ENZYMATIC MICRO REACTORS FORMED BY LIQUID-LIQUID PHASE SEPARATION OF BIOLOGIAL MACROMOLECULES

The primary aim of this study was to produce biocondensates formed via liquid-liquid phase separation of a chimera protein derived from the P450 monooxygenase. The chimera protein is constituted by adding two low-complexity domains (LCDs) to the terminal regions of the P450 protein. An in-depth investigation was conducted utilizing bioinformatics tools to analyze the secondary structures of both the unmodified (wild-type) version of the protein and the experimentally obtained chimera protein. Employing the AlphaFold 2 artificial intelligence tool, the three-dimensional structure of the chimera protein and its associated protein-bound LCDs were elucidated. Furthermore, analysis of circular dichroism (CD) spectra indicates a prevalence of α-helical secondary structures within the protein's architecture. Additionally, differential scanning calorimetry (DSC) resulted in an unfolding temperature of 43ºC, which represents the maximum thermal stability of the protein. Our findings unequivocally demonstrated the viability of constructing an enzymatic microreactor utilizing the P450 chimera version. The capacity to undergo liquid-liquid phase separation (LLPS) not only confirms the structural integrity of the chimera but also opens up a diverse array of potential applications, including but not limited to biosensors and tissue engineering.

This work was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES).