Cellulose is a structural polysaccharide of plants. It is said to be the most abundant organic substance on earth. An estimated 10.sup.11 tons of cellulose are synthesized each year by plants, using photosynthetic energy derived from sunlight. Cellulose is composed essentially of repeating subunits of D-glucose, linked by .beta.-(1.fwdarw.4)-glycosidic bonds. Total hydrolysis yields D-glucose and partial hydrolysis yields the disaccharide cellobiose, which is 0-.beta.-D-glucopyranosyl-(1.fwdarw.4)-.beta.-D-glucopyranose. Therefore, cellulose is a .beta.-1,4-glucan. Although closely related to starch in composition, the glucose units of cellulose are connected differently than in starch, and this fact profoundly affects their properties. Cellulose is more insoluble than starch and is capable of forming long semicrystalline microfibrils. A closely related polymer, .beta.-1,3-glucan, can only form amorphous structures.
Cellulose constitutes the major storage form of photosynthesized glucose, and the major component of solar energy converted to biomass. World wide demand for energy and for food supplies are increasing. Cellulose is an attractive raw material for supplying these needs, because of its abundance. The glucose subunits of cellulose can be used in a variety of processes for production of energy on the one hand or for the production of protein on the other. A major difficulty which has stood athwart the advance of cellulose utilization technology has been the difficulty of obtaining glucose in reasonable yield from cellulose at a reasonable cost in terms of energy input, equipment requirements and the like. Enzyme-catalyzed hydrolysis of cellulose is an attractive potential solution to these difficulties. However, the production of adequate amounts of cellulase is dependent upon obtaining a suitable source of large quantities of the enzyme in a reasonably pure state.
Cellulases are found in the digestive tracts of snails, in certain anaerobic bacteria and in other microorganisms, most notably the rumen microorganisms which inhabit digestive tracts of ruminants. A number of fungal species are known to produce cellulase, including fungi of the class Ascomycetes, such as Neurospora and Trichoderma. The fungal systems are perhaps the most attractive because the organisms can be cultured without resort to special growth conditions such as anaerobiosis, and some, at least, are capable of rapid growth.
The fungal system described herein is derived from Trichoderma reesei, hereinafter T. reesei, an Ascomycete fungus species formerly assigned to the species Trichoderma viride. In general, any Ascomycete fungus capable of synthesizing a complete cellulase could be used to derive a strain having similar properties. T. reesei is presently preferred because large amounts of cellulase are produced extracellularly. See, Simmons, E. G., Abstracts of Second International Mycology Congress, Tampa, Florida, page 618 (1977). The cellulase system produced by this species include an endo-.beta.-glucanase, exo-.beta.-glucanase and .beta.-glucosidase. The first of these enzymes is capable of hydrolyzing .beta.-glucosidic bonds at mainly internal sites on the cellulose molecule. The second is capable of catalyzing the hydrolytic removal of disaccharide subunits from the ends of the cellulose chain, yielding mainly cellobiose as a product. The .beta.-glucosidase catalyzes the hydrolysis of cellobiose to glucose. The term cellulase, as used herein, includes all such enzymes including isozymic forms. The cellulase produced by T. reesei is found in the growth medium. Synthesis of cellulase by the wild type T. reesei is under stringent metabolic and genetic control, in which both induction and repression are observed. The term induction is used herein, as in the art generally, to mean that presence of the substrate to be acted upon by the enzyme, or an analog thereof, is necessary for the synthesis of the enzyme by the organism. Repression is a term used to describe the phenomenon in which the presence of a substance in the growth medium is sufficient to prevent the synthesis of the enzyme. The presence of a repressor substance for a particular enzyme prevents the expression of the gene coding for that enzyme, and the presence of an inducer substance is additionally required for expression of the gene. In cultures of wild type T. reesei, cellulose acts as an inducer of cellulase synthesis and its presence is therefore required in the medium to obtain appreciable levels of cellulase synthesis. A number of substances act as repressors, notably glucose and glycerol. The necessary conditions for cellulase synthesis therefore are the presence of cellulose and the near absence of glucose. However, as cellulase is synthesized and cellulose in the medium is degraded, glucose is produced, which may result in the repression of enzyme synthesis. Consequently, the levels of cellulase produced by the wild type strain are never very great. Furthermore, the synthesis of cellulase is characterized by a post repression lag period. Once the growth medium has been exhausted of glucose, synthesis of cellulase, even in the presence of an inducer, does not begin for several hours. Consequently, maximal enzyme production requires mutational alteration of the wild type strain, to modify the stringent controls normally limiting the rate of expression of the cellulase genes.