Purification and Characterization of Blue Laccase from a Ligninolitic Fungus Trametes gallica

A study on laccase production of Trametes gallica under shaking condition in PAG media was carried out.After 15 d,laccase production reached the highest level,then into the platform.4 liter of 15-day fermentation liquid was used to separate and purify laccase,with the (NH_(4))_(2)SO_(4 )pricepitation,DEAE-Sepharose Fast Flow chromatography and Sephadex G-100 chromatography,laccase was purified by 31.9 times.Purified laccase has a M_r of 60,000 Da,6% and an absorption at 605 nm which is the typical character of blue laccase.The optimal pH of the purified laccase is 2.2 for ABTS,3.0 for DMP and 4.0 for guaiacol,respectively.The K_m of the purified laccase is 0.172 10~(-2) mmol/L for ABTS, 0.51 mmol/L for DMP and 0.48 mmol/L for guaiacol,separatively.The optimal reaction temperature reachies 70℃,using ABTS as substrate.Purified laccase retained fairly high enzymatic activity through 24h treatment in pH 7～9 buffer.EDTA,halogen,NaCN and NaN_(3) have an inhibition on the laccase activity,among which NaN_(3 ) showed as the strongest inhibitor.Purified blue laccase displayed no activity towards tyrosine,but it was able to oxide untraditional substrate phenol red with high high redox potential.