Isolation and partial characterization of laccase from a thermophilic composted municipal solid waste

Abstract

Composted municipal solid waste (CMSW) samples were examined for the presence of laccase in an attempt to further study the properties of this enzyme. Laccase was extracted from CMSW using 50 mM phosphate buffer, pH 6.0 (1:5 w/v compost:buffer) and was partially purified by ammonium-sulfate precipitation (80–90% saturation) followed by DEAE-cellulose chromatography. Of the 18 CMSW samples tested, three exhibited noticeable laccase activity. These samples (designated A, B and C) were taken from the compost at the thermophilic stage of composting. The high oxidation rate of syringaldazine and the absence of activity towards 3,4-dihydroxyphenylalanine and of increased activity in the presence of H 2O 2 indicate that the studied enzymes were laccases. Inhibition studies revealed that laccases B and C show high tolerance to sodium azide, EDTA and thioglycolic acid as compared to extracellular fungal laccases. The optimal temperature range for the activities of all three purified laccases was between 50 and 60°C and the enzymes were active over a wide range of pH (6.0 to 8.0), with a peak at pH 6.0. All three laccases exhibited thermal stability following a 15-min pre-incubation at temperatures of up to 50°C. After pre-incubation at 70°C, laccases A, B and C exhibited 40, 39 and 32% of activity, respectively. Non-denaturing polyacrylamide gel electrophoresis (PAGE) analysis showed differences among the enzymes. Overall, the properties of laccase A matched those of other laccases purified from white-rot fungi, whereas laccases B and C exhibited activity at higher temperatures and a higher tolerance towards common laccase inhibitors. It is suggested that our studies on laccases isolated from CMSW will improve the understanding of the biochemical process leading to the production of humic substances during composting.