Partial purification of laccase and its immobilization of polymer supports

Abstract

Laccase (EC 1.10.3.2 ; Oxygen oxidoreductase) was isolated from Pleurotus sapidus. The enzyme was partially purified by first precipitation with 80% (W/W) ammonium sulfate. After the removal of salts by dialysis the laccase was further purified by ion exchange chromatography using DEAE-Sephadex A-50. The enzyme was concentrated, by ultrafiltration and subjected to further purification by gel permeation chromatography using Sephadex G-100. The pooled, fractions of partially purified laccase was lyophilized and kept at -20°c in dried form. The above procedures resulted in 40 folds purification of laccase resulting in the enzyme having a specific activity of 15 unit/mg. protein. Polyacrylamide gel electrophoresis revealed that laccase consisted of at least 4 isoenzymes. The enzyme solution absorbed light maximally at around. 600 nm and was able to oxidise catechol, hydroquinone as well as syringaldazine. P. sapidus laccase exhibited all optimum pH of around-6 and was relatively stable at 45 °c, loosing only 25% of activity at this temperature. Laccase was immobilized on polymer supports involving two methods. The first method employed epichlorohydrin as linking agent. In this case laccase was covalently linked to the polymer of Amberite IRA-68. The second method involved the entrapment of laccase within the network of polyacrylamide gel. The immobilized laccase in both cases were tested for its stability and were found to function for number of times before the activity was finally lost.