Fig. 3

Hydrolytic efficiency of multi-enzyme complexes on Avicel as substrate. a End-point activity of 1 µg of the enzymes on 0.25% Avicel after 42 h in dependence of the endoglucanase functions present in the complex. The bars represent amount of reducing sugar ends (glucose equivalents) as average values from biological replicates (at least duplicate measurements with standard deviations represented as × 1 SD). The endoglucanase product pattern present (+) or absent (−) in the complex was non-processive (EG) or processive with cellobiose (pEG2) or cellotetraose (pEG4) as intermediate or main product. The complex “all EG types” consists of 9 different enzymes, whereas each cellulase function is present in this complex (cellobiohydrolases, non-processive endoglucanases, members of pEG2 and pEG4, respectively). Each of the complexed mixtures comprised equal stoichiometric loading and statistical distribution of eight single enzymes on CipA8 by cohesin–dockerin protein interaction. The enzyme complexes were purified by gel filtration to exclude the impact of unbound single cellulases. As controls, the activity of complexed and non-complexed enzyme extracts of C. thermocellum mutant SM901 [41] is shown together with the native cellulosome. Abbreviations: Cel8A (A), Cel9D (D), Cel5-26H (H), Cel9-44J (J), Cel9K (K), Cel5L (L), Cel9R (R), and Cel48S (S). b Enzyme kinetics of cellulosomal complexes on 2.5% Avicel. c Electrophoretic mobility shift showing the binding capacity of recombinant scaffolding protein CipA8 made possible by its eight cohesin binding modules. Complex formation by cohesin–dockerin interaction is visible by up-shifted protein bands in the native gel. 10 µM of CipA8 was titrated with 80 µM of a nonavalent cellulase mixture (all EG types + CipA8) for statistically binding all free cohesin modules. As another control, the SM901 enzyme extract was also completely bound (SM901 + CipA8). The 6% native PAGE gel was stained with Coomassie R-250