Production, purification and characterization of a thermostable laccase from a tropical white-rot fungus

A thermostable laccase was isolated from a tropical white-rot fungus Polyporus sp. which produced as high as 69,738 units of laccase l(-1) in an optimized medium containing 20 g of malt extract l(-1), 2 g of yeast extract l(-1), 1.5 mM CuSO4. The laccase was purified to electrophoretic purity with a final purification of 44.70-fold and a recovery yield of 21.04%. The purified laccase was shown to be a monomeric enzyme with a molecular mass of 60 kDa. The optimum temperature and pH value of the laccase were 75A degrees C and pH 4.0, respectively, for 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonate) (ABTS). The Michaelis-Menten constant (K (m) ) of the laccase was 18 mu M for ABTS substrate. The laccase was stable at pH values between 5.5 and 7.5. About 80% of the initial enzyme activity was retained after incubation of the laccase at 70A degrees C for 2 h, indicating that the laccase was intrinsically highly thermostable and with valuable potential applications. The laccase activity was promoted by 4.0 mM of Mg2+, Mn2+, Zn2+ and Ca2+, while inhibited by 4.0 mM of Co2+, Al3+, Cu2+, and Fe2+, showing different profiles of metal ion effects.