Mechanism underlying the tolerance of lipase immobilized in porous organic frameworks in organic solvents.

Enzymes represent promising biocatalysts; however, their industrial application is often limited by instability under harsh conditions, particularly their low tolerance to organic solvents. Although immobilization within porous organic frameworks (POFs) offers a potential solution, systematic investigations remain limited. In this work, Aspergillus niger lipase (ANLP) was immobilized in three POFs (ZIF-8, COF-LZU1, and HOF-101), and the tolerance of these composites was evaluated in six common organic solvents, including n-hexane and dichloromethane. The underlying mechanisms governing solvent tolerance were further investigated. Free ANLP exhibited progressive aggregation with increasing solvent polarity, resulting in reduced activity, whereas immobilization within POFs prevented aggregation through spatial confinement. In addition, hydrophobic carriers promoted interfacial interactions with ANLP that induced opening of the catalytic "lid," thereby enhancing substrate accessibility and enzymatic activity. In contrast, polar solvents promoted lid closure and reduced activity. Hydrophobic interfaces, therefore, contribute to stabilizing the active conformation of ANLP and counteracting the lid-closing effects of polar solvents. Overall, the organic solvent tolerance of immobilized ANLP is governed by both the porosity and hydrophobicity of the POF host. This study provides systematic insight into enzyme-POF-solvent interactions and offers perspectives for the rational design of efficient enzyme catalytic systems tailored to specific industrial environments.