Production, purification and partial characterisation of a novel laccase from the white-rot fungus Panus tigrinus CBS 577.79.
Extracellular laccase from Panus tigrinus CBS 577.79 was produced in a bubble-column reactor using glucose-containing medium supplemented with 2,5-xylidine under conditions of nitrogen sufficiency. The main laccase isoenzyme was purified to apparent homogeneity by ultra-filtration, anion-exchange chromatography and gel filtration that led to a purified enzyme with a specific activity of 317 IU (mg protein)-1 and a final yield of 66%. Laccase was found to be a monomeric protein with a molecular mass of 69.1 kDa, pI of 3.15 and 6.9% N-glycosylation of the high mannose type. Temperature and pH optima were 55 degrees C and 3.75 (2,6-dimethoxyphenol as substrate). At 50 and 60 degrees C, the enzyme half-lives were 281 and 25 min, respectively. The P. tigrinus laccase oxidized a wide range of both naturally occurring and synthetic aromatic compounds: the highest catalytic efficiencies were for 2,2'-azinobis-(3-ethylbenzthiazoline-6-sulfonic) acid and 2,6-dimethoxyphenol (5.99x10(6) and 3.07x10(6) M-1 s-1, respectively). Catalytic rate constants for typical N-OH redox mediators, such as 1-hydroxybenzotriazole (2.6 s-1), violuric acid (8.4 s-1) and 2,2,6,6-tetramethylpiperidin-N-oxide radical (7.8 s-1), were found to be higher than those reported for other high redox potential fungal laccases.
Quaratino
Daniele
D
Dipartimento di Agrobiologia & Agrochimica, Università degli Studi della Tuscia, Via San Camillo de Lellis snc, I-01100, Viterbo, Italy.
Federici
Federico
F
Petruccioli
Maurizio
M
Fenice
Massimiliano
M
D'Annibale
Alessandro
A
D019149:Bioreactors; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D042845:Laccase / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D020074:Lentinula / Q000201:enzymology*; D008970:Molecular Weight; D013379:Substrate Specificity; D013696:Temperature
D042845:Laccase