BIO-007: Recombinant Cytochrome c-type cis/trans Fatty Acid Isomerase from Pseudomonas putida KT2440 and its Catalytic Activity

In the periplasm of Pseudomonas putida strain, there is a cytochrome c-type cis/trans fatty acid isomerase (CTI) containing a heme-binding motif (CXXCH). This enzyme can directly engage in the cis/trans isomerism of unsaturated fatty acids. In this study, a CTI gene of P. putida KT2440 was cloned into the pET26b(+)/pEC86 co-expression system and heterologously expressed in the Escherichia coli system for mass production. The CTI gene with overhangs (2,274 bp) was amplified from the gDNA of P. putida KT2440, and it was in-fusion cloned into a pET26b(+) vector in frame with an N-terminal pelB signal peptide and a C-terminal polyhistidine fusion tag. The cloned pET26b(+) plasmid was co-transformed into E. coli BL21(DE3) with a pEC86 vector for the cytochrome c maturation, and the expression of CTI was induced by adding IPTG and 5-aminolevulinic acid. The pure CTI fractions were obtained at 227.14–277.80 mM imidazole using Ni-NTA affinity chromatography, and a single CTI band (approximately 86 kDa) was confirmed by SDS-PAGE analysis. The CTI activity in the buffer solution was evaluated against three cis-MUFAs (oleic acid, cis-vaccenic acid, and palmitoleic acid), and all their trans-MUFAs (elaidic acid, trans-vaccenic acid, and palmitelaidic acid) were detected after the reaction. These results suggest that the mass production of CTI could be achieved by molecular cloning, and by extension, CTI will be applied to the enzymatic elimination of trans fats in lipid-related foods in the future.