Cellulose is organized into insoluble crystalline ribbons with extensive hydro­gen bonds between strands [98,99]. This structure is not easily hydrated and the fungal cellulase enzymes used for hydrolysis have low catalytic rates in comparison to other glycosidases. Thus, the cost of these enzymes is a ma­jor consideration in cellulose utilization [100]. An additional challenge is the feedback inhibition of cellulose hydrolysis by glucose and cellobiose, the products of the hydrolysis process. Simultaneous saccharification and fer­mentation (SSF), developed by Gulf Oil Company in 1976, combines cellulose saccharification and fermentation of the resultant glucose by Saccharoymyces in a single vessel [ 101,102]. Recent work in this area has focused on reducing the supplemental cellulase demand by engineering the biocatalysts to produce recombinant cellulase enzymes.

Erwinia chysanthemi contains two endoglucanases, CelZ and CelY, which work synergistically to degrade amorphous cellulose and carboxymethyl cel­lulose [103]. In order to effectively reduce the demand for cellulase supple­mentation, CelZ and CelY need to be expressed at high levels and secreted by the biocatalyst. The use of a surrogate Z. mobilis promoter and addition of the E. chrysanthemi out secretion system resulted in high levels of CelZ expres­sion in E. coli and K. oxytoca P2, with active glycan hydrolase representing approximately 5% of total cellular protein in both organisms [104,105]. High endoglucanase activity from recombinantly expressed CelZ and CelY enabled K. oxytoca M5A1 to produce ethanol from amorphous cellulose without the addition of supplemental cellulase enzymes [106,107].

As described above, the primary product of cellulose digestion by en — doglucanase and cellobiohydrolase is cellobiose. Unfortunately, cellobiose is a potent inhibitor of these enzymes [108]. The ability to metabolize cel — lobiose is widespread in prokaryotes [108] and is desirable for biomass­utilizing strains. Ethanologenic K. oxytoca P2 has the native ability to trans­port and metabolize cellobiose, reducing the initial demand for supplemental P-glucosidase [50]. The K. oxytoca cellobiose-utilization operon casAB has been functionally expressed in E. coli KO11, enabling production of ethanol from cellobiose or, with the aid of commercial cellulase, from mixed-waste office paper [50,109].

In the pursuit of a decreased supplemental cellulase demand, an alterna­tive approach to biocatalyst engineering is the use of non-biological processes to improve cellulose hydrolysis. For example, the use of ultrasound during SSF resulted in a 20% increase in ethanol production from mixed-waste office paper by K. oxytoca P2 [110]. Additionally, fungal cellulase demand dur­ing mixed waste office paper fermentation by P2 was reduced by recycling cellulase [54].

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