Novel bilirubin oxidase, its production and use

A bilirubin oxidase M-1 having the following physicochemical properties:

• (1) Action:
• Has an action to oxidize bilirubin in the presence of oxygen far into an almost colorless substance through biliverdin and then a pale violet substance, but forms no hydrogen peroxide;
• (2) Substrate specificity:
• Acts on bilirubin and to some degree on biliverdin as well as on phenol, catechol and hydroquinone;
• (3) Optimum pH and pH stability:
• Has an optimum pH in the vicinity of 5.5, and is stable between pH 5 and pH 9 for 60 minutes treatment at 37.degree. C.
• (4) Optimum temperature and thermal stability:
• Has an optimum temperature in the vicinity of 50.degree. C. and is stable up 55.degree. C. for 10 minutes treatment at a pH of 7.0;
• (5) Molecular weight:
• About 83,000 (gel filtration method);
• (6) Isoelectric point:
• 3.92.+-.0.05;
• (7) Interfering agent:
• Inhibited by sodium azide, L-ascorbic acid, dithiothreitol and potassium cyanide;
• (8) Visible absorption spectrum:
• Has an absorption maximum in the vicinity of 610 nm;
• (9) Sugar content:
• Contains about 4.5% of sugar;
• (10) Copper content:
• Contains 4 moles of copper per mole.