Engineering of Peptide Linkers for Heterogeneous Enzymatic Production of Chiral Amines

Authors: 
Caparco, A. A., Georgia Institute of Technology
Bommarius, A. S., Georgia Institute of Technology
Champion, J. A., Georgia Institute of Technology
Immobilizing enzymes is known to improve their stability and process lifetime.1Working with nano- and microscale immobilization matrices, it is possible to overcome mass transfer resistance that is often associated with macroscopic immobilization materials. The leucine zipper pair, ZE and ZR, allows for high fidelity and high affinity heterodimerization, and may be used to create an affinity immobilization system.2,3 In our work, enzymes are recombinantly fused to ZE with an amino acid linker to provide adequate spacing, while a protein-based calcium-phosphate particle is constructed with ZR. The hierarchically structured porous calcium phosphate supraparticles serve as a modular support for enzyme immobilization.4

A glutamate leucine zipper (ZE) was added to both a chimeric amine dehydrogenase (AmDH)5 and formate dehydrogenase from C. boidinii (cbFDH) using a library of amino acid linkers varying in length, proline content, and terminal orientation. The linker candidate which retained the highest activity for both binding and catalytic activity was identified for each enzyme. Only one linker construct for each enzyme exhibited activity similar to the unfused enzyme, and the preferred linker for each enzyme varied significantly. This highlights the necessity to create a variety of linkers when designing a new fusion protein to find the composition with the highest activity. The enzymes were immobilized in supraparticles through ZE/ZR dimerization, and retained greater than 80% of the soluble activity.

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