Beta-oxidation of fatty acid is the pathway for catabolism of saturated long chain fatty acids such as palmitic acid (C16) or stearic acid (C18) that occurs in mitochondria. In this process two-carbon fragments (as acetyl CoA) are successively removed from carboxyl end of fatty acyl CoA (therefore it is called b-oxidation) and as a result acetyl CoA, NADH and FADH2 are formed. β-oxidation of fatty acids has pre-steps and four steps in one round - step 1: Dehydrogenation (by enzyme acyl CoA dehydrogenase); step 2: Hydration (by enoyl CoA hydratase); step 3: Dehydrogenation (by 3-hydroxyacyl CoA dehydrogenase); step 4: Thiolytic cleavage (by beta-ketoacyl CoA thiolase). The β-oxidation pathway is cyclic. The shortened fatty acyl group is now ready for another round of beta-oxidation. After the fatty acyl CoA has been reduced to acetyl CoA, beta-oxidation is complete. In the result of one cycle of β-oxidation, outgoing fatty acid molecule loses one acetyl-CoA molecule and become shortens by 2 carbon atoms. Therefore, if outgoing fatty acid molecule has n carbon atoms, (n/2 - 1) cycles of beta-oxidation are nesessary to completely splitting of this fatty acid to acetyl-CoA. And this process produces (n / 2) acetyl-CoA molecules.
Oxidation of unsaturated fatty acid occurs in mitochondria and is accompanied by reaction of cis → trans isomerisation. The action of enoyl CoA isomerase is required to handle double bonds at odd-numbered carbons because beta-oxidation requires preexisting double bonds at even-numbered carbons. If there is a double bond at an odd-numbered carbon (e.g., 18:1Δ9), the action of enoyl CoA isomerase is required to move the naturally occurring cis-bond and convert it to the trans- bond used in beta-oxidation. The product, with a trans-double bond, is a substrate for enoyl CoA hydratase, the second enzyme of beta-oxidation. In case of polyunsaturated fatty acids, e.g linoleic acid that is 18:2(9,12), NADPH-dependent Dienoyl CoA Reductase is required in addition to isomerase.