Appl Microbiol Biotechnol (2012) 93:1601–1608 1605 Fig. 2 Endoglucanase CEL7B, endoglucanase CEL12A, and cellobiohydrolase CEL7A pro- duction in the cellulase negative background of T. reesei Δxyr1 transformants. Expression profile of typical Δxyr1 transformants expressing CEL7B (a), CEL12A (b), or CEL7A (c) under the tef1 (t) or cdna1 (c) promoter regions as indicated. Left SDS-PAGE gel of the supernatant of selected transformants after Coomassie blue staining. The amount of supernatant loaded to each gel is indicated in parentheses. Right total extracellular protein concen- tration (in milligrams per liter) of selected transformants expressing the respective cellulase under the cDNA1 (filled diamond) or tef1 promoter region (filled square). Δxyr1 (empty triangle) is included as a negative control values (dotted lines). Solid lines in Fig. 3a show the specific activities in micromoles/minute/milligram extracel- lular protein, illustrating that the specific activity was highest during the early time points (till around 28 h of growth). Thereafter, the specific activities decreased, consis- tent with the delayed secretion of the two faint proteins and other proteins expressed in the Δxyr1 background. Extra sugar addition (10 g/L) after 20 h of growth increased enzyme purity and yield (c-cel7b-6+). CEL7A producing transformants were active on MULAC, and their volumetric and specific activities displayed the same time course as that of CEL7B (Fig. 3c). Activity of CEL12A was quantified by following the release of reducing sugars from CMC (Ghose 1987). The volumetric activities obtained confirmed the produc- tion seen from SDS-PAGE, and the specific activities were—as with CEL7A and CEL7B—also highest during the early cultivation periods (Fig. 3b). It is also evident from a comparison of the activities produced by the different strains that the cdna1 promoter enabled higher protein production when using single-copy transformants and is thus to be preferred over the tef1 promoter under these conditions. Discussion T. reesei is a model system for the degradation of plant biomass to monomeric sugars applied in biofuel production. Its genome was recently sequenced (Martinez et al. 2008) and several sophisticated tools such as strains with improved gene targeting, strain crossing, HTP gene manipulation, and genome sequencing have been devel- oped (reviewed by Seiboth et al. 2011). One step which asks for improvement is the testing of properties of its gene products involved in plant cell wall degradation. Since most of the cellulases and hemicellulases are glycosylated proteins, the use of commonly available hosts may produce misleading results since prokaryotic hosts are not able to perform the necessary posttranslational modifications at all and other eukaryotic hosts produce usually overglycosy- lated proteins with altered activities (Boer et al. 2000;