The enzyme of interest is cellulase, needed for the hydrolysis of the cellulose, that is, cellulolysis. Cellulase is a multicomponent enzyme system consisting of endo-p-1,4-glycanases, exo-^-1,4-glucan glucohydrolases, and exo-p-1,4- glucan cellobiohydrolase. Cellobiose is the dominant product of this system but is highly inhibitory to the enzymes and is not usable by most organisms. Cellobiase hydrolyzes cellobiose to glucose, which is much less inhibitory and highly fermentable. Many fungi produce this cellobiase and most of the work that is presently being conducted is on Trichoderma reesei (viride). The cellulase produced by T. reesei is much less inhibited than other cellulases that have a major advantage for industrial purposes [58].

The type of inhibition exhibited by cellulases is the subject of much debate in research. Although most of the researchers favor competitive inhibition [59-64], some cellulases are noncompetitively [46, 62, 65, 66] or uncompeti­tively inhibited [60]. Uncompetitive inhibition takes place when an enzyme inhibitor binds only to the complex formed between the enzyme and the substrate, whereas noncompetitive inhibition takes place when an enzyme inhibitor and the substrate may both be bound to the enzyme at any given time. On substrates such as Solka Floc® (purified cellulose), wheat straw, and bagasse (biomass remaining after sugarcane stalks are crushed to extract their juice), Trichoderma reesei produced enzyme is competitively inhibited by glucose and cellobiose. On the other hand, some enzymes are noncom­petitively inhibited by cellobiose using other substrates such as rice straw and Avicel®. Avicel is a registered trade name for microcrystalline cellulose that has been partially hydrolyzed with acid and reduced to a fine powder; it is used as a fat replacer. Trichoderma viride is uncompetitively inhibited by glucose in a cotton waste substrate [60].

Many mutants have been produced following Trichoderma reesei. The most prominent among these is the Rut C-30 [67], the first mutant with p-glucosidase production [43]. Other advantages of the strain include its hyperproduc — ing properties and the fact that it is carbolite-repression resistant. The term hyperproduction means excessive production.

Cellulases from thermophilic bacteria have also been extensively exam­ined. Among these, Clostridium thermocellum is perhaps the most extensively characterized organism. C. thermocellum is an anaerobic, thermophilic, cel­lulolytic, and ethanogenic bacterium capable of directly converting cellulosic substrate into ethanol. The enzymes isolated from thermophilic bacteria may have superior thermostability and hence will have longer half-lives at high temperatures. Although this is not always the case, cellulases isolated from Clostridium thermocellum have high specific activities [68], especially against crystalline forms of cellulose that have proven to be resistant to other cel — lulase preparations.

Enzyme production with Trichoderma reesei is difficult because cellulase production discontinues in the presence of easily metabolizable substrates. Thus, most production work has been carried out on insoluble carbon sources such as steam-exploded biomass or Solka-Floc [69]. Solka-Floc is composed of beta-1, 4-glucan units, is white, odorless, and flavorless, and has varying particle sizes [70]. In such systems, the rate of growth and cellulase produc­tion is limited because the fungi must secrete the cellulase and carry out slow enzymatic hydrolysis of the solid to obtain the necessary carbon. Average productivities have been approximately l00 IU/L/hr. [Hydrolytic activity of cellulose is generally in terms of international filter unit (IU). This is a unit defined in terms of the amount of sugar produced per unit time from a strip of Whatman filter paper.] The filter paper unit is a measure of the combined activities of all three enzymes on the substrate. High productivities have been reported with Trichoderma reesei mutant in a fed-batch system using lactose as a carbon source and steam-exploded aspen as an inducer. Although lactose is not available in sufficient quantities to supply a large ethanol industry, this does suggest that it may be possible to develop strains that can produce cel- lulases with soluble carbon sources such as xylose and glucose.

Productivity increases dramatically reduce the size and cost of the fer­menters used to produce the enzyme. More rapid fermentation technolo­gies would also decrease the risk of contamination and might allow for less expensive construction. Alternatively, using a soluble substrate may allow simplification of fermenter design or allow the design of a continuous enzyme production system. Low-cost but efficient enzymes for lignocellu — losic ethanol technology must be developed in order to reduce the opera­tional cost and improve the productivity of the process.