In Japan, there remained only poor instruments and reagents for research immediately after World War II. However, Dr. Hayaishi enthusiastically looked for novel microorganisms that could survive and grow in a simple medium supplemented with several amino acids. Under these adverse conditions, Dr. Hayaishi identified several microorganisms in the soil and started experiments to clarify the mechanisms of metabolism by using tryptophan that was a gift from his mentor, Professor Kotake, at Osaka Imperial University.

Firstly Dr. Hayaishi identified an enzyme that catalyzed the conversion of catechol to muconic acid by oxidative cleavage, and he named the enzyme “Pyrocatechase”. This was the first discovery of an enzyme that cleaved the benzene ring under aerobic conditions. Then he categorized and named such enzymes “oxygenase” by using the strategy of examining the incorporation of a tracer, heavy molecular oxygen, ¹⁸O₂ into the substrate on catalysis.

Almost all organisms have common systems for intake of oxygen into their metabolic pathways. With his colleagues, Dr. Hayaishi expanded the study of oxygenase phylogenetically. They successfully identified a number of oxygenases from animals, plants, and microorganisms, and determined the protein structures of some of them by crystallography. They also elucidated numerous metabolic pathways of physiological importance and demonstrated the ubiquitous presence of oxygenases. For example, they succeeded in demonstrating that the NAD, the well-known coenzyme of many dehydrogenases, could serve as an ADP-ribosyl donor in a unique type of covalent modification of proteins. Moreover, his findings were applied to the analysis of neurotransmitter and local hormones, namely prostaglandins.