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, wh...

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Bibliographic Details
Published in:Chemistry : a European journal 2024-02, Vol.30 (11), p.e202303254-n/a
Main Authors: López‐Domene, Rocío, Manteca, Aitor, Rodriguez‐Abetxuko, Andoni, Beloqui, Ana, Cortajarena, Aitziber L.
Format: Article
Language:English
Subjects:
Biotechnology
Catalytic activity
Combinatorial analysis
Hemin
Histidine
Metalloenzymes
Microfluidics
Nanoclusters
Organic chemistry
Peroxidase
Protein Design
Proteins
Repeat Proteins
Scaffolds
Synthetic Biology
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Summary: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. The development of artificial metalloenzymes (ArMs) is crucial to improve various industrial and biotechnological processes. In this study, we successfully designed a hemin‐based ArM with peroxidase activity. Our results reveal improved properties compared to the activity of free hemin. Moreover, we also achieved the fabrication of this ArM in vitro and in emulsio.
ISSN:0947-6539
1521-3765
DOI:10.1002/chem.202303254