Zhang, X.-J.; Wang, X.-W.; Sun, J.; Su, C.; Yang, S.; Yang, S.;* Zhang, W.-B.* Synergistic Enhancement of Enzyme Performance and Resilience via Orthogonal Peptide-Protein Chemistry Enabled Multilayer Construction. Biomacromolecules 2018, 19 , 2700–2707.

Abstract: Protein immobilization is critical to utilize their unique functions in diverse applications. Herein, we report that orthogonal peptide–protein chemistry enabled multilayer construction can facilitate the incorporation of various folded structural domains, including calmodulin in different states, affibody, and dihydrofolate reductase (DHFR). An extended conformation is found to be the most advantageous for steady film growth. The resulting protein thin films exhibit sensitive and selective responsive behaviors to biosignals, such as Ca²⁺, trifluoperazine, and nicotinamide adenine dinucleotide phosphate (NADPH), and fully maintain the catalytic activity of DHFR. The approach is applicable to different substrates such as hydrophobic gold and hydrophilic silica microparticles. The DHFR enzyme can be immobilized onto silica microparticles with tunable amounts. The multilayer setup exhibits a synergistic enhancement of DHFR activity with increasing numbers of bilayers and also makes the embedded DHFR more resilient to lyophilization. Therefore, this is a convenient and versatile method for protein immobilization with potential benefits of synergistic enhancement in enzyme performance and resilience.