NANOSTRUCTURED PLATFORMS FOR METALLOENZYME BIOCATALYSIS IN OLEFIN PRODUCTION

The industrial production of long-chain olefins relies predominantly on the steam cracking of petrochemical feedstocks, an energy-intensive process associated with high greenhouse gas emissions and significant environmental impacts. Aiming at a transition to a lower-impact economy, fatty acid P450 decarboxylases (CYP152 family), such as the metalloenzyme OleTPRN (Rothia nassimurium), emerge as promising alternatives, as they convert renewable fatty acids into terminal olefins using only H2O2as a cosubstrate. However, the low aqueous solubility of fatty substrates and the irreversible inactivation of the enzyme by H2O2 itself limit their application. To overcome these challenges, this doctoral project proposes the development of a novel enzymatic microreactor through the encapsulation of OleTPRN in cubosomes (bicontinuous lipid nanoparticles). The central hypothesis is that the nanostructure of cubosomes optimizes the enzyme's activity, stability, and selectivity by providing hydrophobic domains to solubilize the substrate and protective aqueous channels that regulate H2O2 diffusion. The methodology will encompass the expression and purification of the enzyme at CNPEM, followed by characterization using CD, DLS, and SEC-MALS. Cubosomes will be prepared at IFUSP (bottom-up method, with phytantriol and Poloxamer 407) and analyzed by SAXS. The enzyme-nanoparticle complex will be evaluated for encapsulation efficiency and structural integrity using biophysical techniques and Cryo-TEM. Catalytic assays quantified by gas chromatography will optimize reaction conditions, system reuse, and validation against crude vegetable oils. Thus, it is expected to consolidate a robust nanobiotechnological platform for the green synthesis of bio hydrocarbons.