In the 10 l packed-bed bioreactor, P. cinnabarinus used glucose at a constant rate. The ammonia content of the medium was exhausted by day 4. Laccase activity was first detected on day 4 and had reached 0.28 U/ml by day 10 when the culture broth was harvested for laccase purification.
The isolation procedure follows that of Schliephake et al. (2000). Laccase was purified from 7.91 of culture fluid in two ultrafiltration steps followed by concentration, dialysis, and column chromatography on Sephadex G 75. Fractions absorbing at 614 nm were analyzed for laccases activity, pooled, dialyzed against 20 mM histidine buffer pH 6, and again concentrated. The concentrate was chromatographed on a 5/5 Mono Q anion exchange column with a sodium chloride gradient of 0-0.5 M over 50 min at 1 ml/min. Fractions absorbing at 614 nm were pooled and stored at — 85°C in the presence of a protease inhibitor mixture. A 45-fold increase in specific activity was obtained on purification. Capillary electrophoresis estimated purity at greater than 95% with minor peaks (areas 0.079 and 4.596%) at longer elution times.
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