Synergism is an enhanced effect of two or more enzymes when acting cooperatively, compared with their additive effect. Giligan and Reese (1954) first demonstrated synergism between cellulase components during the hydrolysis of cellulose. Subsequently several research groups have demonstrated synergism between endo-and exoglucanases during the solubilization of crystalline cellulose (Wood et al., 1988, 1989; Klyosov, 1990; Bhat et al, 1994). Five different types of synergism have been reported between fungal cellulase components: (i) a non-hydrolytic protein called C1 and endoglucanase (Reese et al., 1950); (ii) P-glucosidase and either endoglucanase or CBH (Eriksson and Wood, 1985); (iii) two immunologically related and distinct CBHs (Wood and McCrae, 1986); (iv) endoglucanase and CBH from either the same or different microorganisms (Wood et al., 1989); and (v) between two endoglucanases (Klyosov, 1990). In addition, synergism between bacterial and fungal cellulases, as well as between the subunits of the C. thermocellum cellulosome, has been reported (Bhat et al., 1994; Wood et al., 1994).
Synergism between fungal cellulase components has been studied most extensively (Coughlan and Ljungdahl, 1988; Wood et al., 1988, 1989; Klyosov, 1990). The most interesting types of synergism are between: (i) endoglucanase/ exoglucanase (CBH); (ii) exoglucanase (CBH)/exoglucanase (CBH); and (iii) endoglucanase/endoglucanase. An early model postulated by Wood and McCrae (1972) suggested that cellulose chains cleaved by endoglucanase become the substrate for exoglucanase, and that these two enzymes cooperatively degrade the cellulose. However, this model did not explain the synergism seen between two different CBHs, or the inability of CBH to synergize with endoglucanases from different microorganisms. Subsequently, using highly purified endoglucanases and CBHs from P. pinophilum, it was demonstrated that only two endoglucanases (EGIII and EGV), which were strongly adsorbed on to cellulose, best synergized with CBHs I and II (Fig. 2.4; Wood et al., 1989). The authors explained the observed synergism between CBHs I and II and endoglucanases in terms of the different stereo-specificities of these two enzymes.
The attack of a cellulose chain by a stereospecific endoglucanase will generate only one of two possible types of non-reducing ends, which will be hydrolysed by a stereospecific CBH. The successive removal of cellobiose by CBH will expose another chain-end of a different configuration, which will be attacked by the other stereospecific CBH. Hydrolysis of the two chain-ends by two CBHs acting randomly, together with attack of the cellulose chains by another stereospecific endoglucanase to generate a reducing end of a different configuration, would facilitate the synergism observed between these enzymes. Nevertheless, it was argued that the above synergism could be due to the strong adsorption of CBHs and endoglucanases on to cellulose (Klyosov, 1990). Furthermore, Klyosov (1990) reported that two endoglucanases which adsorbed strongly on to cellulose degraded the cellulose synergistically. Although the adsorption appeared to be important, it is difficult to explain the contribution of adsorption towards synergistic interaction
Fig. 2.4. Synergism between P. pinophilum CBHs (I and II) and endoglucanases (EI to EV) in solubilizing cotton fibre. Reproduced from Biochemical Journal, Vol. 260, Wood, T.M., McCrae, S.I. and Bhat, K.M., The mechanism of fungal cellulase action. Synergism between enzyme components of P. pinophilum cellulase in solubilizing hydrogen bond-ordered cellulose, pp. 37-43, 1989, with permission from Portland Press, London.
between endoglucanases and CBHs. Recent studies (Barr et al., 1996; Teen, 1997) revealed that there are two classes of CBHs (exoglucanases) which attack cellulose chains from both reducing and non-reducing ends. Based on these results, it was speculated that the synergism observed between CBH/CBH (exo/exo) was due to their ability to expose new hydrolysis sites to each other, as well as their ability to act from reducing and non-reducing ends (Barr et al., 1996). Thus, two CBHs acting from reducing and non-reducing ends and an endoglucanase appeared to be essential for the effective hydrolysis of crystalline cellulose. The presence of another endoglucanase with different substrate specificity would increase the synergistic efficiency further, as was observed in the case of the P. pinophilum cellulase system (Wood et al., 1989).
Recent studies of the C. thermocellum cellulase system revealed that two cellulosomal exoglucanases (S5 and S8 subunits) and an endoglucanase (S11 subunit), together with the S1 (scaffoldin) subunit, are essential for maximum synergism during the hydrolysis of crystalline cellulose (Bhat et al., 1994). Furthermore, the S1 subunit mediated the formation of an enzyme complex with the S5, S8 and S11
subunits (Bhat et al., 1994). Using recombinant scaffoldin (CipA) polypeptides and the endoglucanase CelD, it was demonstrated that a truncated scaffoldin polypeptide with a CBD and a single cohesin domain was adequate for the maximum activity of CelD towards Avicel (Kataeva et al., 1997; Beguin et al., 1998). It was also reported that linkage of the CelD-CipA complex with the CBD was critical for maximum synergism during Avicel solubilization, rather than the clustering of catalytic domains (Kataeva et al., 1997; Beguin et al., 1998). These results strongly indicate that, in the case of the aggregated C. thermocellum cellulase system, assembly of an enzyme complex is crucial for the maximum synergistic interaction of subunits during the solubilization of crystalline cellulose. Other studies using recombinant cellulosomal components have confirmed the important role of the CipA CBD in efficient cellulolysis (Ciruela et al., 1998).
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