Recombinant cellulolytic or xylanolytic complex comprising the full-length scaffolding protein RjCipA and cellulase RjCel5B or xylanase RjXyn10C of Ruminiclostridium josui

•Ruminiclostridium josui scaffolding protein (RjCipA) was expressed by E. coli.•RjCel5B and RjXyn10C are cellulosomal endoglucanase and endoxylanase, respectively.•RjCel5B-RjCipA complex showed a strong substrate-targeting effect toward rice straw.•RjXyn10C-RjCipA complex showed a substrate-isolating effect toward rice straw.•Proximity effect by adjoining enzymes was observed in neither of the complexes.

Three cellulosomal subunits of Ruminiclostridium josui, the full-length scaffolding protein CipA (RjCipA), a cellulase Cel5B (RjCel5B) and a xylanase Xyn10C (RjXyn10C), were successfully produced by Escherichia coli recombinant clones. RjCel5B and RjXyn10C were characterized as an endoglucanase and an endoxylanase, respectively. RjCipA, RjCel5B and Xyn10C adsorbed to microcrystalline cellulose (Funacel) and rice straw powder. Interaction between RjCel5B and RjCipA, and RjXyn10C and RjCipA were confirmed by qualitative assays. When a fixed amount of RjCel5B was mixed with different amounts of RjCipA, i.e., at the molar ratio of 6:1 or 6:6, the 6:6 complex showed 6.6-fold higher activity toward Funacel and 11.5-fold higher activity toward rice straw powder than RjCel5B, whereas the 6:1 complex showed only 2.8- and 3.9-folds higher activities toward Funacel and rice straw powder, respectively, than RjCel5B. These results suggest that the family-3 carbohydrate binding module (CBM3) of RjCipA in the RjCel5B-RjCipA complex plays an important role for hydrolysis of cellulose and the substrate-targeting effect of the CBM is more significant than the proximity effect caused by the presence of plural catalytic subunits adjoining each other. In contrast, the 6:1 complex of RjXyn10C and RjCipA showed 45% and 28% of the activities of RjXyn10C toward insoluble wheat arabinoxylan and rice straw powder, respectively. These results suggest that both a negative proximity effect and substrate-isolating effect, but not substrate-targeting effect, are caused by the CBM3 with inappropriate polysaccharide specificity. Substrate-targeting, proximity and substrate-isolating effects are discussed.