Purification and characterization of laccase-1 from Pleurotus florida

Plerotus florida (ITCC 3308) produces two laccase enzymes (L-1 and L-2) in potato-dextrose media containing 0.5% yeast extract. Concentrated culture f iltrate was separated on DEAE-Sephadex (A-50) column into two enzyme peaks, subsequently named L-1 and L-2. The L-1 enzyme has been purified to homoge neity by ion-exchange and gel-permeation chromatography L-1 is a monomeric glycoprotein with a molar mass of 77 and 82 kDa as determined by SDS-PAGE a nd gel-filtration chromatography, respectively. The pI value of L-1 has bee n determined to be 4.1. The optimum reaction temperature of the enzyme is 5 0 degreesC. The K-m and some other kinetic parameters of L-1 have been dete rmined. Cyanide and azide completely inhibit the enzyme activity. The enzym e was fully active in 1 : 1 (V/V) buffer-chloroform for at least 2 h. Spect roscopic analysis revealed that the enzyme has four copper atoms, a type 1 copper, a type 2 copper and a type 3 binuclear copper.