Influence of structure on de degradation with lacase mediator systems

Abstract

A new laccase was purified from Trametes hirsuta IMA2002. The laccase had a molecular mass of 62 kDa and an isoelectric point of pH 7. It had an optimum pH of 3.0 and an optimum temperature of 558C. The laccase was quite stable at 308C and pH 4.0 with a half-life of more than 100 hours. On ABTS, yringaldazide, and DMP the laccase showed KM and Kcat values of 75, 12 and 37 mM and 64, 83 and 54 s_1, respectively. The structurally diverse commercial dyes Indigo Carmine, Lanaset Blue 2R, Diamond Black PV 200 and Diamond Fast Brown were oxidized by the laccase. While the rate and extent of decolorization of the latter dye was significantly enhanced by the presence of different types of mediators, the structurally similar azo-dye Tartrazine was not oxidized. Lanaset Blue 2R, a commercial textile dye containing an anthrachinoid structural fragment acted similarly to anthrachinone sulfonic acid by strongly enhancing the rate of the decolorization reaction. Twenty two model azo-dyes based on the molecular framework of 2,7-dihydroxy-1-phenylazonaphtalene-3,6- disulfonic acid were synthesized and the kinetics of their laccase-catalyzed decolorization was studied. Hydroxy- substituted dyes were the most susceptible to enzyme/mediator action. All reactions were well described by Michaelis_/Menten-like kinetics and the Hammett free energy linear relationship could be successfully applied to describe the influence of dye structure (substituents on the aromatic ring) on decolorization. Strongly electron withdrawing substituents such as a nitro-group in the meta-position (_/0.7) resulted in positive s-constants whereas electron donating groups such as para-methyl (_/0.3) resulted in negative values for s-constants.