Combining directed enzyme evolution and peptide-based nanostructures to increase enzyme`s stability for microbial signal disruption and pollutants decontamination

One of the main challenges in moving towards broader use of enzymes is increasing their production, stability, formulation, and shelf life while maintaining high activity. One option is to improve these characteristics using directed enzyme evolution by randomly mutating genes and screening populations in an iterative process, to select the best performing variants. In doing so, we increased temperature resistance (T50, the temperature at which 50% of activity is retained) by 8◦C of a bacterial lactonase that degrades bacterial quorum sensing molecules and fungal secondary metabolites. An additional approach to stabilizing proteins and extending their durability is encapsulation in nanostructures. Specifically, the low-molecular-weight peptide can undergo spontaneous self-assembly into different structures such as fibrils, tubes, and spheres. We have used an N-terminus-protected peptide tertbutoxycarbonyl-diphenylalanine (BocFF) that can self-assemble into either spheres or tubes. We tested the nanostructured co-assembly with enzymes exhibiting different functionalities. The first is the evolved lactonase that, by encapsulation, its shelf life was extended for more than five weeks, and then we tested its ability to inhibit infection in planta. The second enzyme is a bacterial phosphotriesterase, which its encapsulation within Boc-FF fibrils increased enzyme durability up to 130 days. Moreover, a natural and environmentally friendly one-pot system, including encapsulated enzyme and bacterial culture, was developed to degrade paraoxon, an organophosphate-based pesticide. The combination of directed enzyme evolution and peptide nanostructure encapsulation significantly improved the thermal resistance and durability of the enzymes, respectively, increasing the potential use of enzymes in agriculture and health.