Abstract:

An electrophoretically pure laccase from the fermentation broth of Pleurotus ostreatus heterosis-2 was obtained
through ammonium sulfate fractionation, DEAE-Sepharose fast flow chromatography and Superdex-200 prep grade
chromatography. The results indicated that the specific activity of the purified enzyme reached 115 U/mg. The relative
molecular weight of the laccase was approximately 244.0 kD, with a subunit molecular mass of roughly 85.6 kD. The
enzymatic properties showed that the optimum pH and temperature for the laccase were 5.0 and 55 ℃, respectively. The
enzyme was stabled at pH 6.0–8.0 and 40–55 ℃, and its apparent Km and vmax were 2.1 mmol/L and 0.117 μmol/(min·L),
respectively. Fe2+ and ascorbic acid could completely inactivate the laccase, whereas the enzyme activity was slightly
affected by EDTA, Ag+, Mg2+ and Li+. Cu2+ had little activating effect on laccase activity. The enzyme activity of laccase
could be activated by urea, ethanol, and isopropanol, and inhibited by oxalic acid, methanol, n-butanol, K+, Ca2+, Ba2+,
Zn2+, Cd2+, Pb2+, Mn2+, and Co2+.

Key words: Pleurotus ostreatus heterosis-2, laccase, isolation and purification, enzymatic properties