Abstract

Introduction: Enzymes are highly complex biomolecules with great biological and technological relevance. Laccases are among the most widely used enzymes in the industry , thanks to its copper (Cu2+)-mediated oxidoreductase activity. They catalyze the oxidation of a wide range of toxic phenolic compounds, which makes them suitable for diverse biotechnological applications. However, implementing these and other biocatalysts in the industry typically brings important challenges such as large-scale expression and purification of the recombinant proteins and meeting stability requirements in the intended application site. Thus, a main challenge in modern enzymology is the search for enzymes that are easier to produce and more resistant and flexible to different environmental conditions. In this work, we show how small peptides can produce intermolecular assemblies that are competent in simulating and imitating the catalytic properties of laccases, while overcoming their natural stability and chemical limitations. Using rational design, we generated two peptides that intercalated histidine with nonpolar residues, allowing the assembly to coordinate Cu+2, mimicking the active site of laccases. Materials and Methods: The activity was measured by the chromogenic reaction of 2.4 DP with 4-AAP (aminoantipyrine) at 510 nm, using a 96-well UV transparent plate. Results: Different histidine-containing peptides spontaneously assembled into amyloid-like structures that bind the amyloid-specific probe thioflavin T (Th-T). The assemblies were active against a canonical phenolic pollutant (2,4-Dichlorophenol), catalyzing its oxidation in a similar fashion as laccase. Peptides 1 and 2 showed the highest specific activities, averaging 3.23 μM min-1 mg-1 and 2.36 μM min-1 mg-1 , respectively, surpassing a model thermostable laccase (ID17) that exhibited 1.2 μM min-1 mg-1. The peptide assemblies were active in buffered solutions as well in plain tap water. The activity was retained even upon exposure to high temperature. Conclusion: Self-assembled peptides can exhibit oxidoreductase activity that can mimic laccase under different industrially relevant conditions. The results demonstrate that small peptides can be promising candidates in their assembled state as future minimalistic biocatalysts.