Controlling cis/trans isomerism of monounsaturated fatty acids via a recombinant cytochrome c-type cis/trans fatty acid isomerase

Abstract

Cytochrome c-type cis/trans fatty acid isomerase (CTI) has been proposed to control cis/trans isomerism of unsaturated fats in lipid-related food products. A gene encoding wildtype CTI from Pseudomonas putida KT2440 was introduced into the pET26b/pEC86 co-expression system, and the heme C cofactor was covalently bound into the expressed CTI protein through in vivo cytochrome c maturation. The recombinant CTI, purified from Escherichia coli BL21(DE3), catalyzed the cis/trans isomerization of three edible monounsaturated fatty acids. It exhibited strong substrate selectivity for palmitoleic acid (C16:1, cis-Δ9), reaching an 80.93 ± 1.78% conversion at reaction equilibrium. Notably, its promiscuity for other fatty acids (oleic acid: 29.21 ± 5.01% and cis-vaccenic acid: 51.21 ± 0.05%) was observed. Under the optimum reaction conditions (pH 7.5 and 15 °C), the kinetic parameters (Vmax, Km, and kcat) of CTI were derived as 0.035 mM min−1, 0.267 mM, and 0.141 sec−1, respectively, and the final catalytic efficiency (kcat/Km) was calculated as 5.26 × 102 M−1 s−1. Furthermore, structural properties of CTI were analyzed using deep learning-based protein structure prediction, suggesting the potential for specificity variability by altering loop dynamics and helix interactions surrounding the heme-binding motif. The following results would provide theoretical and practical information for CTI enzymes as novel promising industrial catalysts to control cis/trans isomerism of lipids in food products.