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Magnetic Enzyme Metal Organic Framework Composites

Periodic Reporting for period 1 - MNEMONIC (Magnetic Enzyme Metal Organic Framework Composites)

Reporting period: 2017-11-01 to 2019-10-31

Catalysis accounts for about 90% of worldwide industrial processes, and 35% of the annual world's GDP. Catalysts may be homogeneous or heterogeneous, if dispersed in the same phase of the chemical reaction, or in another one, respectively. Enzymes, homogeneous highly selective biological catalysts, are very attractive to industry, especially pharmaceutical manufacturing. However, they work efficiently within specific ranges of parameters: pH, temperature, etc. Enzyme engineering modifies them to improve stability and activity in unfavorable conditions, e.g. with organic solvents or inhibitors. Another issue is recyclability, as enzymes are often lost after a cycle of reaction. For this reason, there is a fervent research upon their immobilization, especially using porous materials, for example silicate-based nanosystems. This action aims to tackle the previously mentioned issues of stability and recyclability by utilizing metal-organic frameworks (MOFs), a thriving class of porous materials developed from the 1990s. MOFs are made of metal ions and organic ligands, and thanks to the wide range of organic molecules available, they allow for extremely varied architectures. Thousands of MOFs have already been produced, with a plethora of applications especially in gas adsorption. The recently developed combination of MOFs with biomolecules, through a straightforward approach named biomimetic mineralization, produces MOF/enzyme composite biomaterials in a one-pot reaction, in water, at room temperature, in short time. This eliminates organic solvents and compatibility agents, commonly used in other methods. This project also enriched the combination of MOFs and enzymes with magnetic nanoparticles, removing filtration steps in favor of a simple magnetic collection of the catalyst. The interdisciplinary objectives of this action were to design, produce, and study novel ternary systems composed by one or more enzymes, magnetic nanoparticles and the MOFs. We chose a highly porous MOF called ZIF-8, with a surface area of about 1800 m2/g and small pores of about 1 nm in diameter, based on zinc nodes and 2-methylimidazole organic ligand, because of its simple topology, good biocompatibility, and easy synthesis. An initial phase of variables study and optimization of the synthetic conditions was aided by multiple characterization techniques (porosity, structure, size, shape, composition, enzyme loading). Afterwards, the obtained biocomposites were integrated in a customized fluidic device made by 3D printing, to test the catalytic performance of the encapsulated enzyme.
During the first six months of the action, a study assessed the most promising biomacromolecules to be inserted into ZIF-8. Enzymes are usually bigger than the pores, but the selected MOF can self-assemble around the biomolecules, easily integrating them in the framework. The choice of enzyme was influenced by the transport of substrates in and out the MOF pores, thus needing enzymes’ substrates smaller than 1 nm. Horseradish peroxidase (HRP) and glucose oxidase (GOX) were selected, as their substrates (pyrogallol and glucose) are small enough to pass through the pores. In the same period the stability of ZIF-8 to the assay environment was determined, finding out that phosphate-based buffers are not compatible with ZIF-8. However, we implemented a corrective action by using alternative buffers, without interfering with the enzymatic activity and maintaining the MOF structure unchanged. Also, magnetic nanoparticles were produced, based on iron oxide.
The following 12 months regarded the preparation of ternary magnetic enzyme biocomposites. Among all the possible parameters, the concentrations of magnetic particles and of enzymes were optimized to provide the best encapsulation efficiency (EE%) and catalytic activity. In this regard, EE%s above 80% were obtained increasing the magnetic nanoparticle content, indicating a synergistic effect. The HRP enzymatic activity drastically increased with the amount of magnetic nanoparticles, up to 4 times more than without particles. The recyclability of the system was also proven up to 10 reuses, although more efforts are needed to increase this aspect.
We also investigated two other aspects strictly related to the action: the possibility to generate MOFs using alternative pathways, especially when starting from ceramics or raw materials; and the integration of biomolecules other than enzymes with interesting applications. In the first case, MOFs from carbonate-based minerals were successfully prepared, by converting malachite and azurite into HKUST-1, or zinc oxide in ZIF-8. In the second case, promising results were reached using carboxylated dextrans and functional gene sequences, the latter internalized into model cells and expressed in the expected protein.
Fluidic devices were produced in the final six months of the action, in which the previously obtained ternary magnetic enzyme MOF biocomposite were integrated. The devices were prepared by filament 3D printing of sacrificial ABS templates, representing channels and connections. The templates were immersed into silicone elastomer which hardened upon thermal curing. The dissolution of the template left the empty channel, then connected to syringe pumps. A suspension of the magnetic porous biocatalyst was injected and kept in position by a commercial magnet. Further development was possible by 3D stereolithography printing of acrylate resin, providing more rigid devices with better resolution and narrow channels of 1 mm in diameter, along with a faster production process (5 hours vs. 3 days).
This action was disseminated in 8 peer-reviewed scientific articles, gaining 3 cover artworks, and communicated at 5 conferences and 3 internal workshops. Moreover, the results were also divulged on different media (TU Graz University newspaper, Twitter, etc.). The action also attracted students both from local and overseas educational institutions.
Industrial biocatalysts allow for the preparation of specialty chemicals and active pharmaceutical ingredients with high selectivity and yields. The impact of the use of enzymes in price abatement and manufacturing rate will increase with the need of more specific and cleaner production pathways. Enzymes need to match the synthetic conditions, and efforts in engineering their performance and resistance towards unfavorable process parameters may face prohibitive costs. Thus there is the need to protect already available enzymes with currently available methods. A unique aspect developed in this project was the combination of three concepts (catalysis, protection, and recyclability) in a single material composed by enzymes, MOFs, and magnetic nanoparticles. The smart repositionable biocatalysts obtained here may be easily scaled up and adopted in the current industrial technologies, with positive potential impacts on the production of chemicals used in everyday life, such as drugs and agricultural products, and also in their integration in micronized devices where enzymes can be important for sensing, such as in point-of-care health technology.
A synthesized magnetic porous biocatalyst used in flow chemistry