Abstract:
Laccase from Trametes versicolor was crystallized using ammonium sulphate and the resultant crystals on cross-linking with glutaraldehyde produced insoluble and catalytically active enzyme. These cross-linked enzyme crystals (CLEC) of laccase had improved thermal stability (fourfold) than the native enzyme. The half-life of CLEC laccase at 60 degrees C was 123 min compared to 24 min for the soluble enzyme. The kinetics of oxidation reactions catalyzed by CLEC laccase was studied using various substrates 2,2'-azino-bis(3-ethylbenz-thiazoline-6-sulphonic acid) (ABTS), guaiacol, catechol, pyrogallol, syringaldazine and catechin. ABTS was found to be the best substrate for CLEC laccase (K-m = 0.859 mM) and had a catalytic efficiency (k(cat)/K-m = 3.73 x 10(3)) higher than the other substrates. The CLEC laccase showed lower specific activity, V-max and k(cat) values than the native enzyme for all the substrate studied and this may be due to the partial inactivation of laccase crystals by glutaraldehyde, and also the diffusion limitation of the substrate through the channels in the cross-linked crystal structure of laccase enzyme. CLEC laccase had a higher activity in non-polar organic solvents like hexane, toluene, isooctane and cyclohexane. The preparation and characterization of CLEC laccase is reported for the first time.
