The thermostability of endoglucanase Cel8A, a major component of the cellulosome complex from Clostridium thermocellum, was significantly enhanced using a directed evolution strategy. A two-step screening strategy was employed that involved consecutive activity and thermostability assays. We have combined three of the mutations from the thermostability screen to obtain a Cel8A variant with a significant increase in thermal resistance without substantial alteration of kinetic parameters.
One of the three mutations provided the highest contribution to enzyme stability. This single mutation served to increase the Tm by 7.0 °C and the half-life of activity by eight fold at 85 °C.
The mutant was then incorporated into a designer scaffoldin composed of different cohesin modules. The synergistic activity with other enzymes was tested for degradation of a more recalcitrant substrate. It was combined to different exoglucanases: a recombinant form from C.thermocellum (Cel48S) or from Thermobifida fusca (Cel48A).
When it was not connected to a cohesin module, the Cel8A mutant was shown to provide higher activity than the wild-type form after a prolonged period of incubation (3 to 4 days). On the other hand, connection to its respective cohesin module confers higher enzymatic activity to the wild-type form.
These results demonstrate that the cohesin-dockerin binding event provides stability to the wild-type enzyme which eliminates the advantages of the thermostable mutant. In future screening approaches, the directed evolution process should be achieved on the cohesin-bound form of the enzyme.
