To further determine the synergistic effects between CbpC-cellulosomes and mCbpA-cellulosomes, sequential reactions were carried out. PASC was treated with either CbpC or mCbpA cellulosomes for 4 h in the first reaction using a previously described method (Murashima et al., 2003). Next, the reaction mixtures were boiled for 20 min to inactivate the cellulosomes, which were used for the first reactions. By this heat treatment, the cellulosomes were inactivated completely (data not shown). After the reaction mixtures of the first reaction were boiled, the cellulosome that had not been used for the first reactions was added to the reaction mixtures, followed by incubation for an additional 4 h. The amount of liberated reducing sugars was then determined. The results are shown in Table 1. The synergistic degrees of the ONX-0912 hydrolytic activity of EngZ in the “simultaneous reactions” were 1.65 and 1.63. On the other hand, little synergistic effect was observed in the CbpC-cellulosome and mCbpA-cellulosome sequential reactions (1.38-fold) because their synergy degrees were similar to that of the single complex. Interestingly, the sequential reactions of CbpC cellulosome followed by mCbpA cellulosome (1.58-fold) treatment showed increased hydrolytic activity on cellulose compared with the CbpC cellulosome-mCbpA cellulosome sequential reactions. The same tendency was shown with the ExgS-containing cellulosome (Table 1). These results indicated that CbpC cellulosomes and mCbpA cellulosomes degraded PASC most synergistically in a simultaneous manner. Moreover, degradation by the CbpC cellulosome followed by the mCbpA cellulosome on cellulose was more efficient than the single cellulosome reaction.