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Bilirubin oxidase (BOX; EC1.3.3.5) catalyzes the oxidation of bilirubin to biliverdin. It is used to measure the concentration of bilirubin in the serum to diagnose the degree of jaundice. It is also useful to remove bilirubin for the exact measurement of many clinical markers. Filamentous fungus Myrothecium verrucaria (Murao and Tanaka, 1982) and many basidiomycetes (Hiromi et al., 1992;Matsui et al., 1984;Uwajima and Hamamatsu, 1984) have been known as bilirubin oxidase producing fungi. Recently, BOX was purified and also characterized from Penicillium janthinellum (Seki et al., 1996). From Myrothecium, a BOX gene was cloned and expressed in yeast (Koikeda et al., 1993) and in Aspergillus (Xu et al., 1996).Enzyme stable during a long-term storage in solution is generally required for a commercial diagnostic enzyme. We found that BOX from the Pleurotus ostreatus strain Shinshu was more stable than the other previously reported BOXs from other fungi during storage in solution. Since P. ostreatus strain Shinshu produced a small amount of BOX (1 U/ml in 7-day culture), we cloned the cDNA of BOX from P. ostreatus strain Shinshu and tried to express it in Aspergillus sojae.P. ostreatus strain Shinshu was aerobically cultured at 25°C in the BOX production medium (6.0% soluble starch, 0.5% yeast extract, 0.1% sodium citrate, 0.05% MgSO 4 · 7H 2 O, 0.0005% CuSO 4 · 5H 2 O, and 0.008% CaCl 2 ) for 5 days. After centrifugation, the supernatant was adjusted to pH 9.2 and applied to a DEAE-cellulose column equilibrated with 0.01 M carbonic buffer (pH 9.2). The enzyme was eluted by using a linear gradient of 0 M to 0.6 M ammonium sulfate. The fractions containing the bilirubin oxidase activities were concentrated and dialyzed against 0.01 M carbonic buffer (pH 9.2). The concentrated fraction was applied to a Sephadex G-75 column equilibrated with 0.01 M carbonic buffer (pH 9.2). Purified enzyme was obtained by concentrating and dialyzing the peak fractions. The molecular weight of the purified enzyme was 60,000 by SDS-PAGE (data not shown). The standard assay of BOX activity was performed as follows. A standard assay solution was prepared by dissolving 5 mg of crystallized bilirubin in 2 ml of 0.2 M NaOH and diluted to 200 ml by 0.1 M phosphate-buffer (pH 7.5). The reaction was performed at 37°C by adding 0.5 ml of enzyme solution to 2 ml of standard assay solution. The decrease in absorbance at 440 nm was measured. One unit is defined as the amount of enzyme that oxidizes 1 mmol of bilirubin per min (the millimolar extinction coefficient of bilirubin under this assay condition is 40.93).N-terminal amino acid sequence of the purified enzyme was AIGPTGNMYIVNED. The purified BOX stored in the ammonium sulfate solution for a long period was applied to SDS-PAGE after removing the ammonium sulfate by ultrafiltration. We could not observe the band at the position of BOX, 60,000. Instead, we found a few bands at lower molecular weights (Fig. 1A). Long-term storage, the removal of ammonium