Cloning and expression of Clostridium thermocellum genes coding for thermostable exoglucanases (cellobiohydrolases) in Escherichia coli cells

By special screening approach two independent Cl. thermocellum genes directing the synthesis of thermostable glucanases with an exo-mode of action have been isolated from pUC19-based gene bank in E. coli TG1. The genes are located on 3.4 and 11.3 kb DNA fragments showing no homology. E. coli-derived exoglucanases, presumably, cellobiohydrolases, are able to cleave lichenan, carboxymethyl cellulose, xylan and p-nitrophenyl derivatives of cellobioside and lactoside. Cellobiose is the main degradation product of carboxymethyl cellulose, treated with the identified exoglucanases. With p-nitrophenil-beta-D-cellobioside as substrate the enzymes had a pH optimum around 6.5 and a temperature optimum at 65 degrees C. The identified and expressed enzymes differ from all other Cl. thermocellum proteins known to date.