The white-rot fungus Pycnoporus sp. SYBC-L1 produced large amount of laccase in submerged fermentation. Two laccase isozymes (Lad and Lacll) were purified using (NH4)(2)SO4 fractionation, DEAE-cellulose and Sephadex G-100 column chromatography. The molecular masses of Lad l and Lac II were 55.89 and 63.07 kDa, respectively by SDS-PAGE. Both the laccases showed acidic pH optima and high catalytic activities at low temperature for oxidations of 2,6-dimethoxyphenol (DMP), 2,2-azino-bis-(3-ethylbenzothiazoline-6-sulfonate acid) (ABTS), syringaldazine and guaiacol. Lad l and Lacll were not only with high cold adaptation, but also fairly stable at high temperature. The half-lives of Lad l at 50,60 and 70 degrees C were 69.31, 2.58 and 0.13 h, respectively, whereas Lacll was more stable with half-lives of 256.72, 21.00 and 2.06 Is respectively. The best substrates for the enzymes were both found to be ABTS, in which the K-m values of Lacl and Lacll were 0.0166 and 0.0435 mM and the catalytic efficiencies were 19640.36 and 31172.64S(-1) mM(-1), respectively. EDTA and low concentration of Cu2+ and Mn2+ almost had non-inhibitions on their activities. Lacll with syringaldehyde efficiently decolorized Remazol Brilliant Blue R. The high thermostabilities as well as cold adapted properties made Pycnoporus sp. SYBC-L1 laccases to be excellent candidates in harsh industry. (C) 2010 Elsevier Ltd. All rights reserved.