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Characterization of the cellulosomal scaffolding protein CbpC from Clostridium cellulovorans 743B

Nakajima, Daichi, Shibata, Toshiyuki, Tanaka, Reiji, Kuroda, Kouichi, Ueda, Mitsuyoshi, Miyake, Hideo
Journal of bioscience and bioengineering 2017 v.124 no.4 pp. 376-380
Clostridium cellulovorans, DNA, Escherichia coli, adsorption, bacteria, biomass, carbohydrate binding, cellulose, cellulosome, dissociation, genome, polypeptides, recombinant proteins, rice straw, saccharification, scaffolding proteins, synergism, xylanases
Clostridium cellulovorans 743B, an anaerobic and mesophilic bacterium, produces an extracellular enzyme complex called the cellulosome on the cell surface. Recently, we have reported the whole genome sequence of C. cellulovorans, which revealed that a total of 4 cellulosomal scaffolding proteins: CbpA, HbpA, CbpB, and CbpC were encoded in the C. cellulovorans genome. In particular, cbpC encoded a 429-residue polypeptide that includes a carbohydrate-binding module (CBM), an S-layer homology module, and a cohesin. CbpC was also detected in the culture supernatant of C. cellulovorans. Genomic DNA coding for CbpC was subcloned into a pET-22b+ vector in order to express and produce the recombinant protein in Escherichia coli BL21(DE3). Measurement of CbpC adsorption to crystalline cellulose indicated a dissociation constant of 0.60 μM, which is a similar to that of CBM from CbpA. We also subcloned the region encoding xylanase B (XynB) with the dockerin from C. cellulovorans and analyzed the interaction between XynB and CbpC by GST pull-down assay. It was observed that GST-CbpC assembles with XynB to form a minimal cellulosome. The activity of XynB against rice straw tended to be increased in the presence of CbpC. These results showed a synergistic effect on rice straw as a representative cellulosic biomass through the formation of a minimal cellulosome containing XynB bound to CbpC. Thus, our findings provide a foundation for the development of cellulosic biomass saccharification using a minimal cellulosome.