An enzyme preparation containing high cellobiase activity is useful for enzymatic hydrolysis of cellulose, leading to production of glucose, which in turn may be converted to ethanol to be used as an alternative and renewable energy source. Cellulose comprises the most abundant source of fermentable carbohydrates in the world as plant biomass. When biologically converted to fuels, such as ethanol and various other low-value high volumes commodity products, this vast resource can provide environmental, economic and strategic benefits on a large scale unparalleled by any other sustainable resource as referred by—Handbook on Bioethanol Production and utilization. Applied Energy Technology Series, Washington, D.C. Taylor and Francis (1996); and Science (1991), volume 251, pages 1318-1323; and Annual Review of Energy and Environment (1996), volume 21, pages 403-465.
Reese E. T. and Mandels M. during 2nd World war detected cellulase enzyme activity in Trichoderma viride, when the fungus destroyed clothing and plants. Following the war they turned their efforts towards discerning the biochemical mechanisms of cellulase action to convert cellulosic biomass into glucose syrup. Reference may be made to Reese E. T. and Mandels M. (1971). Cellulose and cellulose derivatives (Eds. Bikales, N and Segal. L.) John Wiley & Sons, pages 1079-1094; wherein it was emphasized that cellulase is not a single enzyme but a group of enzymes e.g. endo-glucanase, cellobiohydrolase and cellobiase, which must act synergistically to achieve saccharification of cellulose. Reference may be made to Nisizawa K. (1973) Journal of Fermentation Technology, volume 51, pages 267-304; and Wood T. M. (1975) Biotechnology and Bioengineering Symposium, volume 5, pages 111-137, wherein it was proposed that an initial random attack on the cellulose molecule was achieved by the action of the endo-glucanase (Enzyme Commission number 3.2.1.4) and followed immediately by the action of the cellobiohydrolase (Enzyme Commission Number 3.2.1.91), which released the disaccharide cellobiose, from the non-reducing end of the cellulose. Continued synergism between the two enzymes resulted in solubilization of the cellulose into sugar syrups containing small oligosaccharides and cellobiose. The activity of third enzyme, cellobiase (Enzyme Commission Number 3.2.1.21) is required if glucose syrups are envisaged as the product. Cellobiase converts the oligosaccharides of glucose with degree of polymerization 2 to 7 (G2-G7) into glucose.
There is a great deal of interest in developing methods to produce glucose syrups from cellulose using cellulase preparations. It is generally agreed that cheap glucose is the key intermediate for subsequent chemical and energy products. Activity of endo-glucanase and cellobiohydrolase of cellulolytic enzymes are inhibited by cellobiose. This inhibition is relieved by hydrolysis of cellobiose to glucose by cellobiase. Hence, cellobiase plays an important role in maximizing cellulose hydrolysis. Reference may be made to Archer B. D. and Peberdy J. F. (1997) Current Reviews in Biotechnology, volume 17(41), pages 273-306, wherein it has been stated that action of cellobiase on cellulose is the rate determining step of the entire cellulose hydrolysis, as cellobiase reduces the inhibition of cellulase activity by cellulose derived cellobiose and cellobiase is the key enzyme of cellulose hydrolysis. Reference may also be made to Reczy K., Brumbauer A., Book M., Szengyl Z. S. and Zacchi G. (1998) Applied Biochemistry and Biotechnology, volume 225, pages 70-72, wherein it has been stated that hydrolysis of cellulose by cellulases often results in a mixture of glucose, cellobiose and low molecular weight cellodextrins, and that cellobiose is nonfermentable for most yeasts, and therefore it has to be hydrolyzed to glucose by cellobiase (or β-glucosidase) prior to ethanol fermentation. Reference may be made to Kadam S. K. and Demain A. L. in Biochemical and Biophysical Research Communication (1989) volume 161, pages 706-711, which states that addition of cloned β-glucosidase enhances the degradation of crystalline cellulose by the Clostridium thermocellum cellulose complex.
The cellobiase activity secreted by Trichoderma/Aspergillus and its mutants was sub-optimal for conversion of cellulose to glucose. Efforts have been made to enhance the cellulolytic activity of Trichoderma/Neurospora/Aspergillus by media manipulations, by genetic engineering and by mutagenesis. Reference may be made to Abdel-Fattah A. F., Osman M. Y., Abdel-Naby M. A. Chemical Engineering Journal (1997) volume 68, pages 189-196, wherein a strain of Aspergillus niger A-20 was engineered for cellobiase production and so far the highest activity of cellobiase produced is 27.5 u/ml. Reference may be made to Strauss J. and Kubicek C. P. in Journal of General Microbiology (1991) volume 136, pages 1321-1326, where a mutant strain of fungus Trichoderma reesei M8, obtained by γ-irradiation of Trichoderma reesei QM9414, is said to produce 23 u/ml of cellobiase.
Similarly, efforts have been made to increase the production and secretion of cellobiase activity. Reference may be made to Sternberg D. and Mandels G. R. (1979) Journal of Bacteriology, volume 139, pages 761-769, wherein Sophorose was used for induction of cellulase and cellobiase in Trichoderma reesei. But cellobiase activity remained in the mycelium of the fungus. Reference may be made to Yazdi M. T., Woodward J. R. and Radford A. (1990), Journal of general Microbiology, Volume 136, pages 1313-1319, where effect of addition of some surfactants and amino acids were found to have adverse effect on production of cellobiase in Neurospora crassa. 
Reference may be made to Kubicek C. P., Panda T., Schreferl- Kunar G., Gruber F- and Messner R. (1987), Canadian Journal of Microbiology, Volume 33, pages 698-703, wherein production and secretion of cellobiase in Trichoderma reesei was studied in presence of glycosylation inhibitors Tunicamycin and 2-deoxy-D-glucose. Tunicamycin (0-50 μg/ml) had no effect on secretion of proteins in the fungus and 2-deoxy-D-glucose inhibited the biosynthesis of extracellular as well as intracellular protein over a wide range of concentration (0-150 μg/ml).
The drawback in hydrolysis of cellulose is that by using other methods for production of enhanced cellobiase activity, such as by media manipulations or by genetic engineering or by mutagenesis of the strains used for enzyme production, yield of cellobiase upto the maximum level of 27.5 u/ml could be obtained (in the culture medium of Aspergillus niger A-20 by Abdel-Fattah A. F., Osman M. Y., Abdel-Naby M. A. Chemical Engineering Journal (1997) volume 68, pages 189-196). Cellulolytic enzymes are generally reported to be glycoproteins and glycosylation is known to effect activation or retardation of protein synthesis, protein trafficking and secretion as well as in catalytic function and enzyme stability for extracellular proteins and enzymes. Use of glycosylation inhibitors in the growth medium of Trichoderma reesei however did not improve the production of enzyme, but affected secretion only.