The performance of the thermostable enzymes at a lower temperature, the 35 °C commonly used in SSF, was compared. The T. reesei deletion strains produced only low amounts of background cellulase activities, mainly due to the presence of native EGIII (Cel12A) and EGV (Cel45A). However, the deletion strains used for the production of thermoenzymes produced some hemicellulases. For practical use, any mesophilic background activity en­hancing the hydrolysis can be considered useful, but in order to evaluate the performance of the thermophilic enzymes, the level of remaining background activities was evaluated. The FPU activity in the background was negligible and the endoglucanase activity was very low as compared to the commercial preparations. Most of the endoglucanase activity, 85-90%, was inactivated during the thermal treatment at 60 °C, pH 6.5 for 2 h. Obviously, the EGV ac­tivity was the most stable remaining activity. Thus, the background activities originating from the T. reesei deletion strains had only a minor contribution to the total hydrolysis above 65 °C.

The actual hydrolysis performance of the new thermostable enzyme mix­tures on various pretreated lignocellulose substrates (spruce and corn stover) at 35 °C showed some variations as compared with the T. reesei enzymes: on

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Fig. 7 Hydrolysis of steam pretreated washed spruce (a) and unwashed corn stover (b) by Celluclast (■) and (□) the thermostable enzymes (TM 1 for spruce and TM 2 for corn stover) at 35 °C. Enzyme dosages: Celluclast 5 FPU g-1 substrate, supplemented with 100 nkatNovozym 188 g-1 substrate; thermostable enzymes 5 FPU g-1 substrate, substrate concentration 10 gL-1, hydrolysis time 72 h at pH 5, triplicates with mixing
spruce, the sugar yield obtained by the thermophilic enzymes was generally lower and on corn stover higher than with the commercial T. reesei enzymes (Fig. 7). The result was the same, irrespective of the presence of the thermoxy — lanase in the preparation (TM 1 in Fig. 7 or TM 3 in Fig. 5). Thus, with this substrate the relatively lower cellulase activity at 35 °C is obviously the rea­son for the poorer hydrolysis at the lower temperature. In contrast, on the xylan-containing substrate, corn stover, the additional xylanase activity in the thermostable enzyme mixture had a more profound effect. The total xylanase activity was somewhat higher in the thermostable preparation, emphasising the importance of hemicellulases in the hydrolysis of substrates containing residual xylans. Further research would be needed to study in detail the struc­tural differences of both cellulose and hemicellulose in the two substrates and their impact on the performance of the enzyme patterns used.

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