PURIFICATION AND CHARACTERIZATION OF LACCASE FROM ARTHROGRAPHIS KSF2SHEENA DEVASIA AND A. JAYAKUMARAN NAIRAbstract Laccases are blue copper oxidases (E.C. 1.10.3.2 benzenediol: oxygen oxidoreductase) that catalyze the one-electron oxidation of phenolics, aromatic amines, and other electron-rich substrates with the concomitant reduction of O2 to H2O. They are currently seen as highly interesting industrial enzymes because of their broad substrate specificity. An ascomycetes strain, Arthrographis KSF2 was isolated and enzyme was purified and characterized. Laccase activity was determined using ABTS as substrate. Laccase was purified by ion-exchange and gel filtration chromatography. The purified laccase showed a molecular weight of 55 kDa and pI of 5.5 when analyzed by 2-D PAGE. The purified enzyme was even stable at pH 11 and it was observed that there was 74.4% retention of activity at pH 11 after 24hrs. The K(m) and V(max) values are 15 (μM) and 21.9 (μmol/min), respectively, with ABTS as substrate. The effect of 5mM metal ions such as Mn2+, Mg2+, Na+, Ca2+, Fe2+, Zn2+ and Cu2+ was estimated after incubating the enzyme with the metal ion for 5 min. It was observed that FeCl3 inhibited the laccase activity to the maximum (70.5%) whereas the enzyme was least inhibited by MnSO4 (21%). It was observed that even CuSO4 inhibited the laccase activity in 5 mM concentration. There was 100% denaturation by 1% SDS in 10 min whereas only 8% denaturation was observed by Tween 20. Tween 80 denatured 21% enzyme and Triton X 100 denatured 25% of the total enzyme. The purified laccase on UV-Vis analysis did not show the characteristic laccase peak at 600 nm and so it was confirmed as a typical yellow laccase. |