## 3 Results and Discussion
### Enhanced Crystalline Cellulose Degradation by GH Catalytic Moieties Incorporated with CBM
The original, full images are provided in the Supplementary Material (Figure S2). Assembly of Multiple Catalytic Sites Leads to No Significant Synergistic Effect on Cellulose Digestion  
_A. thermocellus_ expresses several types of cellulosomal GHs with conserved dockerin domains. It also expresses a scaffoldin protein with multiple conserved cohesion domains, which are the counter-binding partners of dockerin, to align a diverse set of the enzymes to the scaffoldin [47]. This fact suggests that the assembly of different types of GHs is beneficial for cellulose degradation. Therefore, artificial cellulosomes composed of various cellulose-digesting enzymes have been developed to achieve synergistic cellulose digestion [48], where EGs open up the polysaccharide strand in between and CBHs are expected to start processing from the newly opened ends. The PCNA heterotrimer can load up to three proteins and is therefore capable of constructing multienzyme complexes in different formats. We assembled the EG-derived catalytic moiety, Cel9* or Cel9**, and the CBH-derived catalytic moiety, Cbh9A*, together with CBM3a using the PCNA heterotrimer to evaluate the effect of clustering different GHs on crystalline cellulose hydrolysis. Unlike the PCNA1 and PCNA2 subunits, which pair equivalently with each other, the PCNA3 subunit is unique in that it can only associate with the PCNA1:PCNA2 heterodimer and not with a PCNA1 or PCNA2 monomer alone. Consequently, CBM3a was fused to PCNA3 for assembly with (i) Cel9* fused to PCNA1 and Cbh9A* fused to PCNA2 and (ii) Cel9** fused to PCNA1 and Cbh9A* fused to PCNA2. Based on the expectation of efficient synergistic multicatalytic reactions even without the incorporation of CBM3a, cellulose degradation was evaluated in combinations of the constructed fusion proteins of the EG-derived catalytic moiety and the CBH-derived catalytic moiety (Figure 4). The bicatalytic complexes composed of Cel9* and Cbh9A* (i.e., P1-Cel9*:P2-Cbh9A* and P1-Cbh9A*:P2-Cel9*) showed higher crystalline cellulose hydrolysis activities than either of their components, Cel9* and Cbh9A*, alone. However, the increase in the total amount of reducing sugars produced by the bicatalytic complexes (1.4-fold) seems to be additive rather than synergistic, compared to the sum of those produced by Cel9* and Cbh9A* separately. Similar results were obtained for the bicatalytic complexes composed of Cel9** and Cbh9A* (i.e., P1-Cel9**:P2-Cbh9A* and P1-Cbh9A*:P2-Cel9**). These results suggest that the two catalytic moieties in the bicatalytic complexes do not, or only to a limited extent, act cooperatively on crystalline cellulose substrates. Figure 4: Cellulose degradation by bicatalytic complexes. The total amounts of reducing sugars after an 18 h incubation at 55 \({}^{\circ}\)C of 8% (\(\omega/\tau\)) Avicel with catalytic moieties (0.1 \(\upmu\)M each) were evaluated. (**A**) P1-Cel9* or P1-Cel9** was incubated with P2-Cbh9A* in the absence or presence of P3-CBM3a (2.5 \(\upmu\)M). (**B**) P2-Cel9* or P2-Cel9** was incubated with P1-Cbh9A* in the absence or presence of P3-CBM3a (2.5 \(\upmu\)M). 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** or P2-Cel9** alone (ND). Similarly to the individual enhancement of Cel9*, Cel9**, and Cbh9A* by assembly with CBM3a, as shown in Figure 3, the activities of the bicatalytic complexes composed of Cel9* and Cbh9A* and of Cel9** and Cbh9A* were significantly enhanced by the assembly with CBM3a through the heterotrim formation of the PCNA subunits (Figure 4). Interestingly, the degree of increase was not affected by the components of the bicatalytic complexes. The increase in the activity of P1-Cel9*:P2-Cbh9A* was not higher than the increase in the activity of P1-Cel9* by assembly with CBM3a.