This study involved an extensive investigation on the cellulase system of the anaerobic fungus Piromyces strain KSI 1, which was isolated from the forestomach of a kangaroo. Considering the harsher conditions the kangaroo endures and lack of nutritional food sources in comparison to fed cattle and sheep, questions were raised as to whether this fungus may have adapted by enhancing its cellulase system. Piromyces KS11 has a relatively high growth rate on a cellulose-based medium, compared tp many other anaerobic fungi (pers. comm. G. Gordon, 1995). Initial experiments involved the determination of cellulase polymorphism of KS11 by zymogram (isozyme pattern) analysis. It was found that Piromyces KS11 cellulase bands ranged in molecular mass from below 30 kDa to above 94 kDa. When compared to other strains of Piromyces (isolated from various rumen and non-rumen sources) and Neocallimastix strains (rumen sources), it was observed that KS11 generally produced not only some unique bands but also more cellulases in total.In order to determine the number and molecular structure of KS11 cellulase genes, a cDNA library was constructed. Prior to construction of the library, medium constituents and optimal growth conditions were determined for large-scale growth and harvesting of Piromyces KS11 for RNA extraction. Optimal medium for maximum growth and hydrolytic enzyme production consisted of a standard rumen fluid based media which had been supplemented with 0.4% (w/v) Avicel, 0.1% (w/v) oatspelt xylan, 0.05% (w/v) citrus pectin, and 0.05% (w/v) cellobiose. Due to optimal conditions being determined for growth and harvesting of the fungi, a good RNA preparation and hence a high quality cDNA library was obtained. After extensive screening for cellulases from the library using CM-cellulose as the substrate, 150 CM-cellulose-positive clones were isolated.Analysis by restriction mapping and partial sequencing revealed twelve unique cellulase cDNAs with catalytic domains belonging to glycoside hydrolase families 5, 6, 9 and 45. Nine of the cDNAs had 5' or 3'-prime non-catalytic docking domains (NCDD), three had regions of sequence with unknown function and one had homology to cellulose-binding domains (CBD). Enzyme properties were analysed from crude extracts and while all showed varying degrees of activity towards soluble cellulose, two clones also exhibited activity towards xylan and another clone expressed activity towards methylumbelliferyl glycopyranoside (MUG), a substrate cleaved by s-glucosidases. The relative activity of each enzyme was calculated by comparing the ratio of activities on Avicel to CM-cellulose and showed that celA, D, and J showed relatively high activity towards Avicel.To determine the primary structure and sequence homology of KS11 cellulases, two cDNA clones were chosen for a more detailed comparison between enzymes produced by this particular anaerobic fungus and with cellulases from other organisms.