Purification and characterisation of naphthalene oxygenase from Corynebacterium renale.

Naphthalene oxygenase has been purified 420-fold from naphthalene-adapted Corynebacterium renale. The purified enzyme was obtained in 32% yield and gave an apparent single band on polyacrylamide gel electrophoresis. It has a molecular weight of approximately 99,000 and contains two non-identical polypeptide chains of molecular weights 43,000 and 56,000. The enzyme requires molecular oxygen for its activity and gave a single products, cis-1,2-dihydroxy-1,2-dihydronaphthalene. The absorption spectrum of the enzyme protein shows a maximum at 278 nm and no absorption in the Soret region, indicating that it is non-heme protein. Absence of cytochrome P-450 was also confirmed when the protein, treated with CO, showed no absorption at 450 nm. The enzyme followed Michaelis-Menten kinetics with Km values of 2.9 mM and 1.42 mM for naphthalene and NADH respectively. It exhibited maximal activity at 30 degrees C and pH 6.4. Studies on the stoichiometry of the reaction showed that one molecule of oxygen is consumed for each molecule of NADH oxidised or product formed.