Synergies between cellulosomal and non-cellulosomal bacterial cellulases complexed in designer cellulosomes

The development of cellulases cocktails to efficiently solubilize cellulose for biotechnological applications is a constant demand. Cellulases exhibit diverse mode of action, operating in the free state or in multi-enzyme complexes (cellulosomes), and potentially acting synergistically for a higher efficiency. The efficacy of three non-cellulosomal bacterial cellulases originating from three different species that naturally act in the free state: Cel9A from Lachnoclostridium phytofermentans, Cel5H from Saccharophagus degradans and two variants of Cel5I from Ruminiclostridium cellulolyticum, were evaluated in bifunctional or trifunctional designer cellulosomes, and in combination with the most efficient pair of cellulases reported in R. cellulolyticum, Cel48F and Cel9G. The activity of the newly tested cellulases in designer cellulosomes, reached a stimulation factor of 1.25. Cel5H and Cel5I can act in a cooperative manner with other cellulases. Low synergy was observed when Cel9A was included in the complexes with the pair Cel48F and Cel9G. However, all the complexes containing Cel9A exhibited the highest activities. Finally, the most efficient enzymatic combination is a bifunctional designer cellulosome comprising Cel48F and Cel9G, in association with free Cel9A. Interestingly this mixture releases high levels of glucose and cellobiose, making this association an especially attractive option for biotechnological conversion of cellulose to chemicals.