The activities of six purified Thermomonospora fusca cellulases and Trichoderma reesei CBHl and CBHll were determined on filter paper, swollen cellulose, and CMC. A simple method t o measure the soluble and insoluble reducing sugar products from the hydrolysis of filter paper was found t o effectively distinguish between exocellulases and endocellulases. Endocellulases produced 34% t o 50% insoluble reducing sugar and exocellulases produced less than 8% insoluble reducing sugar. The ability of a wide variety of mixtures of these cellulases t o digest 5.2% of a filter paper disc in 16 h was measured quantitatively. The specific activities of the mixtures varied from 0.41 to 16.31 pmol cellobiose per minute per micromole enzyme. The degree of synergism ranged from 0.4 t o 7.8. T. reesei CBHll and T. fusca E 3 were found to be functionally equivalent in mixtures. The catalytic domains (cd) of T. fusca endocellulases E2 and E 5 were purified and found to retain 93% and 100% of their CMC activity, respectively, but neither cd protein could digest filter paper to 5.2%. When E2cd and E5cd were substituted in synergistic mixtures for the native proteins, the mixtures containing E2cd retained 60%, and those containing E5cd retained 94% of the original activity. Addition of a P-glucosidase was found to double the activity of the best synergistic mixture. Addition of CBHl to T. fusca crude cellulase increased its activity on filter paper 1.7-fold. 0 1993 John Wiley & Sons, Inc.

One way to improve the economic feasibility of biomass conversion is to enhance the catalytic efficiency of cellulases through protein engineering. This requires that high-resolution structures of cellulases be available. Here we present the structure of E2cd, the catalytic domain of the thermophilic endocellulase E2 from Thermomonospora fusca, as determined by X-ray crystallography. The structure was solved by multiple isomorphous replacement at 2.6-A resolution and has been refined at 1.8-A resolution to an R-value of 18.4% for all reflections between 10- and 1.8-A resolution. The fold of E2cd is based on an unusual parallel beta-barrel and is equivalent to the fold determined for the catalytic domain of cellobiohydrolase II, an exocellulase from Trichoderma reesei [Rouvinen et al. (1990) Science 249, 380-385]. The active site cleft of the enzyme, approximately 11 A deep and running the entire length of the molecule, is seen to be completely free for ligand binding in the crystal. A 2.2-A resolution analysis of crystals of E2cd complexed with cellobiose, an inhibitor, shows how cellobiose binds in the active site and interacts with several residues which line the cleft. Catalytic roles are suggested for three aspartic acid residues at the active site. A comparison of the E2cd and CBHIIcd structures reveals a large difference in their active site accessibilities and supports the hypothesis that the main difference between endo- and exocellulases is the degree to which their active sites are accessible to substrate.

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