The integration of enzymes with solid materials is crucial for promoting their industrialization. Understanding the enzyme behavior upon association within a confined space, though of fundamental importance for biocomposite development, remains a persistent, unresolved challenge. Here, we present a comprehensive elucidation of the spatial environment’s impact on hosted enzymes’ degree of freedom and, consequently, their accompanying reactivity. Site-directed spin labeling in combination with electron paramagnetic resonance spectroscopy allows the direct detection of host-guest interactions at atomic resolution, while the tailorable synthesis of covalent organic frameworks (COFs) enables an evaluation of factors affecting such interactions. Specifically, lysozyme is found to be more constrained and less active along with increasing hydrophilicity of the COFs. These results support the establishment of a connection between the hydrophilicity of the spatial environment and the resulting biocomposites’ reactivity, enabling the prediction of the performance of unknown biocomposites. This study provides a unique insight into the mechanistic pathways underpinning biocatalysis.

酶与固体材料的整合对于促进其工业化至关重要。尽管在密闭空间内缔合后了解酶的行为，尽管对生物复合材料的发展具有根本重要性，但仍然是一个持续存在的，尚未解决的挑战。在这里，我们对空间环境对宿主酶的自由度的影响以及由此产生的伴随反应性的影响进行了全面的阐述。定点自旋标记结合电子顺磁共振波谱可在原子分辨率下直接检测主体与客体的相互作用，而可定制的共价有机骨架（COF）合成则可评估影响此类相互作用的因素。具体来说，发现溶菌酶受到更多的限制，活性降低，同时COFs的亲水性增加。这些结果支持在空间环境的亲水性与所得生物复合材料的反应性之间建立联系，从而能够预测未知生物复合材料的性能。这项研究为支撑生物催化的机制途径提供了独特的见解。