The consequence of these limitations of cellulases as catalysts for the degradation of cellulose is that large amounts of cellulase have been considered necessary to rapidly process pretreated lignocellulosic substrates, for example, 1.5-3% by weight
of the cellulose, thus imposing a high economic cost on cellulose-based ethanol production.91 From the standpoint of the established enzyme manufacturers, this might have been a welcome and major expansion of their market. The multiplicity of other uses of cellulases meant, however, that competition resulted in massive increases in cellulase fermentation productivity — in the 1980s, space-time yields for cellulases increased by nearly tenfold; between 1972 and 1984, total cellulase production doubled every two years by the selection of strains and the development of fed-batch fermentation systems.70 Some trends in Hypocrea jecorina cellulase productivity and costs were published by the Institut Frangais du Petrole from their pilot-plant scale-up work (figure 2.8).92 By 1995, the productivities in industrial fermentations were probably 400% higher, and process intensification has undoubt­edly continued.93 The development of some of the H. jecorina strains from one originally isolated at the U. S. Army Laboratory (Natick, Massachusetts) by physical and chemical mutagenesis treatments has also been described (figure 2.9).94 From a baseline price of $15/kg in 1990, cellulases have decreased markedly, but they still average three to five times the cost of the much more readily produced (and more enzymically active) amylases.95

Major enzyme manufacturers including Genencor (Palo Alto, California), Novozymes (Denmark), Iogen Corporation (Canada), and Rohm (Finland) have long produced cellulases from organisms including H. jecorina, Aspergillus ory — zae, A. niger, Humicola insolens, and Penicillium spp. Different producing organ­isms yield cellulases with different profiles of cellulase components (figure 2.10), and nonbiofuel markets recognize premium products from particular biological sources with optimum properties for particular applications.70

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For all microbial producers of cellulase, however, a key area of knowledge is that of the genetic regulation of cellulase synthesis in the life cycle of the organism and — most acutely — when the producing cells are functioning inside the fermentor. For a fungus such as H. jecorina, the evidence points to the low-level basal production
of cellulase components generating an inducer of rapid cellulase synthesis when cellulose enters the nutritional environment:

• Deletion of the genes coding for discrete cellulase components prevents the expression of other cellulase genes.96,97

• The general carbon catabolite repressor protein CRE1 represses the transcription of cellulase genes, and a hyperproducing mutant has a crel mutation rendering cellulase production insensitive to glucose.98,99

• Production of cellulases is regulated at the transcriptional level, and two genes encoding transcription factors have been identified.100-102

• The disaccharide sophorose (P-1,2-glucopyranosyl-D-glucose) is a strong inducer of cellulases in H. jecorina.103-105

• Cellulases can also catalyze transglycosylation reactions.106

Low activities of cellulase are theorized to at least partially degrade cellulose, liberating cellobiose, which is then transglycosylated to sophorose; the inducer stimulates the transcription of cellulase genes, but this is inhibited if glucose accumulates in the environment, that is, there is a triple level of regulation.107-109 This overall strategy is a typical response of microbes to prevent the “unneces­sary” and energy-dependent synthesis and secretion of degradative enzymes if readily utilizable carbon or nitrogen is already present. Augmenting this elabo­rate mechanism, sophorose also represses P-glycosidase; because sophorose is hydrolyzed by P-glycosidase, this repression acts to maintain sophorose concentra­tions and thus maximally stimulate cellulase formation.105 Another disaccharide, gentiobiose (P-1,6-glucopyranosyl-D-glucose), also induces cellulases in H. jeco­rina.105 In anaerobic cellulolytic bacteria, cellobiose may be the inducer of cellu­lase, but whether induction occurs in anaerobic bacteria is unclear.60 In at least one clostridial species, cell wall-attached cellulosomes are formed during growth on cellulose but not on cellobiose; although the cellulase synthesis is determined by carbon catabolite repression, cellulose (or a breakdown product) is a “signal” that can be readily recognized by the cells.69,110

Can sophorose be used to increase fungal cellulase expression in fermentations to manufacture the enzyme on a large scale? As a fine chemical, sophorose is orders of magnitude more expensive than glucose,[15] and its use (even at low concentrations) would be economically unfeasible in the large fermentors (>50,000 l) mandated for commercial enzyme manufacture. Scientists at Genencor discovered, however, that simply treating glucose solutions with H. jecorina cellulase could generate sophorose — taking advantage of the transglycosylase activity of P-glucosidase — to augment cellulase expression and production in H. jecorina cultures.111 Lactose is used indus­trially as a carbon source for cellulase fermentations to bypass the catabolite repres­sion imposed by glucose but adding cellulase-treated glucose increased both cellulase production and the yield of enzyme per unit of sugar consumed (figure 2.11).

The improving prospects for cellulase usage in lignocellulosic ethanol produc­tion has engendered an intense interest in novel sources of cellulases and in cellu — lase-degrading enzymes with properties better matched to high-intensity cellulose saccharification processes. Both Genencor and Novozymes have demonstrated tangible improvements in the catalytic properties of cellulases, in particular, thermal stability; such enzyme engineering involved site-directed mutagenesis and DNA shuffling (table 2.7). Other discoveries patented by Genencor derived from an extensive and detailed study of gene expression in H. jecorina that revealed 12 previously unrecognized enzymes or proteins involved in polysaccharide degradation; some of these novel proteins may not function directly in cellulose hydrolysis but could be involved in the production and secretion of the cellulase complex or be relevant when other polysaccharides serve as growth substrates.112 The “traditional” and long-established four major components of H. jecorina cellulase — two cellobiohydrolases and two endoglucanases — together constitute more than 50% of the total cellular protein produced by the cells under inducing conditions and can reach more than 40 g/l in contemporary industrial strains that are the products of many years of strain development and selection.112,113 The poor performance of H. jecorina as a cellulase producer — once described as the result of nature opting for an organism secreting very large amounts of enzymically incompetent protein rather than choosing an organism elaborating small amounts of highly active enzymes84 — have engendered many innovative and speculative

TABLE 2.7

Post-2000 Patents and Patent Applications in Cellulase Enzymology and Related Areas

studies on radical alternatives to the “Trichoderma cellulase” paradigm. Proxi­mal to commercially realizable applications are cellulases immobilized on inert carriers that can offer significant cost savings by the repeated use of batches of stabilized enzymes.114115 With a commercial P-glucosidase from Aspergillus niger, immobilization resulted in two important benefits: greatly improved thermal stability at 65°C and a quite unexpected eightfold increase in maximal enzyme activity at saturating substrate concentration, as well as operational stability dur­ing at least six rounds of lignocellulose hydrolysis.116

Evidence for an unambiguously novel type of cellulose-binding protein in H. jecorina has, however, resulted from the discovery of a family of “swollenins,” proteins that bind to macroscopic cellulose and disrupt the structure of the cellulose fibers without any endoglucanase action.117 This fulfils a prediction made by some of the pioneers of cellulase biotechnology who envisaged that “swelling” factors would be secreted by the fungus to render cellulose more susceptible to cellulase-catalyzed attack.118 Fusing cellulose-disrupting protein domains with cellulase catalytic domains could generate more powerful artificial exo — and endoglucanases. Whether cellulose­binding domains/modules in known cellulases disrupt cellulose structures remains unclear.119 Adding a nonionic detergent to steam-pretreated barley greatly increased total polysaccharide saccharification, and the detergent may have been acting partly as a lignocellulose disrupter.120 An unexpected potential resource for laboratory-based evolution of a new generation of cellulases is the very strong affinity for cellulose exhibited not by a cellulase but by a cellobiose dehydrogenase; nothing has yet been disclosed of attempts to combine this binding activity with cellobiohydrolases.121

The relatively low activities of P-glycosidases in fungal cellulase preparations — possibly an unavoidable consequence of the sophorose induction system—have been considered a barrier to the quantitative saccharification of cellulose. Supplementing H. jecorina cellulase preparations with P-glucosidase reduces the inhibitory effects caused by the accumulation of cellobiose.122 Similarly, supplementing cellulase preparations with P-glucosidase eliminated the measured differences in saccharification rates with solvent-pretreated hardwoods.123 Mixtures of cellulase from different cellulolytic organisms have the advantage of maximally exploiting their native traits.124 Additionally, such a methodologically flexible approach could optimize cellulase saccharification for a single lignocellulosic feedstock with significant seasonal or yearly variation in its composition or to process differing feedstock materials within a short or medium-length time frame.

Finally, novel sources of cellulases have a barely explored serendipitous potential to increase the efficiency of saccharification; for example, cellulases from “nonstandard” fungi (Chaetomium thermophilum, Thielavia terrestris, Thermoascus aurantiacus, Corynascus thermophilus, and Mycellophthora thermophila), all thermophiles with high optimum growth temperatures in the range of 45-60oC, improved the sugar yield from steam-pretreated barley straw incubated with a benchmark cellulase/ P-glucosidase mix; the experimental enzyme preparations all possessed active endo — glucanase activities.125 “Biogeochemistry” aims to explore the natural diversity of coding sequences available in wild-type DNA.126 Forest floors are an obvious source of novel microbes and microbial communities adept at recycling lignocelluloses.