## 3 Results and Discussion
### Enhanced Crystalline Cellulose Degradation by GH Catalytic Moieties Incorporated with CBM  
First, the GH catalytic moieties were assembled with a CBM to acquire cellulose-binding activity. PCNA1-fused Cel9* (P1-Cel9*), which lacks a crystalline cellulose-binding module, CBM3b, but has a substrate-feeding module, CBM3c, was found largely in the soluble fraction in the presence of an insoluble crystalline cellulose substrate (Figure 2), indicating the weak binding of P1-Cel9* to the substrate, as expected. In contrast, in the presence of equimolar PCNA2-fused CBM3a (P2-CBM3a), the fusion protein was almost completely co-precipitated with the crystalline cellulose substrate. This result indicates that the P1-Cel9* acquired the crystalline cellulose-binding ability of P2-CBM3a through the stable association of the PCNA1 and PCNA2 moieties. Next, the contribution of the assembled CBM to the cellulose hydrolysis activity of the catalytic GH moieties was evaluated using the DNS assay (Figure 3). The DNS assay was chosen to quantify total reducing sugars because our focus was on the extent of cellulose degradation rather than the identification of specific degradation products. The incorporation of the CBM3a module significantly improved the hydrolysis activity of P1-Cel9*. Furthermore, P1-Cel9**, which showed no detectable hydrolysis activity, probably due to the complete absence of CBMs, apparently acquired the crystalline cellulose hydrolysis activity by assembly with P2-CBM3a. PCNA1-fused Cbh9A* (P1-Cbh9A*), which lacks the two CBMs of the native cellulosomal CBH enzyme, was also stimulated in the presence of P2-CBM3a. These results clearly indicate that the improvement in the  
Figure 1: PCNA fused to functional moieties derived from _A. thermocellus_ cellulose-acting proteins. (**A**) Cel9* and Cel9** are derived from Cel9I. Cbh9A* is derived from Cbh9A. CBM3a is derived from CipA. (**B**) The PCNA subunit proteins, PCNA1, PCNA2, and PCNA3, fused to the functional protein moieties selectively form protein complexes in a stepwise manner. SP, signal peptide; GH9, family-9 GH catalytic domain; N-lg, N-terminal Ig-like domain. association with the crystalline substrate by the incorporated CBM3a is beneficial for the overall reaction rates of hydrolysis. The conserved aromatic amino residues on the surface of CBM3a, which are arranged in a planar geometry, are responsible for adhesion to a single polysaccharide chain. Moreover, the polar aromatic amino residues stabilize the overall association [43]. As a result, CBM3a is known to support the presentation of the substrate surface to the active site of GHs and also to prevent random diffusion [44]. The enhancement of hydrolase activity by CBM3-assisted substrate attachment has also been confirmed in artificial cellulosome studies [20; 45; 46]. Figure 3: The activity of cellulases linked to CBM3a on crystalline cellulose. The activity is presented as the amount of reducing sugars. Avicel (8% (\(w/v\))) was incubated with the PCNA1-fused catalytic moieties in the absence (open bars) or presence (filled bars) of P2-CBM3a (each component 0.1 uM) at 55 \({}^{\circ}\)C for 18 h. Error bars represent the standard deviation of the mean for three replication reactions. Reducing sugar concentration was below the detection limit when Avicel was incubated with P1-Cel9** in the absence of P2-CBM3a (not detectable, ND). Figure 2: Substrate binding of a cellulase catalytic moiety lacking a CBM via strong intermolecular linkage to CBM3a. The substrate association of P1-Cel9* and P2-CBM3a (each at 1 uM) on Avicel (8% (\(w/v\))) was tested. After short incubation at room temperature, the soluble proteins in the supernatant (S) were separated by centrifugation from substrate bound proteins, which remained in the pellet fraction (P). Avicel was washed with buffer (W) to remove nonspecifically bound proteins. Protein samples were subjected to a 10% SDS-PAGE. M, marker proteins. Images from different regions of the sample were juxtaposed.