Engineering of Peptide Linkers for Heterogeneous Enzymatic Production of Chiral Amines Conference: International Conference Biomolecular Engineering ICBEYear: 2018Proceeding: ICBE Asia 2018: International Conference on Biomolecular EngineeringGroup: General SubmissionsSession: Session 6: Translational Biomolecular Engineering Time: Wednesday, January 10, 2018 - 3:00pm-3:25pm 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. Bommarius AS, Paye MF. Stabilizing biocatalysts. Chem Soc Rev Chem Soc Rev. 2013;42(42):6534-6565. doi:10.1039/c3cs60137d. Moll JR, Ruvinov SB, Pastan I, Vinson C. Designed heterodimerizing leucine zippers with a ranger of pIs and stabilities up to 10-15 M. Protein Sci. 2001;10(3):649-655. doi:10.1110/ps.39401. Park WM, Yee CM, Champion JA. Self-assembled hybrid supraparticles that proteolytically degrade tumor necrosis factor-a. J Mater Chem B. 2016;4(4):1633-1639. doi:10.1039/c5tb01647a. Park WM, Champion JA. Colloidal Assembly of Hierarchically Structured Porous Supraparticles from Flower-Shaped ProteinâInorganic Hybrid Nanoparticles. doi:10.1021/acsnano.6b01003. Bommarius BR, SchÃ¼rmann M, Bommarius AS. A novel chimeric amine dehydrogenase shows altered substrate specificity compared to its parent enzymes. Chem Commun. 2014;50(95):14953-14955. doi:10.1039/C4CC06527A.