Cellulose is a linear polymer of 8,000-12,000 D-glucose units linked by 1,4-B-D — glucosidic bonds. The enzyme system for the conversion of cellulose to glucose comprises endo-1,4-B-glucanase (ЕС 3.2.1.4), exo-1, 4-B-glucanase (EC 3.2.1.91) and B-glucosidase (EC 3.2.1.21). Cellulolytic enzymes with B-glucosidase act sequentially and cooperatively to degrade crystalline cellulose to glucose. Endoglucanase acts in a random fashion on the regions of low crystallinity of the cellulosic fiber whereas exoglucanase removes cellobiose (B-l, 4 glucose dimer) units from the non-reducing ends of cellulose chains. Synergism between these two enzymes is attributed to the endo — exo form of cooperativity and has been studied extensively between cellulases in the degradation of cellulose in Trichoderma reesei (11). B-Glucosidase hydrolyzes cellobiose and in some cases cellooligosaccharides to glucose. The enzyme is generally responsible for the regulation of the whole cellulolytic process and is a rate limiting factor during enzymatic hydrolysis of cellulose as both endoglucanase and

cellobiohydrolase activities are often inhibited by cellobiose (12-14). Thus, P — glucosidase not only produces glucose from cellobiose but also reduces cellobiose inhibition, allowing the cellulolytic enzymes to function more efficiently. However, like B-glucanases, most 6-glucosidases are subject to end-product (glucose) inhibition. The kinetics of the enzymatic hydrolysis of cellulose including adsorption, inactivation and inhibition of enzymes have been studied extensively (75). For a complete hydrolysis of cellulose to glucose, the enzyme system must contain the three enzymes in right proportions.

Product inhibition, thermal inactivation, substrate inhibition, low product yield and high cost of cellulase are some barriers to commercial development of the enzymatic hydrolysis of cellulose. Many microorganisms are cellulolytic. However, only two microorganisms (Trichoderma and Aspergillus) have been studied extensively for cellulase. There is an increasing demand for the development of thermostable, environmentally compatible, product and substrate tolerant cellulase with increased specificity and activity for application in the conversion of cellulose to glucose in the fuel ethanol industry. Thermostable cellulases offer certain advantages such as higher reaction rate, increased product formation, less microbial contamination, longer shelf — life, easier purification and better yield.

In our work, forty-eight yeast strains belonging to the genera Candida, Debaryomyces, Kluyveromyces and Pichia (obtained from the ARS Culture Collection, Peoria, IL) were screened for production of extracellular glucose tolerant and thermophilic B-glucosidase activity using p-nitrophenyl-6-D-glucoside as substrate (16). Enzymes from 15 yeast strains showed very high glucose tolerance (< 50% inhibition at 30%, w/v glucose). The optimal temperatures and pH for these B-glucosidase activities varied from 30 to 65°C and pH 4.5 to 6.5. The B-glucosidase from D. yamadae Y — 11714 showed highest optimal temperature at 65°C followed by C. chilensis Y — 17141(60°C) and K. marxianus Y-l 195 (60°C). The optimal pH of these three enzyme preparations were 6.5, 6.0 and 6.5, respectively. The temperature and pH profiles of p-glucosidases from C. chilensis Y-17141, D. yamadae Y-11714 and K. marxianus Y-l 195 are shown in Figure 1. The p-glucosidases from all these yeast strains hydrolyzed cellobiose. Novel glucose tolerance and thermoactivity found in the enzyme preparations from D. yamadae, K. marxianus and C. chilensis are desired

Table П. Biochemical characteristics of thermostable 0-glucosidase from Aureobasidium pullulans NRRL Y-12974 (77)

attributes of a P-glucosidase suitable for industrial application for enzymatic hydrolysis of cellulose to glucose. We have purified and characterized a highly thermophilic P — glucosidase from a color variant strain of Aureobasidium pullulans (17). Some properties of this enzyme are summarized in Table II.

The cellulose hydrolysis step is a significant component of the total production cost of ethanol from wood (18). Achieving a high glucose yield is necessary (>85%

theoretical) at high substrate loading (>10% w/v) over short residence times (< 4 days). It was shown that simultaneous saccharification (hydrolysis) of cellulose to glucose and fermentation of glucose to ethanol (SSF) improve the kinetics and economics of biomass conversion by reducing accumulation of hydrolysis products that are inhibitory to cellulase and P-glucosidase, reducing the contamination risk because of the presence of ethanol, and reducing the capital equipment requirements {19). An important drawback of SSF is that the reaction has to operate at a compromised temperature of around 30°C instead of enzyme optimum temperature of45-50°C. Enzyme recycling, by ultrafiltration of the hydrolyzate, can reduce the net enzyme requirement and thus lower costs {20). Hinman et al. {21) reported that a preliminary estimate of the cost of ethanol production for SSF technology based on wood-to-ethanol process is $ 1.22/gal of which the wood cost is $ 0. 459/gal. Wright et al. (22) evaluated a separate fungal enzyme hydrolysis and fermentation process for converting lignocellulose to ethanol. The cellulase enzyme was produced by the fungal mutant Trichoderma Rut C-30 (the first mutant with greatly increased P-glucosidase activity) in a fed batch production system that is the single most expensive operation in the process. The conversion of lignocellulosic biomass to fermentable sugars requires the addition of complex enzyme mixtures tailored for the process and parallel reuse and recycle the enzymes until the cost of enzymes comes down. Enzyme recycling can increase the rates and yields of hydrolysis, reduce the net enzyme requirements and thus lower costs (23). The first step in cellulose hydrolysis is considered as the adsorption of cellulase onto cellulosic substrate. As the cellulose hydrolysis proceeds, the adsorbed enzymes (endo — and exo-glucanase components) are gradually released in the reaction mixture. The P-glucosidase does not adsorb onto the substrate. These enzymes can be recovered and reused by contacting the hydrolyzate with the fresh substrate. However, the amount of enzyme recovered is limited because some enzymes remain attached to the residual substrate and some enzymes are thermally inactivated during hydrolysis. It has been shown that several substrates containing a high proportion of lignin result in the poor recovery of cellulase {24).

Gusakov et al. (25) found that cellolignin was completely converted to glucose by cellulase from I viride and A. foetidus. Cellolignin was an industrial residue obtained during the production of furfural from wood and com cobs when pretreated by dilute H2S04 at elevated temperature. The concentration of glucose in the hydrolyzate reached 4-5.5%, cellulose conversion being not less than 80%. Kinetic analysis of cellolignin hydrolysis, using a mathematical model of the process, has shown that, with product inhibition, nonspecific adsorption of cellulase onto lignin and substrate induced inactivation seem to affect negatively the hydrolysis efficiency. Borchert and Buchholz (26) investigated the enzymatic hydrolysis of different cellulosic materials (straw, potato pulp, sugar beet pulp) with respect to reactor design. The kinetics was studied including enzyme adsorption, inhibition, and inactivation. The results suggest the use of reactors with plug flow characteristics to achieve high substrate and product concentrations and to avoid back-mixing to limit the effect of product inhibition. For efficient use of cellulases, a reactor with semipermeable hollow fiber or an ultrafilter membrane was used and this allowed cellulases to escape end-product inhibition {27-30). A totally integrated biotechnology of rice straw conversion into ethanol was reported (37). It dealt with (i) ethanol refining of rice straw to segregate cellulose from pentose sugars and lignin, (ii) preparation of highly active mixed cellulase enzymes, (iii) a novel reactor system allowing rapid product formation involving enzymatic hydrolysis of cellulose to sugars followed by microbial conversion of the later into ethanol and its simultaneous flash separation employing a programmed recompression of ethanol vapors and condensation, and (iv) concentration of ethanol via alternative approaches.

In direct microbial conversion of lignocellulosic biomass into ethanol that could simplify the ethanol production process from these materials and reduce ethanol production costs, Clostridium thermocellum, a thermoanaerobe was used for enzyme production, hydrolysis and glucose fermentation (52). Cofermentation with C. thermosaccharolyticum simultaneously converted the hemicellulosic sugars to ethanol. However, the formations of by-products such as acetic acid and low ethanol tolerance are some drawbacks of the system. Several recent reviews have dealt with the molecular biology of cellulose degradation, cellulolytic enzyme systems, and the structure and function of various domains found in the enzymes involved (55-56).