In nature, microbes can efficiently degrade biomass by secreting an array of synergistic enzymes, including cellulases, often from numerous microbes in­termingled in their growth. In an effort to identify new proteins that could work synergistically with those secreted by T. reesei, we conducted mixing ex­periments by supplementing Celluclast 1.5 L with broth from a wide array of cellulolytic fungi grown under cellulase-inducing conditions. By comparing the saccharification of acid pretreated corn stover using equal protein load­ings of either Celluclast alone or mixtures of Celluclast with these broths, fungi secreting components that could work synergistically with the T. ree­sei cellulases could be detected. In Fig. 7, an example of such an experiment shows that a mixture of T. reesei broth and Thielavia terrestris broth has the same level of hydrolyzing activity as twice as much T. reesei or T. terrestris broth alone. These results suggested that some activity present in the T. ter — restris broth was working in synergy with the cellulases present in T. reesei broth to more efficiently degrade the cellulose in the corn stover.

In order to identify the protein or proteins responsible for the observed synergism with the T. reesei cellulases, the T. terrestris broth was fraction­ated and individual fractions were assayed for synergism similarly. Fractions displaying synergism were separated on one — and two-dimensional polyacry­lamide gels, individual proteins were isolated by removal from the gels and subjected to sequencing by tandem mass spectrometry. Several independent chromatographic fractions contained proteins with homology to glycosyl hydrolase family 61A, a protein previously identified in a number of cellu­lolytic fungi. When purified to homogeneity, a number of these proteins were demonstrated to significantly enhance the activity of the T. reesei cellulases in synergism assays. Inclusion of these proteins at less than 5% of the total enzyme dose in some cases could reduce the required cellulase loading by as much as twofold. These results suggested that the GH61 family proteins were the major components responsible for the enhancement of Celluclast 1.5 L activity by crude T. terrestris fermentation broth.

We also tested the cellulase-enhancing effect of GH61 proteins on a var­iety of other lignocellulosic substrates from a variety of pretreatments when

combined with T. reesei cellulases. Those GH61 proteins capable of enhanc­ing hydrolysis of acid pretreated corn stover also enhanced hydrolysis of other substrates, although they differed in their effectiveness by varying amounts. None of the GH61 proteins were able to enhance the hydrolysis of pure cel­lulose in the form of filter paper. This lack of enhancement was also shown with other pure cellulose substrates such as Avicel, phosphoric-acid swollen cellulose, and carboxymethyl cellulose.

The GH61 proteins by themselves showed no significant detectable hy­drolytic activity on PCS or any other lignocellulosic substrate tested, indi­cating that the enhancement was not likely to be the result of any intrinsic endo — or exoglucanase activity of the GH61 proteins. The hydrolytic activity of several GH61 proteins was tested on a variety of model cellulose and hemi — cellulose substrates, but little or no activity was found. These results suggest that the enhancement of cellulolytic activity by GH61 is limited to substrates containing other cell wall-derived material such as lignin or hemicellulose, although there is no clear correlation between the proportions of these ma­terials and the degree of enhancement observed. These findings could be of significant interest for not only the elucidation of the physiological functions of the GH61 protein family, but also the development of a viable enzymatic system to convert biomass to simple platform sugars.

Several of the GH61 genes were transformed into T. reesei, resulting in transformants expressing GH61 at various levels, depending on the number of inserts and site of integration. Fermentation broths produced by these trans­formants were assayed for PCS hydrolysis at various protein loadings to assess their improvement in specific performance relative to control strains not ex­pressing non-native GH61 proteins. The results confirmed that certain GH61 proteins expressed at relatively low levels are capable of significantly enhanc­ing the hydrolysis of cellulose in PCS. For example, expression of T. terrestris GH61B in T. reesei allows for a reduction in protein loading of 1.4-fold to reach 90% conversion of cellulose to glucose in 120 h. The protein loading reduction made possible by GH61 addition becomes more pronounced at longer incubation times and higher levels of hydrolysis, and higher solids loadings.

4.2.3