Production of laccase and xylanase from Coriolus versicolor grown on tomato pomace and their chromatographic behaviour on immobilized metal chelates

A strain of Coriolus versicolor was grown in tomato pomace as sole carbon source for the production of laccase and xylanase. This culture exhibited a peak of laccase (362 U/L of fermentation broth) and xylanase (2565 U/L of fermentation broth) activities on the 3rd and 14th day of cultures with a specific activity of 3.7 and 11.7 U/mg protein, respectively. Differential chromatographic behaviour of xylanase and laccase from C versicolor was investigated on immobilized metal chelates. The effect of pH, length of spacer arm, the presence of imidazole and nature of metal ion was studied for enzyme adsorption on immobilized metal affinity chromatography (IMAC). PDB survey of solvent accessible histidine residues in laccase and xylanase families was carried out by using bioinformatic tools. A one-step purification for laccase from C. versicolor was devised by using Sepharose 6B-EPI 30-IDA-Cu(II) and the purified enzyme was obtained with a specific activity of about 15.0 U/mg protein, a final recovery of enzyme activity of about 60% and a purification factor of about 10. The purified preparation of laccases A exhibited an optimum pH of activity of 5.0 and 3.0 with a dianisidine and ABTS as substrates, respectively. The optimum temperature of activity for this enzyme was found to be 80 degrees C in acetate buffer at pH 4.5 whereas the half-life (t(1/2)) of 19.4 +/- 2.2 h and 0.50 +/- 0.012 h was obtained at 45 and 60 degrees C, respectively. The kinetic parameters (V-max, K-m, K-cat and K-cat/K-m) of the purified enzyme were also obtained with o-dianisidine, guaiacol and ABTS as substrates. By using selective experimental conditions in IMAC, it was possible to separate successfully laccase isoenzymes into two groups, one with low and the other with high pl values. Kinetic characterization of both groups of isoenzymes was also carried out. (C) 2008 Elsevier Ltd. All rights reserved.