In vitro Production of Hemin-Based Artificial Metalloenzymes

Developing enzyme alternatives is pivotal to improving and enabling new processes in biotechnology and industry. Artificial metalloenzymes (ArMs) are combinations of protein scaffolds with metal elements, such as metal nanoclusters or metal-containing molecules with specific catalytic properties, which can be customized. Here, we engineered an ArM based on the consensus tetratricopeptide repeat (CTPR) scaffold by introducing a unique histidine residue to coordinate the hemin cofactor. Our results show that this engineered system exhibits robust peroxidase-like catalytic activity driven by the hemin. The expression of the scaffold and subsequent coordination of hemin was achieved by recombinant expression in bulk and through in vitro transcription and translation systems in water-in-oil drops. The ability to synthesize this system in emulsio paves the way to improve its properties by means of droplet microfluidic screenings, facilitating the exploration of the protein combinatorial space to discover improved or novel catalytic activities.