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As part of the effort to find better cellulases for bioethanol production processes, we were looking for novel GH‐7 family cellobiohydrolases, which would be particularly active on insoluble polymeric substrates and participate in the rate‐limiting step in the hydrolysis of cellulose. The enzymatic properties were studied and are reported here for family 7 cellobiohydrolases from the thermophilic fungi Acremonium thermophilum, Thermoascus aurantiacus, and Chaetomium thermophilum. The Trichoderma reesei Cel7A enzyme was used as a reference in the experiments. As the native T. aurantiacus Cel7A has no carbohydrate‐binding module (CBM), recombinant proteins having the CBM from either the C. thermophilum Cel7A or the T. reesei Cel7A were also constructed. All these novel acidic cellobiohydrolases were more thermostable (by 4–10°C) and more active (two‐ to fourfold) in hydrolysis of microcrystalline cellulose (Avicel) at 45°C than T. reesei Cel7A. The C. thermophilum Cel7A showed the highest specific activity and temperature optimum when measured on soluble substrates. The most effective enzyme for Avicel hydrolysis at 70°C, however, was the 2‐module version of the T. aurantiacus Cel7A, which was also relatively weakly inhibited by cellobiose. These results are discussed from the structural point of view based on the three‐dimensional homology models of these enzymes.

The extracellular bga1-encoded β-galactosidase of Hypocrea jecorina (Trichoderma reesei) was overexpressed under the pyruvat kinase (pki1) promoter region and purified to apparent homogeneity. The monomeric enzyme is a glycoprotein with a molecular mass of 118.8 ± 0.5 kDa (MALDI-MS) and an isoelectric point of 6.6. Bga1 is active with several disaccharides, e.g. lactose, lactulose and galactobiose, as well as with aryl- and alkyl-β-d-galactosides. Based on the catalytic efficiencies, lactitol and lactobionic acid are the poorest substrates and o-nitrophenyl-β-d-galactoside and lactulose are the best. The pH optimum for the hydrolysis of galactosides is ∼ 5.0, and the optimum temperature was found to be 60 °C. Bga1 is also capable of releasing d-galactose from β-galactans and is thus actually a galacto-β-d-galactanase. β-Galactosidase is inhibited by its reaction product d-galactose and the enzyme also shows a significant transferase activity which results in the formation of galacto-oligosaccharides.