Enzymatic hydrolysis of cellulose and hemicellulose can be carried out by highly specific cellulase and hemicellulase enzymes (glycosyl hydro­lases). This group includes at least 15 protein families and some sub­families [15, 27]. Enzymatic degradation of cellulose to glucose is generally accomplished by synergistic action of three distinct classes of enzymes [2]:

■ 1,4-^-D-glucan-4-glucanohydrolases or Endo-1,4-^-glucanases, which are commonly measured by detecting the reducing groups released from carboxymethylcellulose (CMC).

■ Exo-1,4-^-D-glucanases, including both 1,4-^-D-glucan hydrolases and 1,4-^-D-glucan cellobiohydrolases. 1,4-^-D-glucan hydrolases liberate D-glucose and 1,4-^-D-glucan cellobiohydrolases liberate D-cellobiose.

■ ^-D-glucoside glucohydrolases or |3-D-glucosidases, which release D — glucose from cellobiose and soluble cellodextrins, as well as an array of glycosides.

There is a synergy between exo—exo, exo—endo, and endo—endo enzymes, which has been demonstrated several times.

Substrate properties, cellulase activity, and hydrolysis conditions (e. g., temperature and pH) are the factors that affect the enzymatic hydroly­sis of cellulose. To improve the yield and rate of enzymatic hydrolysis, there has been some research focused on optimizing the hydrolysis process and enhancing cellulase activity. Substrate concentration is one of the main factors that affect the yield and initial rate of enzymatic hydrolysis of cellulose. At low substrate levels, an increase of substrate concentration normally results in an increase of the yield and reaction rate of the hydrolysis. However, high substrate concentration can cause substrate inhibition, which substantially lowers the rate of hydrolysis, and the extent of substrate inhibition depends on the ratio of total sub­strate to total enzyme [12].

Increasing the dosage of cellulases in the process to a certain extent can enhance the yield and rate of hydrolysis, but would significantly increase the cost of the process. Cellulase loading of 10 FPU/g (filter paper units per gram) of cellulose is often used in laboratory studies because it provides a hydrolysis profile with high levels of glucose yield in a reasonable time (48-72 h) at a reasonable enzyme cost. Cellulase enzyme loadings in hydrolysis vary from 5 to 33 FPU/g substrate, depend­ing on the type and concentration of substrates. ^-glucosidase acts as a limiting agent in enzymatic hydrolysis of cellulose. Adding supplemental ^-glucosidase can enhance the saccharification yield [28, 29].

Enzymatic hydrolysis of cellulose consists of three steps [12]: (1) adsorp­tion of cellulase enzymes onto the surface of cellulose, (2) biodegrada­tion of cellulose to simple sugars, and (c) desorption of cellulase. Cellulase activity decreases during hydrolysis. Irreversible adsorption of cellulase on cellulose is partially responsible for this deactivation. Addition of surfactants during hydrolysis is capable of modifying the cel­lulose surface property and minimizing the irreversible binding of cel — lulase on cellulose. Tween-20 and Tween-80 are the most efficient nonionic surfactants in this regard. Addition of Tween-20 as an additive in simultaneous saccharification and fermentation (SSF) at 2.5 g/L has several positive effects in the process. It increases the ethanol yield, increases the enzyme activity in the liquid fraction at the end of the process, reduces the amount of enzyme loading, and reduces the required time to attain maximum ethanol concentration [30].