The thermostable enzyme preparations were kindly provided by ROAL, Fin­land. The genes encoding three thermostable enzymes: cellobiohydrolase (CBH/Cel7A) from Thermoascus aurantiacus, fused with the T. reesei CBHI cel­lulose binding domain (CBM), endoglucanase (EG/Cel45A) from Acremonium thermophilum and a xylanase and в-glucosidase (BG/Cel3A) from T. auran­tiacus were inserted under the control of a strong T. reesei cbhl promoter and transformed into a host strain where all the major cellulase genes were deleted (phenotype CBHI/Cel7A — CBHII — Cel6A — EGI/Cel7B — EGII/Cel5A). Fermenter supernatants produced at pilot scale were used to produce vari­ous mixtures of the thermostable enzymes. The background activities in the deletion strains were measured. The composition of the mixture of the three thermostable enzymes was optimised based on the average cellulase compo­sition of T. reesei. The enzyme components were mixed in different ratios and the total cellulase activity of the mixtures was measured at 50 °C(as FPU mL-1) and used as the basis of enzyme dosing. In addition, a family 10 thermostable xylanase from T. aurantiacus, cloned and expressed in the T. reesei deletion strain, was added to some preparations to ensure complete hydrolysis.

Celluclast 1.5 L FG (Novozymes, Denmark) and Econase CE (ABEnzymes, Finland), eventually supplemented with BG from Novozym 188 (Novozymes, Denmark) were used as reference enzymes. The standard enzyme dosage was 10 FPU g-1 cellulose for Celluclast 1.5 L FG, supplemented with additional BG (100-500 nkatg-1 cellulose). Assuming an average 50% cellulose content of the lignocellulose substrates, the enzyme dosage was thus 5 FPU g-1 substrate. For the hydrolysis experiments at elevated temperatures, higher dosage of Celluclast (22 FPU g-1 cellulose) was used.

The total cellulolytic activity used as a basis for dosing of the enzyme mixtures was evaluated using the FPU activity, measured against Whatman no. 1 filter paper [36]. The EG activity was assayed using hydroxyl ethyl cel­lulose as substrate [36]. The CBH activity was determined by using 4-methyl umbelliferyl-b-D-lactoside as substrate, estimating the effect of EGs by carry­ing out the assay with or without 20 mM cellobiose in the reaction [6]. The xy- lanase activity was assayed using birchwood glucuronoxylan as substrate [4] and that of BG using p-nitrophenyl-b-D-glucopyranosidase as substrate [5]. Protein was assayed according Lowry et al. [43]. All the enzyme activity assays were carried out at pH 5.

Substrates

The substrates used were steam pretreated, washed spruce solid fraction kindly provided by Guido Zacchi at the Lund University, Sweden, and steam pretreated corn stover kindly provided by Francesco Zimbardi at ENEA, Italy. The solid fraction of spruce substrate after the pretreatment was sep­arated from the liquid fraction by filtration, washed and used in the hydro­lysis experiments. The composition of the fibre fractions of the substrates is presented in Table 2. In addition to the insoluble fibre fraction, the corn stover substrate contained significant amounts, about 17% (d. w.) of solu­bilised mono — and oligosaccharides, solubilised mainly from hemicelluloses. Based on secondary analytical enzymatic hydrolysis and HPLC analysis, the carbohydrates in the soluble fraction consisted of xylose (74%), arabinose (15%), galactose (5%) and glucose (6%). Comparative hydrolysis experiments were carried out using crystalline cellulose (Avicel, Sigma).