Graduation Year


Document Type




Degree Name

Doctor of Philosophy (Ph.D.)

Degree Granting Department


Major Professor

Shengqian Ma, Ph.D.

Co-Major Professor

Jianfeng Cai, Ph.D.

Committee Member

Li-June Ming, Ph.D.

Committee Member

Anna Pyayt, Ph.D.


Biocatalysis, Enzyme arrangement, Mesoporous matrices, Metal-Organic Frameworks, Structural basics


Enzyme immobilization in metal-organic frameworks (MOFs) as a promising strategy, is attracting the interest of scientists from different disciplines with the expansion of MOF’s development. Different from other traditional host materials, their unique strengths of high surface areas, large yet adjustable pore sizes, functionalizable pore walls, and diverse architectures make MOFs an ideal platform to investigate hosted enzymes, which is critical to the industrial and commercial process. In addition to the protective function of MOFs, the extensive roles of MOFs in the enzyme immobilization are being well-explored by making full use of their remarkable properties like well-defined structure, high porosity, and tunable functionality. Such development shifts the focus from the exploration of immobilization strategies towards functionalization. Meanwhile, this would undoubtedly contribute to a better understanding of enzymes in regard to the structural transformation after hosted in confinement environment, particularly to the orientation and conformation change as well as the interplay between enzyme and matrix MOFs.

Although many indirect methods have been used to investigate the adsorbed biofunctionalities for diverse applications, the understanding of their spatial arrangement in the pores of MOFs, is still preliminary, due to the difficulties in directly monitoring their conformation. Also, the structural flexibility change of immobilized protein in confined environment, is challenging to clarify, but crucial to understanding the existing properties and rationally tailoring the system. The confined environment provides the protective function to stabilize immobilized proteins under denaturation conditions, and optimizes enzymatic performance in many aspects. Molecular-level insights corresponding to via structural investigation is essential to rationalize the tunable performance via adjusting protein-matrix interactions and chemical environment, and understand the structural features influencing protein dynamics within solid matrix supports.

Herein, we introduced a technique using employed site-directed spin labeling in combination with Electron Paramagnetic Resonance spectroscopy (SDSL-EPR) to reveal the structural basics and flexibility of hosted protein in hierarchically isoreticular mesoporous Metal-Organic Frameworks (IRMOFs), and subsequently investigated the impact features on the freedom degree of immobilized protein. The isoreticular matrix MOFs possess variable building components, metal-containing struts (Al, Sc, Fe), and different tritopic linkers with extending length, leading to cage differences in the chemical environment and dimension in cage. Three IRMOFs with zeolite MTN topology, were selected as target matrices to load a model protein, recombinant T4 phage lysozyme (T4L). EPR results give a comprehensive elucidation of interactions between model the protein and internal cavities of MOF, alongside understanding the protein dynamics alternation under different confined environment. Importantly, this approach can be generalized to facilitate such investigations under hierarchical structure of environments.

In addition, we use in situ small-angle neutron scattering (SANS) to characterize deuterated green fluoresce protein (d-GFP) accommodated in mesoporous MOF-919. The empty MOF exhibits hierarchical structure and well-presented according to the SANS profiles. The use of d-GFP not only highlights the scattering contribution from the proteins, also minimizes the incoherent scattering background. The SANS data analysis via combining both model fitting and 3D reconstruction reveals that GFP molecules are spatially correlated in adjacent nanosized cavities of MOF-919 to form “superstructure” through adsorbate-adsorbate interactions across pore apertures. The formation of “superstructure” induced by the confinement is beneficial to stabilize the immobilized proteins to prevent protein leaching from the mesopores. Interestingly, the spatial arrangement of confined proteins depends on the loading concentration, which suggests the passive diffusion is the major driving force for the protein molecules into the MOF pores.

Besides the structural investigation of immobilized protein in confined space of those selected mesoporous host matrices, we also conducted studies on kinetic analysis of co-enzyme dependent AANAT after accommodating in PCN-333s and COF-OMe. The different metal features of PCN-333s (Al/Fe/Sc) influenced the enzymatic performance at different degree, but all matric materials including COF-OMe could provide protective function under elevated temperatures. Nevertheless, those investigation are still preliminary, but also opened up new possibilities to in-dept understand, and explode more novel design of such hybrid functional materials.