10-03: Studies of synergism between two Thermobifida fusca cellulases, Cel48A and Cel9A, via empirical modeling and site directed mutagenesis

Cel48A is an exocellulase secreted by the model bacterium T. fusca.  On crystalline cellulose it has very low activity, but a strong synergistic effect with other cellulases.  It is one of the most abundant cellulases secreted by T. fusca.  The best synergistic partner in binary Cel48A mixtures is Cel9A, a processive endocellulase with high activity on crystalline cellulose.  When Cel48A and Cel9A are mixed together in optimum ratios their activity is several times greater than the sum of their specific activities.  The synergistic interactions between cellulases are only partially understood, and the literature on the topic is sometimes inconsistent.  Different observations in the literature suggest that the synergistic effect is dynamic and that different factors become more or less important as the substrate properties change during enzymatic digestion.  Time course experiments with Cel48A and Cel9A have revealed that the activity of these enzymes on crystalline substrate can be defined mathematically up to high levels of substrate digestion, using only two parameters.  These empirically determined parameters can be used to compare different cellulases to each other and to better understand the interactions that take place in synergistic mixtures.  Double-sequential additions of Cel9A and Cel48A support the results from synergistic time course experiments.  Experiments using wild-type Cel9A in combinations with mutated versions of Cel48A provide additional information about the synergistic interactions between these enzymes.  Combining the results of the above experiments we propose a tentative model of synergism between Cel48A and Cel9A, which is distinct from previously developed models of cellulase synergism.