Purification and characterization of a membrane-bound linoleic acid isomerase from Clostridium sporogenes

Clostridium sporogenes ATCC 25762 converted linoleic acid ( c9, c12, 18:2) to c9, t11 conjugated linoleic acid (CLA, 18:2). The linoleic acid isomerase was membrane-associated and was very unstable, especially after being solubilized by detergents. Isomerase extraction, solubilization and stability were significantly improved by optimizing buffer composition and pH, and by minimizing detergent and protein precipitation. The isomerase was purified by DEAE, chromatofocusing and size exclusion column chromatography, achieving an overall purification of 364-fold and a specific activity of 400 nmol min −1 mg −1 protein. The purified enzyme was a single polypeptide band on native PAGE with an estimated molecular weight of about 190 kDa and a single band of around 45 kDa on SDS-PAGE, suggesting the enzyme is a homotetramer. The optimum pH for isomerase activity was about 7.5. No external cofactors or energy sources were required for catalysis. The isomerase had a definite bias toward substrates containing cis double bonds at the c9 and c12 positions of C18 polyunsaturated fatty acids. A free carboxyl group is absolutely necessary for isomerization. The K m for linoleic acid was 12 μM. The enzyme was subjected to substrate inhibition at linoleic concentrations above 20 μM. Oleic acid, palmitoleic acid and some linoleic acid derivatives also inhibited the isomerase.