When designing novel thermostable enzyme systems, the structural features of the substrates determine the number of enzymes needed for total hydro­lysis. The crystallinity of cellulose, the available surface area and the dis­tribution of lignin and hemicellulose are the major substrate-related factors limiting the hydrolysis rate of cellulose. An efficient pretreatment is the most straightforward solution for improving the hydrolysis rate and decreasing the amount of enzymes needed. Using various pretreatment techniques, either most of the hemicellulose or lignin is removed. It has been observed that the removal of hemicellulose has a direct correlation with the efficiency of the hydrolysis [56]. Even low amounts of residual xylan can limit the extent and rate of the hydrolysis. This can be overcome by addition of suitable hemicel — lulases, especially xylanases, to substrates with high original xylan content. Usually, the xylanase activity in commercial T. reesei preparations has been adequately high to overcome this limitation on xylan-containing substrates. Lignin content and distribution has also been proposed to be a substrate — related factor that affects the efficiency of enzymatic hydrolysis [49]. The close association of lignin and cellulose may prevent swelling of the fibrous substrate and result in limited accessibility of enzymes. The role of lignin or lignin-derived compounds in destabilising or deactivating enzymes is obvi­ously also crucial. High temperature and pressure during the pretreatment result in a variety of soluble inhibitors for the enzymes and the yeast. In this work, it was observed that the inhibition of cellulases on lignin-containing substrates was increased at higher temperatures (Figs. 4 and 5).

The optimal cellulase composition varies depending on the substrate used but usually, the major cellulases comprise two cellobiohydrolases (about 60-70% of total protein), and two major and several minor endoglucanases (about 25% of the total protein). Various models and mechanisms for the syn­ergistic action of cellulases have been proposed. These studies have focused on the T. reesei exo-exo synergism [31,53,77,80] or on the endo-exo syn­ergism [39,46,52,80]. The key role of в-glucosidase in a separate hydrolysis process has been clearly demonstrated, and is due to the end product inhi­bition of especially cellobiohydrolases caused by cellobiose [20,54]. In T. ree — sei this activity is partly mycelium-bound and obviously limits the enzyme performance in commercial T. reesei preparations. Therefore, P-glucosidase is usually supplemented, generally originating from Aspergillus niger. In­terestingly, in this work it was shown that just three major thermostable cellulases, i. e. one cellobiohydrolase and one endoglucanase supplemented by в-glucosidase, used in a preliminarily optimised ratio were able to pro­duce a hydrolysis yield comparable with that obtained with the whole set of cellulolytic and accessory enzymes present in the commercial T. reesei prep­arations. Further research would be necessary to clarify the detailed mech­anisms of these enzymes. Although the endo-exo synergism was obviously efficient enough to result in a high sugar yield, it could be further improved by optimising the thermostable cellulase components. The optimal ratio of the major enzymes was shown to be close to that of T. reesei. In this work, the individual thermostable cellulases were preliminarily screened based on their activity profiles and not based on their synergistic action. Therefore, the hydrolysis result can be considered extremely promising. Previously, thermo­stable enzymes from different organisms have not been combined to form new efficient mixtures. Expectedly, further optimisation, as well as supple­mentation of other synergistically acting enzymes would further improve the hydrolytic efficiency. In the present work, only thermostable xylanase was added to the mixture of the three cellulases (endoglucanase, cellobiohydrolase and P-glucosidase).

Previously, thermostable enzymes have only been studied as individually added proteins to improve the performance of the cellulases from T. ree — sei [62]. The T. reesei cellulase system is rapidly inactivated at temperatures above 45 °C, and the optimal temperature is generally considered to be be­low 45 °C on substrates requiring longer hydrolysis times, e. g. due to higher substrate consistency. Crude culture filtrates from various moderately ther­mophilic fungi (C. thermophilum, T. terrestris, T. aurantiacus, C. thermo — philus, M. thermophila) were added on the protein basis to a commercial T. reesei preparation. Obviously, due to the relatively high proportion of T. reesei enzymes in the mixture, and the consequent inactivation of these en­zymes at elevated temperatures, no improvement of the hydrolysis at higher temperatures could be observed. The main advantage was expected to be due to more active endoglucanases or due to a improved improved ratio of endoglucanase and cellobiohydrolase in the crude fermentation broth. In addition, unidentified enzyme activities in the preparations may also have caused some effects.

In this work, the individual cloned thermostable enzymes were produced with a T. reesei strain where the four genes encoding the major cellulases, i. e. Cel7A, Cel6A, Cel7B and Cel5A, had been deleted. Thus, only the mi­nor endoglucanases Cel12A, Cel61A and Cel45A, as well as xylanases and other accessory enzymes, were present in the T. reesei background. In add­ition, most of these activities were inactivated in a thermal treatment. Only the Cel45A was somewhat more resistant to thermal inactivation and re­tained most activity at higher temperatures. Thus, the hydrolysis results were non-disputably obtained due the cloned thermostable enzymes, and the back­ground activities were negligible. This was also clear from the hydrolysis experiments with the commercial T. reesei enzymes, showing clearly a de­creased performance at temperatures of 50 °C or above.

In addition to improved performance in the hydrolysis of lignocellulosic substrates, thermophilic enzymes allow the design of more flexible process configurations. Traditionally, T. reesei enzymes are used either in a separate hydrolysis and fermentation process (SHF) or in a simultaneous saccharifi­cation and fermentation (SSF) process. It is commonly stated that the major advantage of the SHF is that both process steps (hydrolysis and fermenta­tion) can be run under optimal conditions. Typically, hydrolysis of the SHF is carried out at around 45-50 °C at pH 5, and the fermentation at 35 °C at a lower pH. The SSF, on the other hand is usually carried out at 35 °C at pH 4.5-5. A more efficient hydrolysis is expected to take place at higher tem­peratures. In this work, using thermostable enzymes, it was indeed possible to obtain about 10 °C higher operation temperature than with the present commercial T. reesei enzyme preparations. The applicable hydrolysis tem­perature could be 60-65 °C for the hydrolysis of corn stover substrate and about 55 °C for spruce substrate. The hydrolysis rates at 55 °C were higher than those of the commercial enzymes at 45 °C. The enzymatic hydrolysis at higher temperatures would potentially reduce the reaction time and the enzyme loading.

It can be concluded that the cloned thermostable enzymes in preliminarily optimised preparations clearly demonstrate that the hydrolysis of lignocel — lulosic raw materials can be further improved, leading to potential savings in the hydrolysis costs. Previously, it has been shown that the costs of cel — lulases can be radically decreased, e. g. by improving the specific activity, by omitting the downstream processing of enzyme production or by improving the production process by other means. A mixture of only four thermostable enzymes was shown to be superior to the present commercial T. reesei prep­arations, which are comprised of at least ten enzymes acting synergistically on cellulose and on other components of the lignocellulosic substrates under optimal conditions. Further supplementation of other cellulases or accessory enzymes would expectedly further improve the hydrolysis result and the over­all economy of the process.

Acknowledgements The EU Commission is gratefully acknowledged for the financial sup­port (project number: NNE5-2001-00447, TIME).