Role of scaffolding protein CipC of Clostridium cellulolyticum in cellulose degradation

The role of a miniscaffolding protein, miniCipC(1), forming part of Clostridium cellulolyticum scaffolding protein CipC in insoluble cellulose degradation was investigated, The parameters of the binding of miniCipC(1), which contains a family III cellulose-binding domain (CBD), a hydrophilic domain, and a cohesin domain, to four insoluble celluloses were determined, At saturating concentrations, about 8.2 mu mol of protein was bound per g of bacterial microcrystalline cellulose, while Avicel, colloidal Avicel, and phosphoric acid-swollen cellulose bound 0.25, 0,38, and 0.55 mu mol of miniCipC(1) per g, respectively. The dissociation constants measured varied between 1.3 x 10(-7) and 1.5 x 10(-8) M. These results are discussed with regard to the properties of the various substrates. The synergistic action of miniCipC(1) and two forms of endoglucanase CelA (with and without the dockerin domain [CelA(2) and CelA(3), respectively]) in cellulose degradation was also studied. Although only CelA, interacted with miniCipC(1) (K-d, 7 x 10(-9) M), nonhydrolytic miniCipC(1) enhanced the activities of endoglucanases CelA(2) and CelA(3) with all of the insoluble substrates tested, This finding shows that miniCipC(1) plays two roles: it increases the enzyme concentration on the cellulose surface and enhances the accessibility of the enzyme to the substrate by modifying the structure of the cellulose, leading to an increased available cellulose surface area. In addition, the data obtained with a hybrid protein, CelA(3)-CBDCipC, which was more active towards all of the insoluble substrates tested confirm that the CBD of the scaffolding protein plays an essential role in cellulose degradation.