Purification and characterization of laccase from the white-rot fungus Daedalea quercina and decolorization of synthetic dyes by the enzyme.
The white-rot fungus Daedalea quercina produced the ligninolytic enzymes laccase and Mn-dependent peroxidase. Laccase was purified using anionexchange and size-exclusion chromatographies. SDS-PAGE showed the purified laccase to be a monomeric protein of 69 kDa (71 kDa using gel filtration) with an isoelectric point near 3.0. The optimum pH for activity was below 2.0 for 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (K(m)=38 microM), 4.0 for 2,6-dimethoxyphenol (K(m)=48 microM), 4.5 for guaiacol (K(m)=93 microM) and 7.0 for syringaldazine (K(m)=131 microM). The temperature optimum was between 60 and 70 degrees C depending on the pH and buffer used. The enzyme was stable up to 45 degrees C, and stability was higher at alkaline pH. Enzyme activity was increased by the addition of Cu(2+) and inhibited by Mn(2+), sodium azide, dithiothreitol, and cysteine. Laccase from Daedalea quercina was able to decolorize the synthetic dyes Chicago sky blue, poly B-411, remazol brilliant blue R, trypan blue and reactive blue 2.
D000880:Anthraquinones / Q000378:metabolism; D001391:Azo Compounds / Q000378:metabolism; D052160:Benzothiazoles; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003116:Color; D004396:Coloring Agents / Q000378:metabolism*; D020536:Enzyme Activators / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004795:Enzyme Stability; D006139:Guaiacol / Q000378:metabolism; D006835:Hydrazones / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D042845:Laccase / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D008031:Lignin / Q000378:metabolism; D008670:Metals / Q000494:pharmacology; D008970:Molecular Weight; D010544:Peroxidases / Q000096:biosynthesis; D020072:Polyporales / Q000201:enzymology*; D011748:Pyrogallol / Q000031:analogs & derivatives* / Q000378:metabolism; D013379:Substrate Specificity; D013451:Sulfonic Acids / Q000378:metabolism; D013696:Temperature; D014227:Triazines / Q000378:metabolism; D014343:Trypan Blue / Q000378:metabolism
D000880:Anthraquinones; D001391:Azo Compounds; D052160:Benzothiazoles; D004396:Coloring Agents; D020536:Enzyme Activators; D004791:Enzyme Inhibitors; D006835:Hydrazones; D008670:Metals; D013451:Sulfonic Acids; D014227:Triazines; D011748:Pyrogallol; C009370:syringaldazine; C009000:pontamine sky blue; C002502:2,2'-azino-di-(3-ethylbenzothiazoline)-6-sulfonic acid; C010120:pyrogallol 1,3-dimethyl ether; C012842:Cibacron Blue F 3GA; D006139:Guaiacol; D008031:Lignin; D042845:Laccase; D010544:Peroxidases; C051129:manganese peroxidase; D014343:Trypan Blue; C024758:Remazol Brilliant Blue R