Purification and characterization of two thermostable laccases with high cold adapted characteristics from Pycnoporus sp. SYBC-L1

Abstract

The white-rot fungus Pycnoporus sp. SYBC-L1 produced large amount of laccase in submerged fermentation. Two laccase isozymes (LacI and LacII) were purified using (NH 4) 2SO 4 fractionation, DEAE-cellulose and Sephadex G-100 column chromatography. The molecular masses of LacI 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. LacI and LacII were not only with high cold adaptation, but also fairly stable at high temperature. The half-lives of LacI at 50, 60 and 70 °C were 69.31, 2.58 and 0.13 h, respectively, whereas LacII was more stable with half-lives of 256.72, 21.00 and 2.06 h respectively. The best substrates for the enzymes were both found to be ABTS, in which the K m values of LacI and LacII were 0.0166 and 0.0435 mM and the catalytic efficiencies were 19640.36 and 31172.64 S −1 mM −1, respectively. EDTA and low concentration of Cu 2+ and Mn 2+ almost had non-inhibitions on their activities. LacII 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.