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14 декабря, 2021
Three serious drawbacks have been noted with cellulases — especially of fungal origin — for the efficient saccharification of cellulose and lignocellulosic materials on the industrial and semi-industrial scales. First, cellulases have often been described as being catalytically inferior to other glycosidases. This statement is certainly true when crystalline cellulose is the substrate for cellulase action.84 When more accessible forms of cellulose are hydrolyzed, the catalytic efficiency increases, but comparison with other glycosidases shows how relatively poor are cellulases even with low-molecular-weight substrates (table 2.6).
Second, cellulases from organisms not normally grown at elevated temperatures have poor stability at incubation temperatures higher than 50°C typically used for cellulose digestion: fungal cellulases show half-life times at 65°C as low as 10 minutes, whereas thermophilic clostridial enzymes may be stable for periods longer than ten days.84 This is one of the much-explored causes for the rapid decline of hydrolysis rate when cellulase is mixed with cellulose as a substrate.60 Another possible explanation for this third problem encountered with cellulase-mediated saccharification is the inhibition of cellulases by cellobiose, a major immediate product of cellulase action; a strong product inhibition would be a major drawback, limiting the amounts of soluble sugars that can be produced unless they are rapidly removed; in other words, batch hydrolysis would be inferior to a continuous process with withdrawal of the fermentable sugars or a simultaneous release and utilization of cello — biose would be required. Although such a product inhibition is readily demonstrable
TABLE 2.6 Catalytic Parameters for Fungal Cellulases
Source: Data from Klyosov.84 |
with low-molecular-weight soluble substrates for cellulase, macromolecular cellulose does not show a marked sensitivity to cellobiose.85 The hydrolysis of cellobiose by P-glucosidase to yield free glucose is itself inhibited by glucose.86
The failure to maintain the initial rate of degradation of macromolecular cellulose, however, necessitates an explanation involving the interaction between cellulose and cellulase, and various formulations of a hypothesis have been made in which the “reactivity” of the cellulose decreases during cellulase digestion.60 A more intuitive line of reasoning is simply that, cellulose being macroscopic and inevitably heterogeneous on an enzyme protein scale, cellulases would attack most rapidly any sites on the cellulose substrate that are, by their very nature, most vulnerable (e. g., with directly accessible regions of the glucan polymer chain); once these sites are cleaved, the active sites remain as intrinsically active as they were initially but most then are restricted to operating at less accessible regions of the available surface. The experimental evidence is contradictory: although scanning electron microscopy has revealed changes in the conformations and packing of cellulose microfibers during prolonged cellulase-catalyzed hydrolysis, “restart” experiments (where the cellulase is removed and fresh enzyme added) show that, at least with crystalline cellulose, no rate-limiting decrease in substrate site accessibility occurs while enzyme action continues.8788 Detailed kinetic models have postulated physical hindrance to the movement of exoglucanases along the cellulose and a time variation in the fraction of the P-glucosidic bonds accessible by cellulases bound to the macromolecular cellulose surfaces as factors leading to the failure to maintain hydrolysis rates.89,90