Metal–organic frameworks (MOFs) are advanced platforms for enzyme immobilization. Enzymes can be entrapped via either diffusion (into pre-formed MOFs) or co-crystallization. Enzyme co-crystallization with specific metals/ligands in the aqueous phase, also known as biomineralization, minimizes the enzyme loss compared to organic phase co-crystallization, removes the size limitation on enzymes and substrates, and can potentially broaden the application of enzyme@MOF composites. However, not all enzymes are stable/functional in the presence of excess metal ions and/or ligands currently available for co-crystallization. Furthermore, most current biomineralization-based MOFs have limited (acid) pH stability, making it necessary to explore other metal–ligand combinations that can also immobilize enzymes. Here, we report our discovery on the combination of five metal ions and two ligands that can form biocomposites with two model enzymes differing in size and hydrophobicity in the aqueous phase under ambient conditions. Surprisingly, most of the formed composites are single- or multiphase crystals, even though the reaction phase is aqueous, with the rest as amorphous powders. All 20 enzyme@MOF composites showed good to excellent reusability and were stable under weakly acidic pH values. The stability under weakly basic conditions depended upon the selection of enzyme and metal–ligand combinations, yet for both enzymes, 3–4 MOFs offered decent stability under basic conditions. This work initiates the expansion of the current “library” of metal–ligand selection for encapsulating/biomineralizing large enzymes/enzyme clusters, leading to customized encapsulation of enzymes according to enzyme stability, functionality, and optimal pH.

Proteases are involved in essential biological functions in nature and have become drug targets recently. In spite of the promising progress, two challenges, (i) the intrinsic instability and (ii) the difficulty in monitoring the catalytic process in real time, still hinder the further understanding and engineering of protease functionalities. These challenges are caused by the lack of proper materials/approaches to stabilize proteases and monitor proteolytic products (truncated polypeptides) in real time in a highly heterogeneous reaction mixture. This work combines metal−organic materials (MOMs), site-directed spin labeling-electron paramagnetic resonance (SDSL-EPR) spectroscopy, and mass spectrometry (MS) to overcome both barriers. A model protease, trypsin, which cleaves the peptide bonds at lysine or arginine residues, was immobilized on a Ca-MOM via aqueous-phase, one-pot cocrystallization, which allows for trypsin protection and ease of separation from its proteolytic products. Time-resolved EPR and MS were employed to monitor the populations, rotational motion, and sequences of the cleaved peptide truncations of a model protein substrate as the reaction proceeded. Our data suggest a significant (at least 5−10 times) enhancement in the catalytic efficiency (k cat /k m ) of trypsin@Ca-MOM and excellent reusability as compared to free trypsin in solution. Surprisingly, entrapping trypsin in Ca-MOMs results in cleavage site/region selectivity against the protein substrate, as compared to the near nonselective cleavage of all lysine and arginine residues of the substrate in solution. Remarkably, immobilizing trypsin allows for the separation and, thus, MS study on the sequences of truncated peptides in real time, leading to a time-resolved "movie" of trypsin proteolysis. This work demonstrates the use of MOMs and cocrystallization to enhance the selectivity, catalytic efficiency, and stability of trypsin, suggesting the possibility of tuning the catalytic performance of a general protease using MOMs.

Inquiry-based laboratories were implemented into a General Chemistry Laboratory sequence, and the impact of these exercises on students‘ experimental design skills was assessed using a four-part assessment developed for this study. This assessment contained a multiple-choice section, a section asking students to explain their reasoning behind a subset of the multiple-choice answers, an adapted form of the Experimental Design Ability Test, and a section asking about students’ perceptions of themselves. For two years, pretests were administered before the students‘ first lab exercise, and post-tests were administered at the end of the year of General Chemistry Lab for cohorts of students enrolled in a Revised course and cohorts of students in an unchanged (Traditional) course. Overall, students in both the Traditional and Revised curricula experienced gains in learning outcomes as measured by comparing pre- and post-test scores on the first three sections of the assessment. Importantly, these gains were slightly higher for the Revised cohort on the multiple-choice section in Year 2 of the study. The quality of explanations on the second section of the assessment was also higher for the Revised cohort compared to students in the Traditional course. No significant differences were observed in average Experimental Design Ability Test performance. Student perceptions of confidence in their experimental design ability and their ability to conduct experiments were slightly higher for students who had completed the Revised course when compared to the Traditional course. In total, these modest improvements to students’ experimental design abilities reflect a positive trend that supports implementation of inquiry-based laboratory instruction.

Confining enzymes in well-shaped MOF compartments is a promising approach to mimic the cellular environment of enzymes and determine enzyme structure-function relationship therein. Under the cellular crowding, however, enzymes can...

Aqueous-phase co-crystallization (also known as biomimetic mineralization or biomineralization) is a unique way to encapsulate large enzymes, enzyme clusters, and enzymes with large substrates in metal–organic frameworks (MOFs), broadening the application of MOFs as enzyme carriers. The crystallinity of resultant enzyme@MOF biocomposites, however, can be low, raising a concern about how MOF crystal packing quality affects enzyme performance upon encapsulation. The challenges to overcome this concern are (1) the limited database of enzyme performance upon biomineralization in different aqueous MOFs and (2) the difficulty in probing enzyme restriction and motion in the resultant MOF scaffolds, which are related to the local crystal packing quality/density, under the interference of the MOF backgrounds. We have discovered several new aqueous MOFs for enzyme biomineralization with varied crystallinity [Expanding the Library of Metal–Organic Frameworks (MOFs) for Enzyme BiomineralizationJordahlD.ArmstrongZ.LiQ.GaoR.LiuW.JohnsonK.BrownW.ScheiwillerA.FengL.UgrinovA.MaoH.ChenB.QuadirM.PanY.LiH.YangZ. Jordahl, D. Armstrong, Z. Li, Q. Gao, R. Liu, W. Johnson, K. Brown, W. Scheiwiller, A. Feng, L. Ugrinov, A. Mao, H. Chen, B. Quadir, M. Pan, Y. Li, H. Yang, Z. ACS Appl. Mater. Interfaces2022145161951629]. Here, we address the second challenge by probing enzyme dynamics/restriction in these MOFs at the residue level via site-directed spin labeling (SDSL)–electron paramagnetic resonance (EPR) spectroscopy, a unique approach to determine protein backbone motions regardless of the background complexity. We encapsulated a model large-substrate enzyme, lysozyme, in eight newly discovered MOFs, which possess various degrees of crystallization, via aqueous-phase co-crystallization. Through the EPR study and simulations, we found rough connections between (a) enzyme mobility/dynamics and MOF crystal properties (packing quality and density) and (b) enzyme areas exposed above each MOF and their catalytic performance. This work suggests that protein SDSL and EPR can serve as an indicator of MOF crystal packing quality/density when biomineralized in MOFs. The method can be generalized to probing the dynamics of other enzymes on other solid surfaces/interfaces and guide the rational design of solid platforms (ca. MOF...