金(22108206);中央高校基础研究经费(226-2023-0085);广西重点研发计划项目(GuikeAB21238005);中国博士后科学基金项目(2023T160483);中国博士后科学基金项目(2022M722387);生物催化与酶工程国家重点实验室开放基金(SKLBEE2022004)

Multivariate mesoporous MOFs with regulatable hydrophilic/hydrophobic surfaces as a versatile platform for enzyme immobilization

Yuxiao Feng^a, Qingqing Ma^a, Zichen Wang^a, Qunli Zhang^a, Lixue Zhao^a, Jiandong Cui^a^,^d^,^*(), Yingjie Du^a^,^b^,^c^,^*(), Shiru Jia^a

^aState Key Laboratory of Food Nutrition and Safety, Laboratory of Industrial Fermentation Microbiology, Ministry of Education, Tianjin University of Science and Technology, Tianjin 300457, China
^bState Key Laboratory of Biocatalysis and Enzyme Engineering, School of Life Sciences, Hubei University, Wuhan 430062, Hubei, China
^cTianjin UBasio Biotechnology Group, Tianjin 300457, China
^dKey Laboratory of Biomass Chemical Engineering of Ministry of Education, Zhejiang University, Hangzhou 310027, Zhejiang, China

Received:2024-01-17 Accepted:2024-03-17 Online:2024-05-18 Published:2024-05-20
Contact: *E-mail: jdcui@tust.edu.cn (J. Cui), yingjiedu@tust.edu.cn (Y. Du).
About author:First author contact:¹Contributed to this work equally.
Supported by:
National Natural Science Foundation of China(22108206);Fundamental Research Funds for the Central Universities(226-2023-0085);project of Guangxi Key Research and Development program, China(GuikeAB21238005);China Postdoctoral Science Foundation Project(2023T160483);China Postdoctoral Science Foundation Project(2022M722387);Open Funding Project of the State Key Laboratory of Biocatalysis and Enzyme Engineering(SKLBEE2022004)

摘要/Abstract

摘要：

作为高效的生物催化剂, 酶在食品加工、医药制造、化学工业和能源开发等众多领域发挥着重要作用. 然而, 游离状态的酶由于存在稳定性差、易失活、难以回收利用及不适用于连续化生产等问题, 限制了其广泛应用. 为了解决上述难题, 人们探索发展了固定化酶技术. 固定化酶不仅保留了原有的催化活性, 而且在稳定性方面有了显著提升, 从而极大地提高了酶的利用效率. 随着固定化酶材料和酶工程领域的不断发展, 固定化酶技术为酶的应用开辟了更广阔的前景.

沸石-咪唑骨架材料-8(ZIF-8)作为一种金属-有机骨架(MOFs)材料, 因其独特的结构特性而被广泛用作酶固定化载体. 然而, ZIF-8的微孔结构和固有的疏水性, 以及前体物2-甲基咪唑(2-MeIm)的质子化现象, 常常影响固定化酶的活性. 为克服这些限制, 本文采用多元配体竞争和孔隙工程相结合的策略, 设计并制备了一种具有可调节亲水/疏水表面的新型多元介孔MOFs(mMOFs), 并用作酶固定化载体. 在mMOFs的制备过程中, 采用亲水性和配位能力更强的3-甲基-1H-1,2,4-三唑(3-MTZ)和5-甲基四唑(5-MTA)配体, 以部分替代传统的2-MeIm配体. 随后, 这些多元配体(包括2-MeIm, 3-MTZ和5-MTA)与七水硫酸锌结合, 在甲醇溶液中通过自凝聚作用形成透明的胶体溶液, 进而完全溶解在水中. 在此过程中, 七水硫酸锌发挥了软模板的作用, 引导介孔的形成, 从而成功制备出新型多元介孔mMOFs. 接着, 利用这些mMOFs作为酶固定化的载体, 固定了辣根过氧化物酶(HRP)和葡萄糖氧化酶(GOx), 并详细研究了其催化性能. 结果表明, 与传统的ZIF-8载体相比, 以mMOFs为载体的固定化酶展现出了更高的酶活性和稳定性. 催化性能提升主要归因于mMOFs的亲水性和介孔结构改善了酶和底物间的传质, 同时2-MeIm的质子化效应减弱也为酶提供了优良的微环境, 稳定了酶的构象.

综上, 本文采用多元配体竞争与孔隙工程相结合的策略, 成功构建了一种具有可调节亲水/疏水表面的新型多元介孔mMOFs, 并用于酶固定化, 所得固定化酶表现出较好的催化性能. 通过深入的研究, 揭示了该固定化酶表现出良好催化性能的作用机制, 为可控设计制备性能优越的、以MOFs为载体的固定化酶提供了新思路, 并为进一步拓宽MOFs材料的应用范围提供了参考.

关键词: 介孔金属-有机骨架, 竞争性配体, 软模板, 固定化酶

Abstract:

Zeolitic imidazole frameworks materials-8 (ZIF-8), a member of the metal-organic framework (MOFs) series, have been extensively used as a host matrix for enzyme immobilization. However, the microporous structure and hydrophobicity, as well as the protonation of the precursor 2-methylimidazole (2-MeIm) of ZIF-8, remain considerable challenges in maintaining the activity of immobilized enzymes. Here, novel multivariate mesoporous MOFs (mMOFs) with regulatable hydrophilic/hydrophobic surfaces were designed by the multivariate competitive strategy and pore modification engineering. 3-Methyl-1H-1,2,4-triazole (3-MTZ) and 5-methyltetrazole (5-MTA) were employed to partially replace 2-MeIm. These were then combined with zinc sulfate heptahydrate (soft templates) to yield mMOFs in a methanol solution. As a proof-of-concept application, we used mMOFs as carriers for enzyme immobilization and investigated the properties of the immobilized enzymes. Benefiting from their mesoporous structure, hydrophilic surface, and improved microenvironment, multivariate mMOFs exhibit a strong ability to stabilize enzyme conformation and increase enzyme activity compared with traditional ZIF-8. Our study offers an avenue for the controllable preparation of well-designed MOF structures, which will further broaden the application opportunities of MOF materials for enzyme immobilization.

Key words: Mesoporous metal-organic frameworks, Competitive ligand, Soft template, Immobilized enzyme

冯玉晓, 马庆庆, 王子辰, 张群力, 赵利雪, 崔建东, 杜英杰, 贾士儒. 多元介孔MOFs：通过调节表面亲水/疏水性实现酶固定化的多功能平台[J]. 催化学报, 2024, 60: 386-398.

Yuxiao Feng, Qingqing Ma, Zichen Wang, Qunli Zhang, Lixue Zhao, Jiandong Cui, Yingjie Du, Shiru Jia. Multivariate mesoporous MOFs with regulatable hydrophilic/hydrophobic surfaces as a versatile platform for enzyme immobilization[J]. Chinese Journal of Catalysis, 2024, 60: 386-398.

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图/表 5

Fig. 1. (a) Schematic for the preparation and properties of ZIF-8, HZIF-8, and mMOFs. (b) Schematic for synthesis process of mMOFs and mMOFs/HRP.

Fig. 2. SEM images of ZIF-8 (Scale bar 0.2 μm) (a), mMOFs before removing the template (Scale bar 2 μm) (b), mMOFs (Scale bar 2 μm) (c), surface magnification of mMOFs (Scale bar 0.5 μm) (d). TEM images of ZIF-8 (Scale 0.2 μm) (e), mMOFs before removing the template (f), mMOFs (g), surface magnification of mMOFs (h). (i) N2 adsorption-desorption isotherms of ZIF-8 and the mMOFs. (j) Pore size distributions of ZIF-8 and the mMOFs. (k) FTIR spectra of ZIF-8 and mMOFs. (l) PXRD patterns of ZIF-8 and mMOFs.

Fig. 3. (a) Recovery activity of ZIF-8/HRP, HZIF-8/HRP, mMOFs/HRP, and mMOFs. (b) Immobilization efficiency of ZIF-8/HRP, HZIF-8/HRP, and mMOFs/HRP. (c) Zeta potentials of ZIF-8, HZIF-8, mMOFs, and HRP. (d) Short-term storability of HRP, ZIF-8/HRP, HZIF-8/HRP, and mMOFs/HRP. (e) CD spectra of HRP, ZIF-8/HRP, HZIF-8/HRP and mMOFs/HRP supernatants. (f) UV-vis full wavelength scanning absorption spectra of HRP, ZIF-8/HRP, HZIF-8/HRP and mMOFs/HRP supernatants. (g) pH of 2-MeIm, 3-MTZ, 5-MTA, ZIF-8, HZIF-8 and mMOFs in water. (h) CLSM images of mMOFs/HRP (Scale bar: 10 μm). (i) EDS mapping of mMOFs/HRP (Scale bar: 2 μm).

Fig. 4. (a) EDS mapping of mMOFs/GOxHRP (Scale bar: 2 μm). (b) Schematic of the three immobilization sequences. (c) CLSM images of mMOFs/GOxHRP (HRP labeled with RhB, GOx labeled with FITC, Scale bar: 10 μm).

Fig. 5. (a) Cascade reaction activity of ZIF-8/GOxHRP, HZIF-8/GOxHRP and mMOFs/GOxHRP. (b) UV-vis spectra of the mMOFs/GOxHRP after chromogenic reaction at glucose concentrations of 10-400 μmol/L. (c) Linearity between absorbance values and glucose concentration after mMOFs/GOxHRP detection of glucose. (d) Selectivity of mMOFs/GOxHRP for 50 μmol/L glucose in comparison to 500 μM lactose, maltose, mannose, galactose, and fructose.

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多元介孔MOFs：通过调节表面亲水/疏水性实现酶固定化的多功能平台

Multivariate mesoporous MOFs with regulatable hydrophilic/hydrophobic surfaces as a versatile platform for enzyme immobilization

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