Extracellular proteins from lignocellulose degrading Basidiomycetes: Redox enzymes from Trametes versicolor and Coprinopsis cinerea

Abstract

The extracellular proteome of fungi consists of three different fractions: proteins secreted freely into the medium, the hyphal sheath proteins, and cell wall-associated proteins. Methods for isolation and characterization of secreted enzymes from wood degrading Basidiomycetes were established and/or optimized, including methods for isolation and characterization of cell wall-bound redox enzymes, in particular laccases. Methods established on Ascomycetes were not applicable for the processing of cell walls of the Basidiomycete Trametes versicolor. Grinding of the mycelia in a ball mill with a steel ball resulted in pure cell walls without contamination with cytoplasmic enzymes and the method of the cell wall purification can be transferred to other Basidiomycetes. Proteins isolated from isolated cell walls can be analyzed in 1D- and 2D-electrophoresis just like proteins from a culture supernatant. Fractions of proteins from the hyphal sheath, fractions of cell wall-associated proteins, and fractions of proteins from culture supernatants of T. versicolor differ in charge and size. From 2-D gels of T. versicolor supernatant proteins, seven spots were identified by ESI-LC-MS analysis to represent peroxidases and laccases, respectively. However, the identity of 90 other spots remained unidentified due to missing sequence data. Upon 1D- and 2D-gel electrophoresis, the secreted proteome of T. versicolor and Coprinopsis cinerea as well as cell wall-bound proteins from T. versicolor were analyzed for phenoloxidase activities using several known as well as some new enzyme substrates (alone, with co-substrates and in combinations). While the combination TMA (N,N,N´,N´-tetramethyl-1,4-phenyldiammonium dichloride) + alpha-naphthol was most sensitive in detecting phenoloxidase activities in the gels, MBTH (3-methyl-2-benzothiazolinone-hydrazone hydrochloride monohydrate) + DHPPA (3,4-dihydroxyhydrocinnamic acid) resulted in the sharpest and most stable bands and spots. By ESI-LC-MS analysis, some of phenoloxidase-reactive bands were identified to contain laccase of T. versicolor and C. cinerea, respectively. Laccases from T. versicolor were isolated by treating isolated cell walls with different lytic enzymes. Released enzymes were separated by native PAGE and phenoloxidase activities detected by enzymatic staining reactions. By ESI-LC-MS analysis of stained bands, laccase III was identified besides a pyranose oxidase. Laccase III is also present in high concentrations in culture supernatants of T. versicolor after induction by 2,5-xylidine. Laccase from culture supernatants showed different characteristics compared to the cell wall-bound laccase. The supernatant and the cell wall-bound laccases differed in their pH optima and in sensitivity towards high temperatures and the cell wall-bound enzymes were more resistant to enzyme inhibitors than the free laccases.