Laccase (Benzenediol-oxygen oxidoreductase)

Laccase, a fungal enzyme, consists of a group of multi-copper proteins of low specificity acting on both o- and p-quinols, often acting also on aminophenols and phenylenediamine. The semiquinone may react further either enzymatically or non-enzymatically. Laccase can be used for enzymatic detoxification of lignocellulosic hydrolysates. A Saccharomyces cerevisiae strain with enhanced resistance to phenolic inhibitors and thereby improved ability to ferment lignocellulosic hydrolysates would presumably be obtained by heterologous expression of laccase. Sequencing of the cDNA for the novel laccase gene lcc2 from the lignin-degrading basidiomycete Trametes versicolor showed that it encodes an isoenzyme of 499aa preceded by a 21-residue signal peptide. By comparison with Edman degradation data, it was concluded that lcc2 encodes an isoenzyme corresponding to laccase A. The gene product of lcc2 displays 71% identity with the previously characterized T. versicolor lcc1 gene product. An alignment of laccase sequences revealed that the T. versicolor isoenzymes in general are more closely related to corresponding isoenzymes from other white-rot fungi than to the other T. versicolor isoenzymes. The multiplicity of laccase is thus a conserved feature of T. versicolor and related species of white-rot fungi. When the T. versicolor lcc2 cDNA was expressed in S. cerevisiae, the production of active enzyme was strongly dependent on the temperature. After 3 days of incubation, a 16-fold higher laccase activity was found when a positive transformant was kept at 19 degrees C instead of 28 degrees C. Similar experiments with Pichia pastoris expressing the T. versicolor laccase gene lcc1 also showed that the expression level was favored considerably by lower cultivation temperature, indicating that the observation made for the S. cerevisiae expression system is of general significance. Systematic name: Benzenediol-oxygen oxidoreductase Reaction: 4-benzenediol + O2 -> 4-benzosemiquinone + 2 H2O Unit Definition: One unit will oxidize 1.0umole of Catechol/min at pH 4.5 and 25 degrees C. Optimum pH: ABTS-oxidation (50mM citrate/phosphate buffer pH4.5) Activity: (same/more than)10U/mg Storage and Stability: Lyophilized powder may be stored at -20 degrees C. Stable for 12 months at -20 degrees C. For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap.