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er was replaced with 0.1 m K phosphate buffer (KPB), pH 7.0. Protein in the eluate was monitored by absorbance at 280nm. Pycnoporus coccineus IFO-4923 was cultured in the mediumof Fahraeus and Reinhammar3)on a reciprocal shaker (120 rev./min) at 25°C for 2 weeks. One active band of P-laccase was shown by the PAGE6)of this culture mediumfrom which the mycelia were removed by filtration (Fig. 1A). The culture medium (980ml) was concentrated to 1/10 volume by ultra filtration (exclusion Mr, lOkDa) and put on a column of DEAE-cellulose (4.5x40cm) equilibrated with 50mMKPB, pH 6.0. Protein was eluted stepwise with 50mM(2 1) and 100mM (1 1) ofKPB (pH 6.0), successively. P-laccase was eluted with 100mM KPB.

Purification and properties of laccase excreted by Pycnoporus coccineus.