(727d) Optimizing a Porous Calcium-Phosphate Supraparticle for Enzyme Immobilization

Caparco, A. A. - Presenter, Georgia Institute of Technology
Bommarius, A., Georgia Institute of Technology
Immobilizing enzymes can significantly improve their process lifetime, stability, and recoverability.[1] By shifting the scale of the immobilization materials to the nanometer and micron range, it is possible to overcome the mass transfer resistances that can plague the efficiency of enzyme immobilization in porous, millimeter-scale particles, thus improving the prospective process economics. To further enhance their efficacy, it is possible to take advantage of affinity binding domains, such that the enzyme interacts with the material in a controlled fashion, with limited effects on its structure or function.[2] Using a heterodimerizing leucine zipper pair, ZE and ZR, it is possible to take advantage of the high fidelity and high affinity binding of the leucine zippers. An enzyme can be tagged with ZE into a fusion protein, while a protein-based immobilization material can be synthesized using ZR. This is the basis for our work, in which hierarchically structured porous supraparticles made of calcium phosphate and leucine-zipper fusion protein serve as a modular support for enzyme immobilization. In our system, we co-immobilize an amine dehydrogenase (cFL1AmDH) and a formate dehydrogenase (FDH) in the supraparticles for cofactor regeneration and improved productivity of the amine product.[3–5]

We sought to optimize the synthesis of the supraparticles to maximize enzyme loading, minimize polydispersity of the size distribution, and show immobilized enzyme activity. Supraparticles were synthesized with several calcium concentrations, rotational speeds, and fluid viscosities. After synthesis, these samples were mixed with eGFP- ZE, a fluorescent protein, and imaged using fluorescence microscopy. Image segmentation and analysis were then used to quantify the number density and size distribution of supraparticles loaded with protein. The samples’ loading capacities were determined using isothermal titration calorimetry. After selecting the supraparticles with the most desirable characteristics, they were loaded with AmDH- ZE and FDH- ZE. The conversion of ketone to amine in the biocatalyst was determined using HPLC, and the optimal loading ratio of the two enzymes was determined to maximize conversion with respect to time. Immobilization of the enzymes in the supraparticles did not significantly reduce their specific activity. These results indicate supraparticles can immobilize multiple enzymes with high activity, laying the framework for their use in industrial enzyme cascades.

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