Acyl-CoA oxidase plays a critical role in the peroxisomal beta-oxidation pathway, where it catalyzes the first step in the breakdown of fatty acids. This enzyme is essential for converting acyl-CoA into trans-2-enoyl-CoA, generating hydrogen peroxide as a byproduct. Its primary application lies in metabolic research, particularly in studying lipid metabolism and disorders related to fatty acid oxidation, such as peroxisomal biogenesis disorders. Additionally, it is used in biochemical assays to measure fatty acid oxidation rates and to investigate the impact of genetic mutations on metabolic pathways. In biotechnology, acyl-CoA oxidase is employed in the development of diagnostic tools for detecting metabolic diseases and in the production of biofuels, where understanding fatty acid degradation is crucial for optimizing microbial lipid metabolism.