Biochemical and molecular characterization of a cellobiohydrolase from Trametes versicolor

A cellobiohydrolase-encoding cDNA, Tvcel7a, from Trametes versicolor has been cloned and expressed in Aspergillus niger. The deduced amino acid sequence shows that Tvcel7a encodes a 456-amino acid polypeptide belonging to glycosyl hydrolase family 7. TvCel7a possesses a 19-amino acid secretion signal but does not possess a linker region nor a carbohydrate-binding domain. Two peaks of activity were obtained after TvCel7a was purified to apparent homogeneity by gel-filtration followed by anion-exchange chromatography. Mass spectrometry performed on the purified proteins confirmed that both peaks corresponded to the predicted sequence of the T versicolor cellulase. The biochemical properties of the purified TvCel7a obtained from both peaks were studied in detail. The pH and temperature optima were 5.0 and 40 degrees C, respectively. The enzyme was stable over a pH range extending from pH 3.0 to 9.0 and at temperatures lower than 50 degrees C. The kinetic parameters with the substrate p-nitrophenyl beta-D-cellobioside (pNPC) were 0.58 mM and 1.0 mu mol/min/mg protein for the purified TvCel7a found in both peaks I and 2. TvCe17a catalyzes the hydrolysis of pNPC, filter paper, beta-glucan, and avicel to varying extents, but no detectable hydrolysis was observed when using the substrates carboxymethylcellulose, laminarin and pNPG.